IL323044A - Prognostic and therapeutic methods for non-small cell lung cancer - Google Patents

Description

WO 2024/186790 PCT/US2024/018472 PROGNOSTIC AND THERAPEUTIC METHODS FOR NON-SMALL CELL LUNG CANCER SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on February 14, 2024, is named 50474-320WO2_Sequence_Listing_2_14_24 and is 6,606 bytes in size.

FIELD OF THE INVENTION The present invention relates to prognostic and therapeutic methods for the treatment of non- small cell lung cancer (NSCLC) using expression levels of CD8+ T cell-associated genes. In particular, the invention provides methods for patient selection and treatment.

BACKGROUND Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer, accounting for approximately 80%-85% of all cases. For advanced disease, the overall five-year survival rate is 2%-4%. Despite improvements in the first-line treatment of patients with advanced NSCLC that have resulted in longer survival times and reduced disease-related symptoms, nearly all patients experience disease progression.Programmed cell death-1/programmed cell death ligand-1 (PD-1/PD-L1) blockade is efficacious across a broad range of malignancies. However, not all patients benefit, and a significant fraction of initial responders eventually relapse. One approach to extend and expand the impact of cancer immunotherapy has been to target additional immune checkpoints. One such co-inhibitory checkpoint is TIGIT (T cell immunoreceptor with ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain).Thus, there is an unmet need in the field for robust prognostic methods that identify patients likely to benefit from a treatment comprising atezolizumab and/or tiragolumab for more effective management of the disease.

SUMMARY OF THE INVENTION In one aspect, the invention provides a method of identifying an individual having a non-small cell lung cancer (NSCLC) who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual, wherein an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method for selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual, wherein an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.1 WO 2024/186790 PCT/US2024/018472 In some aspects, the individual has an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual, wherein the expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCRthat is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the benefit is a clinical response. In some aspects, the clinical response is a complete response (CR) or a partial response (PR).In some aspects, the individual has an expression level of one or more of CCL5, CXCR3, and CCR7 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CCR7 and the benefit is an increase in overall survival (OS) hazard ratio (HR).In some aspects, the individual has an expression level of one or more of CCL5, CXCR3, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CXCR6 and the benefit is an increase in overall survival (OS).In some aspects, the reference expression level of CCL5, CXCR3, or CXCR6 is a pre-assigned expression level.In some aspects, the reference expression level of CCL5, CXCR3, or CXCR6 is an expression level in a reference population. In some aspects, the expression level in the reference population is the median expression level of CCL5, CXCR3, or CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method for selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

WO 2024/186790 PCT/US2024/018472 In some aspects, the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In some aspects, the method comprises further detecting the expression level of CCR7 in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the expression level of CCR7 has been detected in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the benefit is a clinical response. In some aspects, the clinical response is a CR or a PR.In some aspects, the benefit is an increase in OS HR.In some aspects, the benefit is an increase in OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a referencepopulation. In some aspects, the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising:(a) detecting an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual and determining a Ccr7.2 gene signature score therefrom;(b) detecting an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB in a sample from the individual and determining a Ccr7.3 gene signature score therefrom; WO 2024/186790 PCT/US2024/018472 (c) detecting an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual and determining a Cxcr3 gene signature score therefrom;(d) detecting an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual and determining a Ccl5.1 gene signature score therefrom;(e) detecting an expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1 in a sample from the individual and determining an Ifit gene signature score therefrom;(f) detecting an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual and determining a Mitotic gene signature score therefrom;(g) detecting an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT in a sample from the individual and determining a Cytotox.2 gene signature score therefrom; or(h) detecting an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual and determining a Cytotox.4 gene signature score therefrom;wherein (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab,and (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method for selecting a therapy for an individual having a NSCLC, the method comprising:(a) detecting an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual and determining a Ccr7.2 gene signature score therefrom;(b) detecting an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB in a sample from the individual and determining a Ccr7.3 gene signature score therefrom;(c) detecting an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual and determining a Cxcr3 gene signature score therefrom;(d) detecting an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual and determining a Ccl5.1 gene signature score therefrom; WO 2024/186790 PCT/US2024/018472 (e) detecting an expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1 in a sample from the individual and determining an Ifit gene signature score therefrom;(f) detecting an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual and determining a Mitotic gene signature score therefrom;(g) detecting an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT in a sample from the individual and determining a Cytotox.2 gene signature score therefrom; or(h) detecting an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual and determining a Cytotox.4 gene signature score therefrom;wherein (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab,and (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the individual has (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising:(i) (a) detecting an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual and determining a Ccr7.2 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(b) detecting an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB in a sample from the individual and determining a Ccr7.3 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(c) detecting an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual and determining a Cxcr3 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; WO 2024/186790 PCT/US2024/018472 (d) detecting an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HOST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual and determining a Ccl5.1 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(e) detecting an expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1 in a sample from the individual and determining an Ifit gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(f) detecting an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual and determining a Mitotic gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(g) detecting an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT in a sample from the individual and determining a Cytotox.2 gene signature score therefrom, wherein the gene signature score is below a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; or(h) detecting an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual and determining a Cytotox.4 gene signature score therefrom, wherein the gene signature score is below a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and(II) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have:(a) a Ccr7.2 gene signature score based on an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual that is at or above a reference Ccr7.2 gene signature score;(b) a Ccr7.3 gene signature based on an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB in a sample from the individual that is at or above a reference Ccr7.3 gene signature score;(c) a Cxcr3 gene signature score based on an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual that is at or above a reference Cxcr3 gene signature score; WO 2024/186790 PCT/US2024/018472 (d) a Ccl5.1 gene signature score based on an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HOST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual that is at or above a reference Ccl5.1 gene signature score;(e) an Ifit gene signature score based on an expression level of each of ISG15, IFIT1B, I FITS, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 in a sample from the individual that is at or above a reference Ifit gene signature score;(f) a Mitotic gene signature score based on an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS1 2, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual that is at or above a reference Mitotic gene signature score;(g) a Cytotox.2 gene signature score based on an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1 B, LY6E, and DUT in a sample from the individual that is below a reference Cytotox.2 gene signature score; or(h) a Cytotox.4 gene signature score based on an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual that is below a reference Cytotox.gene signature score;thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.In some aspects, the individual has a Ccr7.3, Cxcr3, or Ccl5.1 gene signature score in the sample that is at or above a reference gene signature score and the benefit is a clinical response. In some aspects, the clinical response is a CR or a PR.In some aspects, the individual has (i) a Ccr7.2, Cxcr3, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score and the benefit is an increase in OS HR.In some aspects, the individual has a Ccr7.2, Ccr7.3, or Cxcr3 gene signature score in the sample that is at or above a reference gene signature score and the benefit is an increase in OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a reference population. In some aspects, the gene signature score in the reference population is the median Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene WO 2024/186790 PCT/US2024/018472 signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab.In another aspect, the invention provides a method for selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab.In some aspects, the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab; and (b) administering an effective amount of atezolizumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCRdetected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In some aspects, the method comprises further detecting the expression level of CCR7 in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the expression level of CCR7 has been detected in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the benefit is an increase in progression-free survival (PFS) or OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a reference population. In some aspects, the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In some aspects, the treatment comprising atezolizumab is atezolizumab monotherapy.In another aspect, the invention provides use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in WO 2024/186790 PCT/US2024/018472 a sample from the individual that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the benefit is a clinical response. In some aspects, the clinical response is a complete response (CR) or a partial response (PR).In some aspects, the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and CCR7 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CCR7 and the benefit is an increase in overall survival (OS) hazard ratio (HR).In some aspects, the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CXCR6 and the benefit is an increase in overall survival (OS).In some aspects, the reference expression level of CCL5, CXCR3, or CXCR6 is a pre-assigned expression level.In some aspects, the reference expression level of CCL5, CXCR3, or CXCR6 is an expression level in a reference population. In some aspects, the expression level in the reference population is the median expression level of CCL5, CXCR3, or CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In some aspects, the expression level of CCR7 has been detected in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the benefit is a clinical response. In some aspects, the clinical response is a CR or a PR.In some aspects, the benefit is an increase in OS HR.In some aspects, the benefit is an increase in OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a referencepopulation. In some aspects, the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have: WO 2024/186790 PCT/US2024/018472 (i)(a) a Ccr7.2 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X detected in a sample from the individual;(b) a Ccr7.3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB detected in a sample from the individual;(c) a Cxcr3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 detected in a sample from the individual;(d) a Ccl5.1 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 detected in a sample from the individual;(e) an Ifit gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 detected in a sample from the individual;(f) a Mitotic gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL detected in a sample from the individual;(g) a Cytotox.2 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT detected in a sample from the individual; or(h) a Cytotox.4 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of S100A6, WO 2024/186790 PCT/US2024/018472 NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.In some aspects, the individual has been determined to have a Ccr7.3, Cxcr3, or Ccl5.1 gene signature score in the sample that is at or above a reference gene signature score and the benefit is a clinical response. In some aspects, the clinical response is a CR or a PR.In some aspects, the individual has been determined to have (i) a Ccr7.2, Cxcr3, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.or Cytotox.4 gene signature score that is below a reference gene signature score and the benefit is an increase in OS HR.In some aspects, the individual has been determined to have a Ccr7.2, Ccr7.3, or Cxcr3 gene signature score in the sample that is at or above a reference gene signature score and the benefit is an increase in OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a reference population. In some aspects, the gene signature score in the reference population is the median Ocr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides use of atezolizumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In some aspects, the expression level of CCR7 has been detected in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the benefit is an increase in progression-free survival (PFS) or OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a reference population. In some aspects, the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In some aspects, the treatment comprising atezolizumab is atezolizumab monotherapy.In another aspect, the invention provides atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual that is at or above a WO 2024/186790 PCT/US2024/018472 reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the benefit is a clinical response. In some aspects, the clinical response is a complete response (CR) or a partial response (PR).In some aspects, the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and CCR7 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CCR7 and the benefit is an increase in overall survival (OS) hazard ratio (HR).In some aspects, the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CXCR6 and the benefit is an increase in overall survival (OS).In some aspects, the reference expression level of CCL5, CXCR3, or CXCR6 is a pre-assigned expression level.In some aspects, the reference expression level of CCL5, CXCR3, or CXCR6 is an expression level in a reference population. In some aspects, the expression level in the reference population is the median expression level of CCL5, CXCR3, or CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In some aspects, the expression level of CCR7 has been detected in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the benefit is a clinical response. In some aspects, the clinical response is a CR or a PR.In some aspects, the benefit is an increase in OS HR.In some aspects, the benefit is an increase in OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a referencepopulation. In some aspects, the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have: WO 2024/186790 PCT/US2024/018472 (i)(a) a Ccr7.2 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X detected in a sample from the individual;(b) a Ccr7.3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB detected in a sample from the individual;(c) a Cxcr3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 detected in a sample from the individual;(d) a Ccl5.1 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 detected in a sample from the individual;(e) an Ifit gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 detected in a sample from the individual;(f) a Mitotic gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL detected in a sample from the individual;(g) a Cytotox.2 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT detected in a sample from the individual; or(h) a Cytotox.4 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of S100A6, WO 2024/186790 PCT/US2024/018472 NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.In some aspects, the individual has been determined to have a Ccr7.3, Cxcr3, or Ccl5.1 gene signature score in the sample that is at or above a reference gene signature score and the benefit is a clinical response. In some aspects, the clinical response is a CR or a PR.In some aspects, the individual has been determined to have (i) a Ccr7.2, Cxcr3, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.or Cytotox.4 gene signature score that is below a reference gene signature score and the benefit is an increase in OS HR.In some aspects, the individual has been determined to have a Ccr7.2, Ccr7.3, or Cxcr3 gene signature score in the sample that is at or above a reference gene signature score and the benefit is an increase in OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a reference population. In some aspects, the gene signature score in the reference population is the median Ocr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In another aspect, the invention provides atezolizumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In some aspects, the expression level of CCR7 has been detected in the sample from the individual. In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.In some aspects, the benefit is an increase in progression-free survival (PFS) or OS.In some aspects, the reference gene signature score is a pre-assigned gene signature score.In some aspects, the reference gene signature score is a gene signature score in a reference population. In some aspects, the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population. In some aspects, the reference population is a population of individuals having the NSCLC.In some aspects, the treatment comprising atezolizumab is atezolizumab monotherapy.In some aspects, the expression level is a nucleic acid expression level or a protein expression level.

WO 2024/186790 PCT/US2024/018472 In some aspects, the expression level is a nucleic acid expression level. In some aspects, the nucleic acid expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof.In some aspects, the nucleic acid expression level is an mRNA expression level. In some aspects, the mRNA expression level is determined by RNA-seq.In some aspects, the expression level is a protein expression level. In some aspects, the protein expression level is determined by mass spectrometry.In some aspects, the sample is obtained from the individual prior to treatment with atezolizumab and/or tiragolumab.In some aspects, the sample is a tissue sample, a tumor sample, a blood sample, a plasma sample, a serum sample, or a combination thereof.In some aspects, the sample is a tissue sample. In some aspects, the tissue sample is a tumor tissue sample. In some aspects, the tumor tissue sample is a biopsy.In some aspects, the tissue sample is a tumor draining lymph node (dLN) sample.In some aspects, the sample is a blood sample.In some aspects, the sample is an archival sample, a fresh sample, or a frozen sample.In some aspects, the individual has a PD-L1-positive NSCLC. In some aspects, the PD-L1- positive NSCLC has been determined to have a PD-L1-positive tumor cell fraction by an immunohistochemical (IHC) assay. In some aspects, the PD-L1 -positive tumor cell fraction is determined by positive staining with an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is SP263, 22C3, SP142, or 28-8.In some aspects, the individual is a human.In some aspects, the individual has not previously been treated for NSCLC.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1Ais a set of graphs showing tumor volume (mm3) (Iog2 scale) over time in BALB/c mice that were inoculated subcutaneously with syngeneic CT26 tumor cells and treated with an isotype control, anti-PD-L1, anti-TIGIT, or the combination of anti-PD-L1 and anti-TIGIT antibodies, with or without FTY720, an agent that blocks T cell egress from tumor draining lymph nodes (dLN). Grouped analysis (left panel) and growth curves for each individual animal (n = 10 per group) (right panels) are shown. Tumor growth efficacy study is representative of three independent experiments. FIG. 1Bis a set of bar graphs showing the frequency of CD8+ T cells with positive staining for the gp70-specific tetramer in dLN (left panel); the number of CD8+ T cells with positive staining for the gp70- specific tetramer in blood (right panel); and the frequency of CD8+ T cells with positive staining for the gp70-specific tetramer in tumor (right panel) in BALB/c CT26 tumor model mice treated with an isotype control, anti-PD-L1, anti-TIGIT, and/or FTY720. p-values are indicated where differences between two groups were determined by two-way unpaired Student’s t-test to be statistically significant. FIG. 1Cis a bar graph showing quantitation of CD8+ T cells producing IFN-g and TNF-a as a percentage of total CD8+ tumor-infiltrating lymphocytes (TILs) in BALB/c CT26 tumor model mice treated with an isotype control, anti-PD-L1, anti-TIGIT, and/or FTY720. Pharmacodynamic data are WO 2024/186790 PCT/US2024/018472 representative of three independent experiments (n = 5 per group), p-values are indicated where differences between two groups were determined by two-way unpaired Student’s t-test to be statistically significant. FIG. 1Dis a set of graphs showing tumor volume (mm3) (Iog2 scale) over time in BALB/c mice that were inoculated subcutaneously with syngeneic CT26 tumor cells, treated with an isotype control or the combination of anti-PD-L1 and anti-TIGIT antibodies, and treated with FTY720 at day 0, one day prior to initiation of therapy, or on day 7 after one week of therapy (delayed FTY720). Grouped analysis (left panel) and growth curves for each individual animal (n = 10 per group) (right panel) are shown. Tumor growth efficacy study is representative of three independent experiments. FIG. 2Ais a Uniform Manifold Approximation and Projection (UMAP) showing 174,514 CD8+ T cells isolated from tumor, dLN, and blood of CT26 tumor model mice shaded by cluster. FIG. 2B is a heatmap showing the relative average expression of selected marker genes associated with CD8+ T cell phenotype, function, or differentiation state in each cluster identified in the UMAP shown in Fig. 2A. FIG. 2Cis a set of stacked bar graphs of CD8+ T cell cluster composition in lymph nodes and blood under the indicated treatment condition. In each stacked bar, an open bar denotes singletons, a solid bar denotes numbers for clones with less than 100 cells, and a hatched bar denotes numbers for clones with 100 or more cells. FIG. 2Dis a set of stacked bar graphs of CD8+ T cell cluster composition in blood (Day 7) and tumor under the indicated treatment condition. In each stacked bar, an open bar denotes singletons, a solid bar denotes numbers for clones with less than 100 cells, and a hatched bar denotes numbers for clones with 100 or more cells. FIG. 3Ais an image showing relative clone sizes projected on the CD8+ T cell UMAP shown in Fig. 2A. Clonal diversity was determined by T cell receptor sequencing (TCR-seq). FIG. 3B is an image showing antibody-derived tag (ADT) counts measured from cellular indexing of transcriptomes and epitopes (CITE-seq) projected on the CD8+ T cell UMAP shown in Fig. 2A. FIG. 3Cis an image showing the specificity of clones for gp70 subdivided by high (> 100) or low (< 100) ADT count projected on the CD8+ T cell UMAP shown in Fig. 2A and a stacked bar graph showing the fraction of cells in each cluster that are gp70+, gp70-, and have a high (> 100) or low (< 100) ADT count. FIG. 3Dis a set of scatterplots showing primary clusters of each individual clonotype in dLN (upper panels) or tumor (lower panels). Shade of circles denote cluster designation. Size of circles is representative of clonotype numbers detected in blood at day 7. FIG. 3Eis a set of scatterplots showing gp70 specificity and ADT count for individual clones. FIG. 3Fis a set of stacked bar graphs showing the cluster composition of the top 50 largest clones in tumor with matching clonotypes, based on identical TOR usage, in dLN and blood, in absolute numbers. Cluster identity is indicated by shade. FIG. 4Ais a set of UMAPs showing a cluster co-occurrence analysis in dLN (lymph), blood, and tumor samples from CT26 tumor model mice treated with a control anti-gp120 treatment. Lines within UMAPs denote co-occurrence between different clusters within the indicated tissue. Lines between WO 2024/186790 PCT/US2024/018472 UMAPs denote co-occurrence between the same cluster in different tissues. Thickness of line denotes relative strength of co-occurrence, with thickest lines indicating strongest co-occurrence. For lines between tissues, shade of the line indicates the cluster. FIG. 4B is a set of UMAPs showing a cluster co-occurrence analysis in dLN (lymph), blood, and tumor samples from CT26 tumor model mice treated with an anti-PD-L1 antibody. FIG. 4Cis a set of UMAPs showing a cluster co-occurrence analysis in dLN (lymph), blood, and tumor samples from CT26 tumor model mice treated with an anti-TIGIT antibody. FIG. 40 is a set of UMAPs showing a cluster co-occurrence analysis in dLN (lymph), blood, and tumor samples from CT26 tumor model mice treated with an anti-TIGIT antibody and an anti-PD-Lantibody. FIG. 4Eis a set of UMAPs showing a cluster co-occurrence analysis in dLN (lymph), blood, and tumor samples from CT26 tumor model mice treated with an anti-TIGIT antibody, an anti-PD-L1 antibody, and FTY720. FIG. 5Ais a plot showing the proportion of gp70+CD8+ T cells from dLN of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT with or without FTY720 (FTY) that expressed CD226. p-values are indicated where differences between two groups were determined by two-way unpaired Student’s t-test to be statistically significant. FIG. 5Bis a set of plots showing the proportion of CD226+ (left panel) or CD226- (right panel) gp70+CD8+ T cells from dLN of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD- L1 + anti-TIGIT with or without FTY720 (FTY) that expressed Ki67. FIG. 5Cis a set of plots showing the proportion of CD226+ (left panel) or CD226 (right panel) gp70+CD8+ T cells from dLN of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD- L1 + anti-TIGIT with or without FTY720 (FTY) that had a naive phenotype. FIG. 50is a set of plots showing the proportion of CD226+ (left panel) or CD226- (right panel) gp70+CD8+ T cells from dLN of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD- L1 + anti-TIGIT with or without FTY720 (FTY) that had a cytotoxic CD8+ T effector / memory cell (Teff/Tem) phenotype. FIG. 5Eis a set of plots showing the proportion of CD226+ (left panel) or CD226 (right panel) gp70+CD8+ T cells from dLN of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD- L1 + anti-TIGIT with or without FTY720 (FTY) that expressed PD-1. FIG. 5Fis a set of plots showing the proportion of CD226+ (left panel) or CD226 (right panel) gp70+CD8+ T cells from dLN of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD- L1 + anti-TIGIT with or without FTY720 (FTY) that expressed TCF1 and Tim3. FIG. 5Gis a set of plots showing the proportion of CD226+ (left panel) or CD226 (right panel) gp70+CD8+ T cells from dLN of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD- L1 + anti-TIGIT with or without FTY720 (FTY) that expressed Tox. FIG. 5His a plot showing the proportion of gp70+CD8+ T cells from tumor tissue of CT26 tumor- bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT with or without FTY720 (FTY) that expressed CD226.

WO 2024/186790 PCT/US2024/018472 FIG. 51is a set of plots showing the proportion of CD226+ (left panel) or CD226 (right panel) gp70+CD8+ T cells from tumor tissue of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT with or without FTY720 (FTY) that expressed Ki67. FIG. 5Jis a set of plots showing the proportion of CD226+ (left panel) or CD226- (right panel) gp70+CD8+ T cells from tumor tissue of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT with or without FTY720 (FTY) that expressed TCF1 and Tim3. FIG. 5Kis a set of plots showing the proportion of CD226+ (left panel) or CD226 (right panel) gp70+CD8+ T cells from tumor tissue of CT26 tumor-bearing mice treated with anti-PD-L1, anti-TIGIT, or anti-PD-L1 + anti-TIGIT with or without FTY720 (FTY) that expressed Tox. FIG. 5Lis a set of plots showing the proportion of dLN (left panel) or tumor (right panel) gp70+CD8+ T cells from CT26 tumor-bearing mice treated with anti-PD-L1 + anti-TIGIT, anti-PD-L1 + anti- TIGIT + anti-CD226, or a control that expressed TCF1 and Tim3. FIG. 5Mis a set of plots showing the proportion of dLN (left panel) or tumor (right panel) gp70+CD8+ T cells from CT26 tumor-bearing mice treated with anti-PD-L1 + anti-TIGIT, anti-PD-L1 + anti- TIGIT + anti-CD226, or a control that expressed Tox. FIG. 6Ais a set of box-and-whisker plots showing associations between levels of the indicated human gene signature scores in baseline tumor bulk RNA-seq samples from the Phase 2 CITYSCAPE non-small cell lung cancer (NSCLC) trial and clinical response to tiragolumab plus atezolizumab. Patients, irrespective of treatment arm, were separated on the basis of clinical response (CRPR, complete response/partial response; SDPD, stable disease/progressive disease), p-values are indicated for statistically significant differences by two-tailed t-test. FIG. 6B is a set of box-and-whisker plots showing associations between expression levels of the indicated individual human genes in baseline tumor bulk RNA-seq samples from Phase 2 CITYSCAPE NSCLC trial patients that were treated with tiragolumab plus atezolizumab or placebo plus atezolizumab, separated on the basis of clinical response. FIG. 6Cis a forest plot showing association of high or low expression of indicated individual human gene with overall survival (OS) hazard ratio (HR) in tiragolumab plus atezolizumab (T+A) or placebo plus atezolizumab (P+A) treatment groups. Mean HR with 95% confidence intervals and p- values are shown. FIG. 60is a set of Kaplan-Meier (K-M) curves showing the probability of OS in P+A or T+A treatment groups dichotomized on the basis of high or low expression of indicated gene, p-value is from a log-rank test with the null hypothesis that there is no difference between the groups. FIG. 6Eis a set of box-and-whisker plots showing a gene score calculated using the average expression of the CDS gene panel comprised of CCR7, CXCR3, CXCR6 and CCL5 (top) or CXCR3, CXCR6 and CCL5 (bottom) in tumor bulk RNA-seq samples from patients treated with tiragolumab plus atezolizumab separated on the basis of clinical response. FIG. 6Fis a forest plot showing association of high or low expression of the indicated composite gene scores with OS HR in T+A or P+A treatment groups. Mean HR with 95% confidence intervals and p- values are shown.

WO 2024/186790 PCT/US2024/018472 FIG. 6Gis a set of K-M curves showing the probability of OS in P+A or T+A treatment groups dichotomized on the basis of high or low composite gene score, p-value is from a log-rank test with the null hypothesis that there is no difference between the groups. FIG. 7Ais a set of diagrams showing experimental schema for tumor growth and pharmacodynamic (PD) analysis for the CT26 and EO771 studies (left); delayed FTY720 treatment in the CT26 model (middle); and multi-omic analysis of T cells in the CT26 tumor model (right). FIG. 7Bis a set of graphs showing tumor volume (mm3) (Iog2 scale) over time in C57BL/6 mice that were inoculated in mammary fat pad with EO771 tumor cells, treated with an isotype control anti-PD- L1, anti-TIGIT, or the combination of anti-PD-L1 and anti-TIGIT antibodies, with or without FTY720. Grouped analysis (left panel) and growth curves for each individual animal (n = 10 per group) (right panel) are shown. Data shown are representative of two independent experiments. FIG. 7Cis a set of plots showing the total numbers of CD8+ T cells, CD4+ T cells, or Tregs in dLN (top panels) or tumor (bottom panels) of CT26 tumor-bearing mice treated with isotype control, anti-PD- L1, anti-TIGIT, or the combination, with or without FTY720. n = 5 mice per group, mean ± s.d. are represented by bars and whiskers. Data are representative of three independent experiments. FIG. 70is a set of bar graphs showing the frequency of CD8+ T cells that had positive staining for the gp70-specific tetramer in dLN (left panel); the number of CD8+ T cells with positive staining for the gp70-specific tetramer in blood (right panel); and the frequency of CD8+ T cells with positive staining for the gp70-specific tetramer in tumor (right panel) in BALB/c CT26 tumor model mice treated with isotype control, anti-PD-L1 (denoted as aPDL1 or aP), anti-TIGIT (denoted as aTIGIT or aT), or the combination of anti-PD-L1 and anti-TIGIT antibodies, with or without FTY720. Data are a compilation of 2 (blood) or (dLN, tumor) independent experiments (n = 5 per group per experiment), with each dot representing one animal. Bars represent mean, whiskers represent s.d. p-values are indicated where differences between two selected groups were determined by Mann-Whitney test to be statistically significant. *, p < 0.05; ", p < 0.01; p < 0.001; p < 0.0001. Fig. 7Eis a bar graph showing quantitation of CD8+ T cells producing IFN-g and TNF-a as a percentage of total CD8+ tumor-infiltrating lymphocytes (TILs) in BALB/c CT26 tumor model mice treated with an isotype control, anti-PD-L1, anti-TIGIT, and/or FTY720. Data are a compilation of 2 independent experiments (n = 5 per group per experiment), p-values are indicated where differences between two selected groups were determined by Mann-Whitney test to be statistically significant. *, p < 0.05; **, p < 0.01; p < 0.001; p < 0.0001. FIG. 8Ais a set of representative dot plots showing IFN-g and TNF-a intracellular staining in CD8+ T cells from tumor. FIG. 8Bis a set of representative dot plots for CD226 and PD-1 co-expression on CD8+ T cells in dLN (top) and for Tim3 and PD-1 co-expression on CD8+ T cells in dLN (bottom). FIG. 8Cis a set of representative dot plots for CD226 and LAGS co-expression on CD8+ T cells in tumor (top) and for Tim3 and PD-1 co-expression on CD8+ T cells in tumor (bottom). FIG. 80is a set of representative dot plots for TCF1 and Tox co-expression on CD8+ T cells in dLN (top) or in tumor (bottom).

WO 2024/186790 PCT/US2024/018472 FIG. 8Eis a set of representative dot plots for SLAMF6 and TCF1 co-expression on CD8+ T cells in tumor. FIG. 8Fis a set of representative dot plots for CD226 and gp70 tetramer co-expression on CD8+ T cells in dLN (top) or tumor (middle), or gp70 tetramer staining on CD8+ T cells in blood (bottom). FIG. 9Ais a set of images showing T cell clusters representing CD8+ T cells, CD4+ T cells, and regulatory T cells in 305,908 total T cells pooled from CT26 tumor, dLN, and blood from 31 mice. CD8+ T cell clusters (Ccl5-1, Ribo-2, Ccl5-3, CD8-4, CD8-5, Mitotic-9, Mitotic-12, Xcl1 -13, lfn-15, and Ly6a-16), four conventional CD4+ T cell clusters (CD40lg-0, CD40lg-7, Vim-11, and Statl -14), two regulatory T (Treg) cell clusters (Treg-6, Treg-8), three clusters of mitotic cells (Mitotic-9, Mitotic-12, and Mitotic-20), two clusters of dying cells (Dying-10 and Dying-17), and several clusters with no clear phenotype (Gxp1-19, CD74-18, and CD74-21) were classified. CD8a expression, granzyme B expression, tissue source, treatment group (Group), clone size, ADT counts, and gp70 antigen specificity projections on UMAR are shown as indicated. FIG. 9B is a set of images showing CITE-seq relative expression levels for the indicated markers projected on a UMAR. FIG. 10Ais a set of images showing relative expression levels of the indicated markers, as measured by CITE-seq, projected on UMAPs comprised of CD8+ T cells from CT26 tumor, dLN, and blood. FIG. 10B is a heatmap showing relative CITE-seq marker expression levels in the indicated tissues under various treatment conditions. FIG. 11Ais a set of heatmaps showing cross-labelling of CD8+ T cell clusters (rows) to reference gene signatures (columns), taken from the analyses of Huang et al., Cell, 185:1-18, 2022; Deak et al., Nature, 610: 173-181,2022); Hashimoto et al., Nature, 610: 173-181,2022; and Daniel et al., Nat Immunol, 23: 1614-1627, 2022, with intensities indicating normalized frequency. FIG. 11B is a set of UMAPs showing cluster composition in dLN (lymph), tumor, and blood from CT26 tumor-bearing mice treated with isotype control, anti-PD-L1, anti-TIGIT, combination, or combination with FTY720. FIG. 12Ais a set of scatterplots showing primary clusters of each of the indicated individual clonotypes in dLN and blood at day 7. The shade of the circles denotes cluster designation as shown in Fig. 3D. FIG. 12B is a set of stacked bar graphs showing cluster composition for the 50 largest clonotypes from tumor (upper panels), lymph node (middle panels), or blood at day 7 (lower panels) of CT26 tumor- bearing mice treated with isotype control, anti-PD-L1, anti-TIGIT, combination, or combination with FTY720, shown as absolute numbers. Cluster identity is indicated by shade. FIG. 12Cis a set of stacked bar graphs showing cluster composition for the 50 largest clonotypes from tumor (upper panels), lymph node (middle panels), or blood at day 7 (lower panels) of CT26 tumor- bearing mice treated with isotype control, anti-PD-L1, anti-TIGIT, combination, or combination with FTY720, normalized against the total number of cells for each individual clonotype (total cell number = 1). Cluster identity is indicated by shade.

WO 2024/186790 PCT/US2024/018472 FIG. 13Ais a heatmap showing relative co-occurrence relationships within or between dLN, blood, and tumors for CD8+ T cells clusters from CT26 tumor-bearing mice. Greater intensity of co- occurrence between two clusters indicates greater similarity, indicative of shared differentiation trajectory. FIG. 13Bis a heatmap showing clusters defined using metadata from Li et al., J Exp Med, 219: 620210749, 2022, wherein CD8T1 are likely naive, CD8T2 has Tscm/memory properties, CD8T4 has activated effector cell properties, and CD8T5 has an exhausted phenotype. CD8T1 and CD8T2 were described as T cells that recently entered the tumor, while CD8T4 and CD8T5 were cells resident in tumor. CD8T2 also have the capacity to recirculate from tumor to dLN. FIG. 14Ais a set of charts showing the frequencies of CD8+ T cells in dLN (left) or tumor (right) as a percentage of CD45+ cells in mice with established EO771 tumors that were treated with an isotype control Ab or anti-TIGIT combined with anti-PD-L1. dLN and tumors were collected on day 7 post- treatment for phenotypic characterization of CD8+ T cells by flow cytometry, n = 4 per group; mean and s.d. are shown as bar and whiskers; data are representative of one of two independent experiments, p- values are indicated where differences were determined by unpaired t-test to be statistically significant. FIG. 14Bis a set of charts showing the frequencies of CD8+ T cells expressing CD226 in dLN (left) or tumor (right) as a percentage of CD45+ cells in mice with established EO771 tumors that were treated with an isotype control Ab or anti-TIGIT combined with anti-PD-L1. dLN and tumors were collected on day 7 post-treatment for phenotypic characterization of CD8+ T cells by flow cytometry, n = per group; mean and s.d. are shown as bar and whiskers; data are representative of one of two independent experiments, p-values are indicated where differences were determined by unpaired t-test to be statistically significant. FIG. 14Cis a set of charts showing the frequencies of CD8+ T cells positively stained with tetramer against EO771-specific antigen p15E in dLN (left) or tumor (right) as a percentage of CD45+ cells in mice with established EO771 tumors that were treated with an isotype control Ab or anti-TIGIT combined with anti-PD-L1. dLN and tumors were collected on day 7 post-treatment for phenotypic characterization of CD8+ T cells by flow cytometry, n = 4 per group; mean and s.d. are shown as bar and whiskers; data are representative of one of two independent experiments, p-values are indicated where differences were determined by unpaired t-test to be statistically significant. FIG. 14Dis a set of charts showing the frequencies of p15E+CD8+ T cells expressing CD226 in dLN (left) or tumor (right) as a percentage of CD45+ cells in mice with established EO771 tumors that were treated with an isotype control Ab or anti-TIGIT combined with anti-PD-L1. dLN and tumors were collected on day 7 post-treatment for phenotypic characterization of CD8+ T cells by flow cytometry, n = per group; mean and s.d. are shown as bar and whiskers; data are representative of one of two independent experiments, p-values are indicated where differences were determined by unpaired t-test to be statistically significant. FIG. 14Eis a set of charts showing the frequencies of CD226+ (left) or CD226 (right) p15E+CD8+ Teff/Tem cells co-expressing TCF1 and Tim3 in dLN (left) or tumor (right) as a percentage of CD45+ cells in mice with established EO771 tumors that were treated with an isotype control Ab or anti-TIGIT combined with anti-PD-L1. dLN and tumors were collected on day 7 post-treatment for phenotypic characterization of CD8+ T cells by flow cytometry, n = 4 per group; mean and s.d. are shown as bar and WO 2024/186790 PCT/US2024/018472 whiskers; data are representative of one of two independent experiments, p-values are indicated where differences were determined by unpaired t-test to be statistically significant. FIG. 14Fis a set of charts showing the frequencies of CD226+ (left) or CD226- (right) p15E+CD8+ Teff/Tem cells co-expressing Tox in dLN (left) or tumor (right) as a percentage of CD45+ cells in mice with established EO771 tumors that were treated with an isotype control Ab or anti-TIGIT combined with anti- PD-L1. dLN and tumors were collected on day 7 post-treatment for phenotypic characterization of CD8+ T cells by flow cytometry, n = 4 per group; mean and s.d. are shown as bar and whiskers; data are representative of one of two independent experiments, p-values are indicated where differences were determined by unpaired t-test to be statistically significant. FIG. 15Ais an image showing scRNA-seq of 144,413 human CD8+ T cells from blood of patients in a Phi b NSCLC study of tiragolumab plus atezolizumab (T+A) in which human genes were renamed to their mouse ortholog (if present) and gene expression was normalized before sample integration and projection onto a mouse CD8+ T cell reference UMAP. FIG. 15B is a set of plots showing predicted cell type scores for mapped human CD8+ T cells for each assigned mouse CD8+ T cell reference cluster. FIG. 15Cis a set of box-and-whisker plots showing frequencies of the indicated human predicted clusters in patients with complete response or partial response (CRPR) compared to stable disease or progressive disease (SDPD) on cycle 2 day 1 of treatment with T+A in the Phi b study. Percent total was calculated as the percentage of the cluster in total CD8+ T cells for each patient. FIG. 15Dis a forest plot showing the association between high or low expression of the top corresponding human 18-20 signature genes (signature gene score) from each mouse CD8+ T cell cluster or CD8A and overall survival (OS) hazard ratio (HR) T+A or placebo plus atezolizumab (P+A) treatment groups in the Ph2 CITYSCAPE study. Mean HR with 95% confidence intervals and p-values are shown. FIG. 15Eis a set of Kaplan-Meier curves showing OS probability in P+A or T+A treatment groups dichotomized on the basis of high or low CD8+ T cell cluster gene scores from each reference cluster, p- value is from a log-rank test with the null hypothesis that there is no difference between the groups. FIG. 15Fis a set of Kaplan-Meier curves comparing progression-free survival (PFS, left) or OS (right) in patients from the phase 3 NSCLC OAK study who received atezolizumab monotherapy. Patients were dichotomized by median gene score calculated using the average expression of the CDS gene panel comprised of CCR7, CXCR3, CXCR6 and CCL5. FIG. 16 is a diagram showing a model of anti-TIGIT plus anti-PD-L1 combination effects on tumor-specific CD8+ T cell differentiation in the syngeneic mouse CT26 tumor model. Combination treatment drives dual expansion of tumor-specific gp70+CD8+ T cell clones in both dLN and tumor, and egress from dLN and trafficking to tumor is required initially until the tumor is sufficiently seeded by infiltrating cells. Dual expanded gp70+CD8+ T cell clones highly express CD226 and Ccl5 and have properties of stem cell-like memory / precursor exhausted (Tscm/Tpex) cells such as TCF-1 expression and reduced exhaustion programming as marked by reduction in Tox expression, thereby increasing the pool of CD8+ T cells with antitumor effector activity at the expense of exhaustion. Anti-TIGIT alone phenocopies gp70+CD8+ T cells responding to combination treatment but does not drive robust dual WO 2024/186790 PCT/US2024/018472 expansion. Anti-PD-L1 alone does not promote dual expansion nor does it confer protection from the exhaustion pathway. FIG. 17 is a set of Kaplan-Meier curves showing progression-free survival (PFS, left) or OS (right) in the PD-L1 -positive subset (TPS > 1 %) of patients from the IMpowerl 10 study who received atezolizumab monotherapy. Patients were dichotomized by median gene score calculated using the average expression of the CDS gene panel comprised of CCR7, CXCR3, CXCR6 and CCL5.

DETAILED DESCRIPTION 1. GENERAL TECHNIQUES AND DEFINITIONS The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (F.M. Ausubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney), ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. De Vita et al., eds., J.B. Lippincott Company, 1993).It is to be understood that aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments. As used herein, the singular form "a," "an," and "the" includes plural references unless indicated otherwise.The term "about" as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X."The "amount," "level," or "expression level," used herein interchangeably, of a biomarker is a detectable level in a biological sample. "Expression" generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into the structures present and operating in the cell.23 WO 2024/186790 PCT/US2024/018472 Therefore, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). Expression levels can be measured by methods known to one skilled in the art and also disclosed herein.The terms "detecting" and "detection" are used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels. Detecting may be direct or indirect.The presence and/or expression level/amount of various biomarkers described herein in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, quantitative blood based assays (e.g., Serum ELISA), biochemical enzymatic activity assays, in situ hybridization, fluorescence in situ hybridization (FISH), Southern analysis, Northern analysis, whole genome sequencing, massively parallel DNA sequencing (e.g., next-generation sequencing), NANOSTRING®, polymerase chain reaction (PCR) including quantitative real time PCR (qRT-PCR) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like, RNA-seq, microarray analysis, gene expression profiling, and/or serial analysis of gene expression ("SAGE"), as well as any one of the wide variety of assays that can be performed by protein, gene, and/or tissue array analysis. Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD") may also be used.The term "C-C motif chemokine 5" or "CCL5," as used herein, broadly refers to any native CCLfrom any mammalian source, including primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses full-length CCL5 and isolated regions or domains of CCL5, e.g., the CCL5 ECD. The term also encompasses naturally occurring variants of CCL5, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CCL5 is shown under UniProt Accession No. P13501. Minor sequence variations, especially conservative amino acid substitutions of CCL5 that do not affect CCL5 function and/or activity, are also contemplated by the invention.The term "C-X-C chemokine receptor type 3" or "CXCR3," as used herein, broadly refers to any native CXCR3 from any mammalian source, including primates (e.g., humans) and rodents (e.g., mice WO 2024/186790 PCT/US2024/018472 and rats), unless otherwise indicated. The term encompasses full-length CXCR3 and isolated regions or domains of CXCR3, e.g., the CXCR3 ECD. The term also encompasses naturally occurring variants of CXCR3, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CXCR3 is shown under UniProt Accession No. P49682. Minor sequence variations, especially conservative amino acid substitutions of CXCR3 that do not affect CXCR3 function and/or activity, are also contemplated by the invention.The term "C-X-C chemokine receptor type 6" or "CXCR6," as used herein, broadly refers to any native CXCR6 from any mammalian source, including primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses full-length CXCR6 and isolated regions or domains of CXCR6, e.g., the CXCR6 ECD. The term also encompasses naturally occurring variants of CXCR6, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CXCR6 is shown under UniProt Accession No. 000574. Minor sequence variations, especially conservative amino acid substitutions of CXCR6 that do not affect CXCR6 function and/or activity, are also contemplated by the invention.The term "C-C chemokine receptor type 7" or "CCR7," as used herein, broadly refers to any native CCR7 from any mammalian source, including primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses full-length CCR7 and isolated regions or domains of CCR7, e.g., the CCR7 ECD. The term also encompasses naturally occurring variants of CCR7, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human CCR7 is shown under UniProt Accession No. P32248. Minor sequence variations, especially conservative amino acid substitutions of CCR7 that do not affect CCR7 function and/or activity, are also contemplated by the invention.The term "TIGIT" or "T-cell immunoreceptor with ig and ITIM domains" as used herein refers to any native TIGIT from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. TIGIT is also known in the art as DKFZp667A205, FLJ39873, V-set and immunoglobulin domain-containing protein 9, V-set and transmembrane domain-containing protein 3, VSIG9, VSTM3, and WUCAM. The term encompasses "full-length," unprocessed TIGIT (e.g., full-length human TIGIT having the amino acid sequence of SEQ ID NO: 1), as well as any form of TIGIT that results from processing in the cell (e.g., processed human TIGIT without a signal sequence, having the amino acid sequence of SEQ ID NO: 2). The term also encompasses naturally occurring variants of TIGIT, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human TIGIT may be found under UniProt Accession Number Q495A1.The term "PD-L1" or "Programmed Cell Death Ligand 1" refers herein to any native PD-L1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. PD-L1 is also known in the art as CD274 molecule, CD274 antigen, Bhomolog 1, PDCD1 Ligand 1, PDCD1LG1, PDCD1L1, B7H1, PDL1, programmed death ligand 1, B7-H1, and B7-H. The term also encompasses naturally occurring variants of PD-L1, e.g., splice variants, or allelic variants. The amino acid sequence of an exemplary human PD-L1 may be found under UniProt Accession Number Q9NZQ7 (SEQ ID NO: 3).As used herein, the term "atezolizumab" refers to anti-PD-L1 antagonist antibody having the WO 2024/186790 PCT/US2024/018472 International Nonproprietary Names for Pharmaceutical Substances (INN) List 112 (WHO Drug Information, Vol. 28, No. 4, 2014, p. 488), or the CAS Registry Number 1380723-44-3.As used herein, "tiragolumab" is a fully human IgG1/kappa MAb that binds TIGIT and comprises the heavy chain sequence of SEQ ID NO: 4 and the light chain sequence of SEQ ID NO: 5. Tiragolumab comprises two N-linked glycosylation sites (N306) in the Fc domain. Tiragolumab is also described in WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Proposed INN: List 117, Vol. 31, No. 2, published June 9, 2017 (see page 343). Tiragolumab (Genentech) is also known as MTIG7192A, RG6058, or RO7092284. Tiragolumab is described in PCT Pub. Nos. WO2003072305A8, WO2004024068A3, WO2004024072A3, WO2009126688A2, WO2015009856A2, WO2016011264A1, WO2016109546A2, WO2017053748A2, and WO2019165434A1; US Pub. Nos. 2017/0044256, 2017/0037127, 2017/0145093, 2017/260594, 2017/0088613, 2018/0186875, 2019/0119376; and US Pat. Nos. US9873740B2, US10626174B2, US10611836B2, US9499596B2, US8431350B2, US10047158B2, and US10017572B2.As used herein, "administering" is meant a method of giving a dosage of a compound (e.g., tiragolumab and/or atezolizumab) or a composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition including an tiragolumab and/or atezolizumab) to a subject. The compounds and/or compositions utilized in the methods described herein can be administered, for example, intravenously (e.g., by intravenous infusion), subcutaneously, intramuscularly, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated).As used herein, "systemic treatment" refers to a treatment that travels through the bloodstream and is capable of contacting multiple organ systems upon a single administration. The term "systemic treatment" is well understood by those skilled in the art and is equivalent to systemic therapy.A "fixed" or "flat" dose of a therapeutic agent (e.g., tiragolumab or atezolizumab) herein refers to a dose that is administered to a patient without regard for the weight or body surface area (BSA) of the patient. The fixed or flat dose is therefore not provided as a mg/kg dose or a mg/m2dose, but rather as an absolute amount of the therapeutic agent (e.g., mg).As used herein, the term "treatment" or "treating" refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include delaying or decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, WO 2024/186790 PCT/US2024/018472 decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.As used herein, "in conjunction with" refers to administration of one treatment modality in addition to another treatment modality. As such, "in conjunction with" refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.A "disorder" or "disease" is any condition that would benefit from treatment including, but not limited to, disorders that are associated with some degree of abnormal cell proliferation, e.g., cancer, e.g., lung cancer, e.g., non-small cell lung cancer (NSCLC).The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to lung cancer, such as non-small cell lung cancer (NSCLC), which includes squamous NSCLC or non-squamous NSCLC, including locally advanced unresectable NSCLC (e.g., Stage IHB NSCLC), or recurrent or metastatic NSCLC (e.g., Stage IV NSCLC).The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.As used herein, "metastasis" is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream, and stopping at a distant site. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior, and interactions between the tumor cell and host cells in the distant site are also significant.An "effective amount" of a compound, for example, tiragolumab or atezolizumab, or a composition (e.g., pharmaceutical composition) thereof, is at least the minimum amount required to achieve the desired therapeutic result, such as a measurable increase in overall survival or progression- free survival of a particular disease or disorder (e.g., cancer, e.g., lung cancer (e.g., NSCLC)). An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the subject. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease (e.g., reduction or delay in cancer-related pain, symptomatic skeletal-related events (SSE), reduction in symptoms per the European Organization for Research and Treatment of Cancer Quality-of-Life WO 2024/186790 PCT/US2024/018472 Questionnaire (EORTC QLQ-C30, e.g., fatigue, nausea, vomiting, pain, dyspnea, insomnia, appetite loss, constipation, diarrhea, or general level of physical emotional, cognitive, or social functioning), reduction in pain as measured by, e.g., the 10-point pain severity (measured at its worst) numerical rating scale (NRS), and/or reduction in symptoms associated with lung cancer per the health-related quality of life (HRQ0L) questionnaire as assessed by symptoms in lung cancer (SILC) scale (e.g., time to deterioration (TTD) in cough dyspenea and chest pain), increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease (e.g. progression-free survival or radiographic progression-free survival (rPFS); delay of unequivocal clinical progression (e.g., cancer-related pain progression, symptomatic skeletal-related event, deterioration in Eastern Cooperative Group Oncology Group (ECOG) Performance Status (PS) (e.g., how the disease affects the daily living abilities of the patient), and/or initiation of next systemic anti-cancer therapy), and/or delaying time to lung-specific antigen progression), and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved."Individual response" or "response" can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of disease progression (e.g., progression of cancer, e.g., lung cancer (e.g., NSCLC)), including slowing down and complete arrest; (2) a reduction in tumor size; (3) inhibition (i.e., reduction, slowing down or complete stopping) of cancer cell infiltration into adjacent peripheral organs and/or tissues; (4) inhibition (i.e., reduction, slowing down or complete stopping) of metastasis; (5) relief, to some extent, of one or more symptoms associated with the disease or disorder (e.g., cancer, e.g., lung cancer (e.g., NSCLC)); (6) increase or extension in the length of survival, including overall survival and progression-free survival; and/or (9) decreased mortality at a given point of time following treatment.As used herein, "complete response" or "CR" refers to disappearance of all target lesions.As used herein, "partial response" or "PR" refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD.As used herein, "objective response rate" (ORR) refers to the sum of complete response (CR) rate and partial response (PR) rate.

WO 2024/186790 PCT/US2024/018472 An "effective response" of a subject or a subject’s "responsiveness" to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a subject as risk for, or suffering from, a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.A subject who "does not have an effective response" to treatment refers to a subject who does not have any one of extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.As used herein, "survival" refers to the patient remaining alive, and includes overall survival as well as progression-free survival.As used herein, "overall survival" (OS) refers to the percentage of subjects in a group who are alive after a particular duration of time, e.g., 1 year or 5 years from the time of diagnosis or treatment.As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer, e.g., lung cancer (e.g., NSCLC)) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.As used herein, "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started.As used herein, "progressive disease" or "PD" refers to at least a 20% increase in the SLD of target lesions, taking as reference the smallest SLD recorded since the treatment started or the presence of one or more new lesions.As used herein, "delaying progression" of a disorder or disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease or disorder (e.g., cancer, e.g., lung cancer (e.g., NSCLC)). This delay can be of varying lengths of time, depending on the history of the disease and/or subject being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the subject does not develop the disease. For example, in a late stage cancer, development of central nervous system (CNS) metastasis, may be delayed.By "extending survival" is meant increasing overall or progression free survival in a treated patient relative to an untreated patient (e.g., relative to a patient not treated with the medicament), or relative to a patient who does not express a biomarker at the designated level, and/or relative to a patient treated with an approved anti-tumor agent. An objective response refers to a measurable response, including complete response (CR) or partial response (PR).As used herein, "hazard ratio" or "HR" is a statistical definition for rates of events. For the purpose of the invention, hazard ratio is defined as representing the probability of an event (e.g., PFS or OS) in the experimental (e.g., treatment) group/arm divided by the probability of an event in the control group/arm at any specific point in time. An HR with a value of 1 indicates that the relative risk of an WO 2024/186790 PCT/US2024/018472 endpoint (e.g., death) is equal in both the "treatment" and "control" groups; a value greater than indicates that the risk is greater in the treatment group relative to the control group; and a value less than indicates that the risk is greater in the control group relative to the treatment group. "Hazard ratio" in progression-free survival analysis (i.e., PFS HR) is a summary of the difference between two progression- free survival curves, representing the reduction in the risk of death on treatment compared to control, over a period of follow-up. "Hazard ratio" in overall survival analysis (i.e., OS HR) is a summary of the difference between two overall survival curves, representing the reduction in the risk of death on treatment compared to control, over a period of follow-up.As used herein, the "Ventana SP263 IHC assay" (also referred to herein as the Ventana SP2CDx assay) is conducted according to the Ventana PD-L1 (SP263) Assay package insert (Tucson, AZ: Ventana Medical Systems, Inc.), which is incorporated herein by reference in its entirety.As used herein, the "Ventana SP142 IHC assay" is conducted according to the Ventana PD-L(SP142) Assay package insert (Tucson, AZ: Ventana Medical Systems, Inc.), which is incorporated herein by reference in its entirety.As used herein, the "pharmDx 22C3 IHC assay" is conducted according to the PD-L1 IHC 22CpharmDx package insert (Carpinteria, CA: Dako, Agilent Pathology Solutions), which is incorporated herein by reference in its entirety.A "tumor-infiltrating immune cell," as used herein, refers to any immune cell present in a tumor or a sample thereof. Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, other tumor stroma cells (e.g., fibroblasts), or any combination thereof. Such tumor-infiltrating immune cells can be, for example, T lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes), B lymphocytes, or other bone marrow-lineage cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells.The term "biomarker," as used herein, refers to an indicator, e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample. In some embodiments, a biomarker is a gene. Biomarkers include, but are not limited to, polypeptides, polynucleotides (e.g., DNA, and/or RNA), polynucleotide copy number alterations (e.g., DNA copy numbers), polypeptide and polynucleotide modifications (e.g., posttranslational modifications), carbohydrates, and/or glycolipid-based molecular markers.As used herein, "subject" or "individual" means a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. In some embodiments, the subject is a human. Patients are also subjects herein.The term "sample," as used herein, refers to a composition that is obtained or derived from a subject and/or individual of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "tumor sample," "disease sample," and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized. In some embodiments, the sample is a tumor tissue sample (e.g., a lung cancer sample (e.g., a NSCLC sample)). Other samples include, WO 2024/186790 PCT/US2024/018472 but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, stool, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, cellular extracts, and combinations thereof. In some aspects, the sample is a tumor draining lymph node (dLN) sample.The terms "tissue sample" and "cell sample" mean a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen, and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.A "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue," as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject. For example, healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of a subject who is not the subject. In even another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the subject.The term "protein," as used herein, refers to any native protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length," unprocessed protein as well as any form of the protein that results from processing in the cell. The term also encompasses naturally occurring variants of the protein, e.g., splice variants or allelic variants."Polynucleotide" or "nucleic acid," as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single- stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, WO 2024/186790 PCT/US2024/018472 the term "polynucleotide" as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. The terms "polynucleotide" and "nucleic acid" specifically includes mRNA and cDNAs.A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, "caps," substitution of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-O- methyl-, 2’-O-allyl-, 2’-fluoro-, or 2’-azido-ribose, carbocyclic sugar analogs, a-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR’, CO or CH2 ("formacetal"), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA."Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; WO 2024/186790 PCT/US2024/018472 monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. 11. PROGNOSTIC METHODS AND ASSAYS FOR TIRAGOLUMAB PLUS ATEZOLIZUMAB A. CCL5, CXCR3, CCR7, and CXCR6 Methods of identifying individuals who may benefit from treatmentIn one aspect, the invention provides a method of identifying an individual having a non-small cell lung cancer (NSCLC) who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR(e.g., one, two, three, or all four of CCL5, CXCR3, CCR7, and CXCR6) in a sample from the individual, wherein an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 (e.g., an expression level of one, two, three, or all four of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6) identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.For example, in one aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CCL5 in a sample from the individual, wherein an expression level of CCL5 that is at or above a reference expression level of CCL5 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CXCR3 in a sample from the individual, wherein an expression level of CXCR3 that is at or above a reference expression level of CXCR3 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CCR7 in a sample from the individual, wherein an expression level of WO 2024/186790 PCT/US2024/018472 CCR7 that is at or above a reference expression level of CCR7 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CXCR6 in a sample from the individual, wherein an expression level of CXCR6 that is at or above a reference expression level of CXCR6 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the individual has an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab (e.g., an effective amount of atezolizumab and tiragolumab as described in Section III herein).

Methods of selecting a therapyIn another aspect, the invention provides a method of selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 (e.g., one, two, three, or all four of CCL5, CXCR3, CCR7, and CXCR6) in a sample from the individual, wherein an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 (e.g., an expression level of one, two, three, or all four of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6) identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.For example, in one aspect, the invention provides a method of selecting a therapy for an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CCL5 in a sample from the individual, wherein an expression level of CCL5 that is at or above a reference expression level of CCL5 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of selecting a therapy for an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CXCR3 in a sample from the individual, wherein an expression level of CXCR3 that is at or above a reference expression level of CXCR3 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of selecting a therapy for an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CCR7 in a sample from the individual, wherein an expression level of CCR7 that is at or above a reference expression level of CCR7 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of selecting a therapy for an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of CXCR6 in a sample from the individual, wherein an WO 2024/186790 PCT/US2024/018472 expression level of CXCR6 that is at or above a reference expression level of CXCR6 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the individual has an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab (e.g., an effective amount of atezolizumab and tiragolumab as described in Section III herein).

Methods of treatmentIn another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 (e.g., one, two, three, or all four of CCL5, CXCR3, CCR7, and CXCR6) in a sample from the individual, wherein the expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.For example, in one aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of CCL5 in a sample from the individual, wherein the expression level of CCL5 is at or above a reference expression level of CCL5 and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of CXCR3 in a sample from the individual, wherein the expression level of CXCR3 is at or above a reference expression level of CXCR3 and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of CCR7 in a sample from the individual, wherein the expression level of CCR7 is at or above a reference expression level of CCR7 and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of CXCR6 in a sample from the individual, wherein the expression level of CXCR6 is at or above a reference expression level of CXCR6 and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR(e.g., an expression level of one, two, three, or all four of CCL5, CXCR3, CCR7, and CXCR6) that is at or WO 2024/186790 PCT/US2024/018472 above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.For example, in one aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have an expression level of CCL5 that is at or above a reference expression level of CCL5, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have an expression level of CXCR3 that is at or above a reference expression level of CXCR3, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have an expression level of CCR7 that is at or above a reference expression level of CCR7, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have an expression level of CXCR6 that is at or above a reference expression level of CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In another aspect, the invention provides atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

WO 2024/186790 PCT/US2024/018472 BenefitAn individual who benefits from receiving treatment with atezolizumab and tiragolumab may experience, for example, a delay or prevention in the occurrence or recurrence of NSCLC, alleviation of symptoms of the cancer, diminishment of any direct or indirect pathological consequences of the cancer, prevention of metastasis, decrease in the rate of disease progression, amelioration or palliation of the disease state, or remission or improved prognosis.In some aspects, the benefit achieved by the treatment comprising atezolizumab and tiragolumab is a clinical response, e.g., a complete response (CR) or a partial response (PR) (e.g., an individual treated by the method has an increased likelihood of achieving a CR or PR and/or the frequency of CR or PR is increased in the population of individuals treated according to the method).In some aspects, the individual has an expression level of one or more of CCL5, CXCR3, and CCR7 (e.g., an expression level of one, two, or all three of CCL5, CXCR3, and CCR7) in the sample that is at or above a reference expression level of CCL5, CXCR3, or CCR7 and the benefit is an increase in overall survival (OS) hazard ratio (HR) (e.g., an increase in the average OS HR of a population of individuals treated according to the method).In some aspects, the individual has an expression level of one or more of CCL5, CXCR3, and CXCR6 (e.g., an expression level of one, two, or all three of CCL5, CXCR3, and CXCR6) in the sample that is at or above a reference expression level of CCL5, CXCR3, or CXCR6 and the benefit is an increase in overall survival (OS) (e.g., an increase in the duration of OS experienced by an individual treated according to the method or an increase in the average OS of a population of individuals treated according to the method).An increased clinical response likelihood, OS HR, and/or OS may be determined by comparison to, e.g., an untreated reference individual and/or a reference population of individuals; a reference individual and/or a reference population of individuals who have received a control treatment, such as one or more previously approved treatments or marketed products for treatment of the cancer; and/or a reference individual and/or a reference population of individuals who have been treated with atezolizumab or tiragolumab as a monotherapy. In some aspects, the increased clinical response likelihood, OS HR, and/or OS is determined relative to a reference individual and/or a reference population of individuals having cancer that have been treated with a treatment comprising atezolizumab and tiragolumab, wherein the reference individual and/or each individual in the reference population has an expression level of each of CCL5, CXCR3, CCR7, and CXCR6 that is below a respective reference expression level. Reference expression levels are described herein and may be, for example, a median expression level of CCL5, CXCR3, CCR7, or CXCR6 in a reference population of individuals having a NSCLC.The skilled person is readily able to decide whether a given clinical outcome is improved in accordance with the invention. For example, "improved" in this context means that the clinical outcome resulting from the treatment of an individual having an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is above a respective reference expression level with a treatment comprising atezolizumab and tiragolumab is at least 3% higher, at least 5% higher, at least 7% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 40% WO 2024/186790 PCT/US2024/018472 higher, at least 50% higher, at least 75% higher, at least 100% higher, or at least 120% higher, as compared to the clinical outcome resulting from a comparator treatment as described above.For example, in some aspects, the duration of OS experienced by an individual treated according to the method or the average OS of a population of individuals treated according to the method is increased by at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, or at least 120%.The time at which the clinical outcome/clinical endpoint is assessed can readily be determined by the skilled person. In principle, it is determined at a timepoint when the difference in the clinical outcome/clinical endpoint between the two treatments becomes evident. This time may, for example, be at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least months after the beginning of the treatment.

B. Gene signatures comprising CCL5, CXCR3, and CXCR6 Methods of identifying individuals who may benefit from treatment with atezolizumab and tiragolumabIn one aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab (e.g., an effective amount of atezolizumab and tiragolumab as described in Section III herein).In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In other aspects, the method comprises further detecting the expression level of CCR7 in the sample from the individual, and the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual. Accordingly, in one aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of each of CCL5, CXCR3, CXCR6, and CCR7 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

WO 2024/186790 PCT/US2024/018472 Methods of identifying individuals who may benefit from treatment with atezolizumabIn another aspect, the invention provides a method of identifying an individual having a NSCLC (e.g., a PD-L1-positive NSCLC, e.g., a NSCLC having a PD-L1 tumor proportion score (TPS) >1% as determined using the Ventana (SP263) PD-L1 IHC assay) who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy), the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab.In some aspects, the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab (e.g., an effective amount of atezolizumab as described in Section III herein).In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In other aspects, the method comprises further detecting the expression level of CCR7 in the sample from the individual, and the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual. Accordingly, in one aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy), the method comprising detecting an expression level of each of CCL5, CXCR3, CXCR6, and CCR7 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab.In some aspects, the individual has not previously been treated for NSCLC (e.g., has not previously been treated for Stage IV NSCLC).

Methods of selecting a therapy for an individual who may benefit from treatment with atezolizumab and tiragolumabIn another aspect, the invention provides a method for selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab (e.g., an effective amount of atezolizumab and tiragolumab as described in Section III herein).

WO 2024/186790 PCT/US2024/018472 In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In other aspects, the method comprises further detecting the expression level of CCR7 in the sample from the individual, and the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual. Accordingly, in one aspect, the invention provides a method of selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of each of CCL5, CXCR3, CXCR6, and CCR7 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

Methods of selecting a therapy for an individual who may benefit from treatment with atezolizumabIn another aspect, the invention provides a method for selecting a therapy for an individual having a NSCLC (e.g., a PD-L1-positive NSCLC, e.g., a NSCLC having a PD-L1 tumor proportion score (TPS) >1% as determined using the Ventana (SP263) PD-L1 IHC assay), the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy).In some aspects, the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab (e.g., an effective amount of atezolizumab as described in Section III herein).In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In other aspects, the method comprises further detecting the expression level of CCR7 in the sample from the individual, and the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual. Accordingly, in one aspect, the invention provides a method of selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of each of CCL5, CXCR3, CXCR6, and CCR7 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy).In some aspects, the individual has not previously been treated for NSCLC (e.g., has not previously been treated for Stage IV NSCLC).

Methods of treatment comprising atezolizumab and tiragolumabIn another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample WO 2024/186790 PCT/US2024/018472 from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In other aspects, the expression level of CCR7 has been detected in the sample from the individual, and the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual. Accordingly, in one aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of each of CCL5, CXCR3, CXCR6, and CCR7 in a sample from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (b) administering an effective amount of atezolizumab and tiragolumab to the individual. In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, CXCR6, and CCR7 detected in a sample from the individual.In another aspect, the invention provides use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In another aspect, the invention provides atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

WO 2024/186790 PCT/US2024/018472 Methods of treatment comprising atezolizumabIn another aspect, the invention provides a method of treating an individual having a NSCLC (e.g., a PD-L1-positive NSCLC, e.g., a NSCLC having a PD-L1 tumor proportion score (TPS) >1% as determined using the Ventana (SP263) PD-L1 IHC assay), the method comprising (a) detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy); and (b) administering an effective amount of atezolizumab to the individual.In another aspect, the invention provides a method of treating an individual having a NSCLC (e.g., a PD-L1-positive NSCLC), the method comprising administering atezolizumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy), and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.In some aspects, the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.In other aspects, the expression level of CCR7 has been detected in the sample from the individual, and the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual. Accordingly, in one aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising (a) detecting an expression level of each of CCL5, CXCR3, CXCR6, and CCR7 in a sample from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy); and (b) administering an effective amount of atezolizumab to the individual. In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab (e.g., atezolizumab monotherapy), and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, CXCR6, and CCR7 detected in a sample from the individual.In some aspects, the individual has not previously been treated for NSCLC (e.g., has not previously been treated for Stage IV NSCLC).In another aspect, the invention provides use of atezolizumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

WO 2024/186790 PCT/US2024/018472 In another aspect, the invention provides atezolizumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

Benefit of treatment comprising atezolizumab and tiragolumabIn some aspects, the benefit achieved by the treatment comprising atezolizumab and tiragolumab is a clinical response, e.g., a complete response (CR) or a partial response (PR) (e.g., an individual treated by the method has an increased likelihood of achieving a CR or PR and/or the frequency of CR or PR is increased in the population of individuals treated according to the method).In some aspects, the benefit achieved by the treatment comprising atezolizumab and tiragolumab is an increase in overall survival (OS) hazard ratio (HR) (e.g., an increase in the average OS HR of a population of individuals treated according to the method).In some aspects, the benefit achieved by the treatment comprising atezolizumab and tiragolumab is an increase in overall survival (OS) (e.g., an increase in the duration of OS experienced by an individual treated according to the method or an increase in the average OS of a population of individuals treated according to the method).An increased clinical response likelihood, OS HR, and/or OS may be determined by comparison to, e.g., an untreated reference individual and/or a reference population of individuals; a reference individual and/or a reference population of individuals who have received a control treatment, such as one or more previously approved treatments or marketed products for treatment of the cancer; and/or a reference individual and/or a reference population of individuals who have been treated with atezolizumab or tiragolumab as a monotherapy. In some aspects, the increased clinical response likelihood, OS HR, and/or OS is determined relative to a reference individual and/or a reference population of individuals having cancer that have been treated with a treatment comprising atezolizumab and tiragolumab, wherein the reference individual and/or each individual in the reference population has a gene signature score is based on the expression level of (a) each of CCL5, CXCR3, and CXCR6 or (b) each of CCL5, CXCR3, CXCR6, and CCR7 that is below a respective reference gene signature score.The skilled person is readily able to decide whether a given clinical outcome is improved in accordance with the invention. For example, "improved" in this context means that the clinical outcome resulting from the treatment of an individual having a gene signature score is based on the expression level of (a) each of CCL5, CXCR3, and CXCR6 or (b) each of CCL5, CXCR3, CXCR6, and CCR7 that is above a respective reference expression level with a treatment comprising atezolizumab and tiragolumab is at least 3% higher, at least 5% higher, at least 7% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 75% higher, at least 100% higher, or at least 120% higher, as compared to the clinical outcome resulting from a comparator treatment as described above.For example, in some aspects, the duration of OS experienced by an individual treated according to the method or the average OS of a population of individuals treated according to the method is WO 2024/186790 PCT/US2024/018472 increased by at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, or at least 120%.The time at which the clinical outcome/clinical endpoint is assessed can readily be determined by the skilled person. In principle, it is determined at a timepoint when the difference in the clinical outcome/clinical endpoint between the two treatments becomes evident. This time may, for example, be at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least months after the beginning of the treatment.

Benefit of treatment comprising atezolizumabIn some aspects, the benefit achieved by the treatment comprising atezolizumab (e.g., atezolizumab monotherapy) is an increase in overall survival (OS) (e.g., an increase in the duration of OS experienced by an individual treated according to the method or an increase in the average OS of a population of individuals treated according to the method).In some aspects, the benefit achieved by the treatment comprising atezolizumab (e.g., atezolizumab monotherapy) is an increase in progression-free survival (PFS) (e.g., an increase in the duration of PFS experienced by an individual treated according to the method or an increase in the average PFS of a population of individuals treated according to the method).An increased OS and/or PFS may be determined by comparison to, e.g., an untreated reference individual and/or a reference population of individuals; a reference individual and/or a reference population of individuals who have received a control treatment, such as one or more previously approved treatments or marketed products for treatment of the cancer. In some aspects, the increased OS and/or PFS is determined relative to a reference individual and/or a reference population of individuals having cancer that have been treated with a treatment comprising a PD-1 axis binding antagonist (e.g., atezolizumab), wherein the reference individual and/or each individual in the reference population has a gene signature score is based on the expression level of (a) each of CCL5, CXCR3, and CXCR6 or (b) each of CCL5, CXCR3, CXCR6, and CCR7 that is below a respective reference gene signature score.The skilled person is readily able to decide whether a given clinical outcome is improved in accordance with the invention. For example, "improved" in this context means that the clinical outcome resulting from the treatment of an individual having a gene signature score is based on the expression level of (a) each of CCL5, CXCR3, and CXCR6 or (b) each of CCL5, CXCR3, CXCR6, and CCR7 that is above a respective reference expression level with a treatment comprising atezolizumab and tiragolumab is at least 3% higher, at least 5% higher, at least 7% higher, at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 75% higher, at least 100% higher, or at least 120% higher, as compared to the clinical outcome resulting from a comparator treatment as described above.For example, in some aspects, the duration of OS experienced by an individual treated according to the method or the average OS of a population of individuals treated according to the method is WO 2024/186790 PCT/US2024/018472 increased by at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, or at least 120%.in some aspects, the duration of PFS experienced by an individual treated according to the method or the average PFS of a population of individuals treated according to the method is increased by at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, or at least 120%.The time at which the clinical outcome/clinical endpoint is assessed can readily be determined by the skilled person. In principle, it is determined at a timepoint when the difference in the clinical outcome/clinical endpoint between the two treatments becomes evident. This time may, for example, be at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least months after the beginning of the treatment.

C. Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, If it, Mitotic Cytotox.2, and Cytotox.4 gene signatures Methods of identifying individuals who may benefit from treatmentIn another aspect, the invention provides a method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising:(a) detecting an expression level of at least two of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X, e.g., each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X) in a sample from the individual and determining a Ocr7.2 gene signature score therefrom;(b) detecting an expression level of at least two of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB, e.g., each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB) in a sample from the individual and determining a Ccr7.3 gene signature score therefrom;(c) detecting an expression level of at least two of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7, e.g., each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7) in a sample from the individual and determining a Cxcr3 gene signature score therefrom;(d) detecting an expression level of at least two CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 (e.g., at least 2, 3, 4, 5, 6, WO 2024/186790 PCT/US2024/018472 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HOST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3, e.g., of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HOST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3) in a sample from the individual and determining a Ccl5.1 gene signature score therefrom;(e) detecting an expression level of at least two of ISG15, IFIT1B, I FITS, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, I Fl 16, and IFIH1, e.g., each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1) in a sample from the individual and determining an Ifit gene signature score therefrom;(f) detecting an expression level of at least two of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1 B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS1 2, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL, e.g., each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL) in a sample from the individual and determining a Mitotic gene signature score therefrom;(g) detecting an expression level of at least two of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15 of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT, e.g., each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT) in a sample from the individual and determining a Cytotox.2 gene signature score therefrom; or(h) detecting an expression level of at least two of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2, e.g., each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2) in a sample from the individual and determining a Cytotox.4 gene signature score therefrom;wherein (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab,and (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the individual has (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score, and the method further WO 2024/186790 PCT/US2024/018472 comprises administering to the individual an effective amount of atezolizumab and tiragolumab (e.g., an effective amount of atezolizumab and tiragolumab as described in Section III herein).In some aspects, the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.In some aspects, the reference gene signature score is a pre-assigned gene signature score and/or a gene signature score in a reference population (e.g., a population of individuals having the NSCLC). In some aspects, the gene signature score in the reference population is the median Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population.

Methods of selecting a therapyIn another aspect, the invention provides a method for selecting a therapy for an individual having a NSCLC, the method comprising:(a) detecting an expression level of at least two of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X, e.g., each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X) in a sample from the individual and determining a Ccr7.2 gene signature score therefrom;(b) detecting an expression level of at least two of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB, e.g., each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB) in a sample from the individual and determining a Ccr7.3 gene signature score therefrom;(c) detecting an expression level of at least two of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7, e.g., each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7) in a sample from the individual and determining a Cxcr3 gene signature score therefrom;(d) detecting an expression level of at least two CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3, e.g., of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3) in a sample from the individual and determining a Ccl5.1 gene signature score therefrom; WO 2024/186790 PCT/US2024/018472 (e) detecting an expression level of at least two of ISG15, IFIT1B, I FITS, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, I Fl 16, and IFIH1, e.g., each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1) in a sample from the individual and determining an Ifit gene signature score therefrom;(f) detecting an expression level of at least two of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1 B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS1 2, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL, e.g., each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL) in a sample from the individual and determining a Mitotic gene signature score therefrom;(g) detecting an expression level of at least two of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15 of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT, e.g., each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT) in a sample from the individual and determining a Cytotox.2 gene signature score therefrom; or(h) detecting an expression level of at least two of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2, e.g., each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2) in a sample from the individual and determining a Cytotox.4 gene signature score therefrom;wherein (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab,and (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the individual has (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab (e.g., an effective amount of atezolizumab and tiragolumab as described in Section III herein).In some aspects, the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.

WO 2024/186790 PCT/US2024/018472 In some aspects, the reference gene signature score is a pre-assigned gene signature score and/or a gene signature score in a reference population (e.g., a population of individuals having the NSCLC). In some aspects, the gene signature score in the reference population is the median Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population.

Methods of treatmentIn another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising:(i)(a) detecting an expression level of at least two of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X, e.g., each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X) in a sample from the individual and determining a Ccr7.2 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(b) detecting an expression level of at least two of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB, e.g., each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB) in a sample from the individual and determining a Ccr7.3 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(c) detecting an expression level of at least two of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7, e.g., each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7) in a sample from the individual and determining a Cxcr3 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(d) detecting an expression level of at least two CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, WO 2024/186790 PCT/US2024/018472 TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3, e.g., of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3) in a sample from the individual and determining a Ccl5.1 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(e) detecting an expression level of at least two of ISG15, IFIT1B, I FITS, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, I Fl 16, and IFIH1, e.g., each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1) in a sample from the individual and determining an Ifit gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(f) detecting an expression level of at least two of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1 B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS1 2, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL, e.g., each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL) in a sample from the individual and determining a Mitotic gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(g) detecting an expression level of at least two of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15 of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT, e.g., each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT) in a sample from the individual and determining a Cytotox.2 gene signature score therefrom, wherein the gene signature score is below a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; or(h) detecting an expression level of at least two of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of S100A6, NRN1, CXCR6, KLRC1 , PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2, e.g., each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2) in a sample from the individual and determining a Cytotox.4 gene signature score therefrom; and (ii) administering an effective amount of atezolizumab and tiragolumab to the individual.

WO 2024/186790 PCT/US2024/018472 In another aspect, the invention provides a method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have:(a) a Ccr7.2 gene signature score based on an expression level at least two of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X, e.g., each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X) in a sample from the individual that is at or above a reference Ocr7.2 gene signature score;(b) a Ocr7.3 gene signature based on an expression level of at least two of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or all 17 of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB, e.g., each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB) in a sample from the individual that is at or above a reference Ccr7.3 gene signature score;(c) a Cxcr3 gene signature score based on an expression level at least two of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7, e.g., each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7) in a sample from the individual that is at or above a reference Cxcr3 gene signature score;(d) a Ccl5.1 gene signature score based on an expression level of at least two of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or all 16 of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3, e.g., of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3) in a sample from the individual that is at or above a reference Ccl5.1 gene signature score;(e) an Ifit gene signature score based on an expression level of at least two of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, I F116, and I FI H1, e.g., each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1) in a sample from the individual that is at or above a reference Ifit gene signature score; WO 2024/186790 PCT/US2024/018472 (f) a Mitotic gene signature score based on an expression level of at least two of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or all 19 of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL, e.g., each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS1 2, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL) in a sample from the individual that is at or above a reference Mitotic gene signature score;(g) a Cytotox.2 gene signature score based on an expression level of at least two of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1 B, LY6E, and DUT (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or all 15 0f HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT, e.g., each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT) in a sample from the individual that is below a reference Cytotox.2 gene signature score; or(h) a Cytotox.4 gene signature score based on an expression level of at least two of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or all 18 of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2, e.g., each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2) in a sample from the individual that is below a reference Cytotox.4 gene signature score;thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.In some aspects, the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.In some aspects, the reference gene signature score is a pre-assigned gene signature score and/or a gene signature score in a reference population (e.g., a population of individuals having the NSCLC). In some aspects, the gene signature score in the reference population is the median Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population.In another aspect, the invention provides use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have: WO 2024/186790 PCT/US2024/018472 (i)(a) a Ccr7.2 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X detected in a sample from the individual;(b) a Ccr7.3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB detected in a sample from the individual;(c) a Cxcr3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 detected in a sample from the individual;(d) a Ccl5.1 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 detected in a sample from the individual;(e) an Ifit gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 detected in a sample from the individual;(f) a Mitotic gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL detected in a sample from the individual;(g) a Cytotox.2 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT detected in a sample from the individual; or(h) a Cytotox.4 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of S100A6, WO 2024/186790 PCT/US2024/018472 NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 detected in a sample from the individual.In another aspect, the invention provides atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have: (i)(a) a Ccr7.2 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X detected in a sample from the individual;(b) a Ccr7.3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB detected in a sample from the individual;(c) a Cxcr3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 detected in a sample from the individual;(d) a Ccl5.1 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 detected in a sample from the individual;(e) an Ifit gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 detected in a sample from the individual;(f) a Mitotic gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL detected in a sample from the individual;(g) a Cytotox.2 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT detected in a sample from the individual; or WO 2024/186790 PCT/US2024/018472 (h) a Cytotox.4 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 detected in a sample from the individual.

BenefitIn some aspects, the individual has a Ccr7.3, Cxcr3, or Ccl5.1 gene signature score in the sample that is at or above a reference gene signature score and the benefit achieved by the treatment comprising atezolizumab and tiragolumab is a clinical response, e.g., a complete response (CR) or a partial response (PR) (e.g., an individual treated by the method has an increased likelihood of achieving a CR or PR and/or the frequency of CR or PR is increased in the population of individuals treated according to the method).In some aspects, the individual has (i) a Ccr7.2, Cxcr3, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score and the benefit achieved by the treatment comprising atezolizumab and tiragolumab is an increase in overall survival (OS) hazard ratio (HR) (e.g., an increase in the average OS HR of a population of individuals treated according to the method).In some aspects, the individual has a Ccr7.2, Ccr7.3, or Cxcr3 gene signature score in the sample that is at or above a reference gene signature score and the benefit achieved by the treatment comprising atezolizumab and tiragolumab is an increase in overall survival (OS) (e.g., an increase in the duration of OS experienced by an individual treated according to the method or an increase in the average OS of a population of individuals treated according to the method).An increased clinical response likelihood, OS HR, and/or OS may be determined by comparison to, e.g., an untreated reference individual and/or a reference population of individuals; a reference individual and/or a reference population of individuals who have received a control treatment, such as one or more previously approved treatments or marketed products for treatment of the cancer; and/or a reference individual and/or a reference population of individuals who have been treated with atezolizumab or tiragolumab as a monotherapy. In some aspects, the increased clinical response likelihood, OS HR, and/or OS is determined relative to a reference individual and/or a reference population of individuals having cancer that have been treated with a treatment comprising atezolizumab and tiragolumab, wherein the reference individual and/or each individual in the reference population has (i) a Ccr7.2, Ocr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is below a reference gene signature score and/or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is at or above a reference gene signature score.The skilled person is readily able to decide whether a given clinical outcome is improved in accordance with the invention. For example, "improved" in this context means that the clinical outcome resulting from the treatment of an individual having (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is at or above a reference gene signature score and/or (ii) a Cytotox.2 or Cytotox.gene signature score that is below a reference gene signature score with a treatment comprising atezolizumab and tiragolumab is at least 3% higher, at least 5% higher, at least 7% higher, at least 10% WO 2024/186790 PCT/US2024/018472 higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 75% higher, at least 100% higher, or at least 120% higher, as compared to the clinical outcome resulting from a comparator treatment as described above.For example, in some aspects, the duration of OS experienced by an individual treated according to the method or the average OS of a population of individuals treated according to the method is increased by at least 3%, at least 5%, at least 7%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 75%, at least 100%, or at least 120%.The time at which the clinical outcome/clinical endpoint is assessed can readily be determined by the skilled person. In principle, it is determined at a timepoint when the difference in the clinical outcome/clinical endpoint between the two treatments becomes evident. This time may, for example, be at least 1 month, at least 2 months, at least 3 months, at least 6 months, at least 12 months, at least months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, or at least months after the beginning of the treatment.

D. Samples, expression levels, and PD-L1 status SamplesAn expression level of one or more of CCL5, CXCR3, CCR7, or CXCR6; a member of any one of the Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, and Cytotox.4 gene signatures and/or a gene signature score may be determined from any suitable sample. Exemplary sample types include, without limitation, a tissue sample, a tumor sample, a blood sample (e.g., a whole blood sample), a plasma sample, a serum sample, and combinations thereof. Samples may be fresh, archival, or frozen.In some aspects, the sample is a tissue sample, e.g., a tumor tissue sample. In some aspects, the tumor tissue sample is a biopsy, e.g., a biopsy of the NSCLC.In some aspects, the sample is a tumor draining lymph node (dLN) sample.In some aspects, the sample is obtained from the individual prior to treatment with atezolizumab and tiragolumab, e.g., is obtained immediately prior to the first administration of atezolizumab and/or tiragolumab, or is obtained at least one day, at least one week, or at least one month prior to the first administration of atezolizumab and/or tiragolumab.

Expression levelsThe expression levels of the one or more genes detected in the methods provided herein may be, e.g., nucleic acid expression levels or protein expression levels.In some aspects, the expression levels are nucleic acid expression levels, e.g., mRNA expression levels. Nucleic acid expression levels may be detected using any suitable method known in the art, e.g., may be determined by RNA-seq, reverse transcriptase quantitative PCR (RT-qPCR), quantitative PCR (qPCR), real-time PCR, quantitative real-time PCR (qRT-PCR), multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, in situ hybridization (ISH), or a combination thereof. Other amplification-based methods include, for example, transcript-mediated amplification (TMA), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), and signal amplification methods such as bDNA.

WO 2024/186790 PCT/US2024/018472 In some instances, nucleic acid expression levels of the genes described herein may be measured by sequencing-based techniques, such as, for example, RNA-seq, serial analysis of gene expression (SAGE), high-throughput sequencing technologies (e.g., massively parallel sequencing), and Sequenom MassARRAY® technology. Nucleic acid expression levels also may be measured by, for example, NanoString nOounter, and high-coverage expression profiling (HiCEP). Additional protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), (Immunoblotting) and 18 (PCR Analysis).Other methods for detecting nucleic acid levels of the genes described herein include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies.Other methods to detect nucleic acid expression levels of the genes described herein include electrophoresis, Northern and Southern blot analyses, in situ hybridization (e.g., single or multiplex nucleic acid in situ hybridization), RNAse protection assays, and microarrays (e.g., Illumina BEADARRAY™ technology; Beads Array for Detection of Gene Expression (BADGE)).In some aspects, the expression level is a protein expression level, e.g., a protein expression level determined by mass spectrometry, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, surface plasmon resonance, optical spectroscopy, mass spectrometry, or HPLC.

Normalization of expression levelsIn some aspects, the expression levels of one or more genes detected in the methods provided herein are normalized expression levels, e.g., gene signature score is an average of the normalized expression levels of the one or more genes in the sample from the individual.The detected expression level of a gene may be normalized using any one of the standard normalization methods known in the art. One of skill in the art will appreciate that the normalization method used may depend on the gene expression methodology used (e.g., one or more housekeeping genes may be used for normalization in the context of an RT-qPCR methodology, but a whole genome or substantially whole genome may be used as a normalization baseline in the context of an RNA-seq methodology). For example, the detected expression level of each gene assayed can be normalized for differences in the amount of the gene(s) assayed, variability in the quality of the samples used, and/or variability between assay runs.In some instances, normalization may be accomplished by detecting expression of certain one or more normalizing gene(s), including reference gene(s) (e.g., a housekeeping gene (e.g., 3-actin)). For example, in some instances, the nucleic acid expression levels detected using the methods described herein may be normalized to the expression level of one or more reference genes (e.g., one, two, three, four, five, six, seven, eight, nine, or more reference genes, e.g., a housekeeping gene (e.g., 3-actin)). Alternatively, normalization can be based on the average signal or median signal of all of the assayed genes. On a gene-by-gene basis, a measured normalized amount of an mRNA can be compared to the amount found in a reference expression level. The presence and/or expression level/amount measured WO 2024/186790 PCT/US2024/018472 in a particular subject sample to be analyzed will fall at some percentile within this range, which can be determined by methods well known in the art.In other instances, to determine an expression level, the detected expression level of each assayed gene is not normalized.Any statistical approaches known in the art may be used to determine the expression level of each gene. For example, the expression level may reflect the median expression level, median normalized expression level, or mean expression level, or mean normalized expression level.

Reference expression levels and gene signature scoresThe terms "reference expression level" and "reference gene signature score" refer to an expression level or a gene signature score against which another expression level or gene signature score is compared, e.g., to make a diagnostic, predictive, prognostic, and/or therapeutic determination.In some aspects, the reference expression level or reference gene signature score is a pre- assigned reference expression level or reference gene signature score.In some aspects, the reference expression level or reference gene signature score is an expression level or a gene signature score in a reference population (e.g., a population of individuals having the NSCLC).In some aspects, the expression level or gene signature score in the reference population is a median expression level or gene signature score of the reference population.In other aspects, the expression level or gene signature score in the reference population is a mean expression level or gene signature score of the reference population.In still other aspects, the expression level or gene signature score is defined as the 25th percentile, the 26th percentile, the 27th percentile, the 28th percentile, the 29th percentile, the 30th percentile, the 31st percentile, the 32nd percentile, the 33rd percentile, the 34th percentile, the 35th percentile, the 36th percentile, the 37th percentile, the 38th percentile, the 39th percentile, the 40th percentile, the 41st percentile, the 42nd percentile, the 43rd percentile, the 44th percentile, the 45th percentile, the 46th percentile, the 47th percentile, the 48th percentile, the 49th percentile, the 50th percentile, the 51st percentile, the 52nd percentile, the 53rd percentile, the 54th percentile, the 55th percentile, the 56th percentile, the 57th percentile, the 58th percentile, the 59th percentile, the 60th percentile, the 61st percentile, the 62nd percentile, the 63rd percentile, the 64th percentile, the 65th percentile, the 66th percentile, the 67th percentile, the 68th percentile, the 69th percentile, the 70th percentile, the 71st percentile, the 72nd percentile, the 73rd percentile, the 74th percentile, the 75th percentile, the 76th percentile, the 77th percentile, the 78th percentile, the 79th percentile, the 80th percentile, the 81st percentile, the 82nd percentile, the 83rd percentile, the 84th percentile, the 85th percentile, the 86th percentile, the 87th percentile, the 88th percentile, the 89th percentile, the 90th percentile, the 91st percentile, the 92nd percentile, the 93rd percentile, the 94th percentile, the 95th percentile, the 96th percentile, the 97th percentile, the 98th percentile, or the 99th percentile of expression levels or TAM signature scores in the reference population.In some instances, the reference expression level or reference gene signature score is a cut-off value that significantly separates a first and a second subset of individuals who have been treated with atezolizumab and tiragolumab in the same reference population based on a significant difference WO 2024/186790 PCT/US2024/018472 between an individual’s responsiveness to treatment with atezolizumab and tiragolumab above the cut-off value or at or below the cut-off value. In some aspects, the individual’s responsiveness to treatment with atezolizumab and tiragolumab is significantly improved relative to the individual’s responsiveness to treatment with atezolizumab and tiragolumab at or above the cut-off value.In some instances, the reference expression level or reference gene signature score is a cut-off value that significantly separates a first and a second subset of individuals who have been treated with atezolizumab (e.g., atezolizumab monotherapy) in the same reference population based on a significant difference between an individual’s responsiveness to treatment with atezolizumab above the cut-off value or at or below the cut-off value. In some aspects, the individual’s responsiveness to treatment with atezolizumab is significantly improved relative to the individual’s responsiveness to treatment with atezolizumab at or above the cut-off value.

PD-L1 statusIn some aspects, the expression level of PD-L1 has been assessed in the sample from a subject described herein. In some aspects, the sample has been determined to have a PD-L1-positive tumor cell fraction (e.g., by an immunohistochemical (IHC) assay, e.g., by positive staining with an anti-PD-Lantibody, wherein the anti-PD-L1 antibody is SP263, 22C3, SP142, or 28-8).Exemplary methods for assessing the expression level of PD-L1 are provided in Section II(F).

E. Assessment of TIGIT expression In some aspects, the expression of TIGIT is assessed in an individual described herein. The methods provided herein may include determining the expression level of TIGIT in a biological sample (e.g., a tumor sample) obtained from the individual. In other examples, the expression level of TIGIT in a biological sample (e.g., a tumor sample) obtained from the individual has been determined prior to initiation of treatment or after initiation of treatment. TIGIT expression may be determined using any suitable approach. Any suitable tumor sample may be used, e.g., a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.For example, TIGIT expression may be determined in terms of the percentage of a tumor sample comprised by tumor-infiltrating immune cells expressing a detectable expression level of TIGIT, as the percentage of tumor-infiltrating immune cells in a tumor sample expressing a detectable expression level of TIGIT, and/or as the percentage of tumor cells in a tumor sample expressing a detectable expression level of TIGIT. It is to be understood that in any of the preceding examples, the percentage of the tumor sample comprised by tumor-infiltrating immune cells may be in terms of the percentage of tumor area covered by tumor-infiltrating immune cells in a section of the tumor sample obtained from the individual, for example, as assessed by IHC using an anti-TIGIT antibody. Any suitable anti-TIGIT antibody may be used. In some examples, the anti-TIGIT antibody is 10A7 (WO 2009/126688A3; U.S. Patent No: 9,499,596). In other examples, the anti-TIGIT antibody is the anti-human TIGIT rabbit monoclonal antibody clone SP410 (Roche Tissue Diagnostics, Pleasanton, CA). In some aspects, the anti-TIGIT antibody (e.g., SP410) is detected using the VENTANA OptiView DAB IHC Detection Kit on the automated VENTANA BenchMark ULTRA platform.

WO 2024/186790 PCT/US2024/018472 F. Assessment of PD-L1 expression In some aspects, the expression of PD-L1 is assessed in an individual described herein. The methods provided herein may include determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the individual. In other examples, the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the individual has been determined prior to initiation of treatment or after initiation of treatment. PD-L1 expression may be determined using any suitable approach. For example, PD-L1 expression may be determined as described in U.S. Patent Application Publication Nos. US20180030138A1 and US20180037655A1. Any suitable tumor sample may be used, e.g., a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.For example, PD-L1 expression may be determined in terms of the percentage of a tumor sample comprised by tumor-infiltrating immune cells expressing a detectable expression level of PD-L1, as the percentage of tumor-infiltrating immune cells in a tumor sample expressing a detectable expression level of PD-L1, and/or as the percentage of tumor cells in a tumor sample expressing a detectable expression level of PD-L1. It is to be understood that in any of the preceding examples, the percentage of the tumor sample comprised by tumor-infiltrating immune cells may be in terms of the percentage of tumor area covered by tumor-infiltrating immune cells in a section of the tumor sample obtained from the individual, for example, as assessed by IHC using an anti-PD-L1 antibody (e.g., the SP142 antibody). Any suitable anti-PD-L1 antibody may be used, including, e.g., SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28- (Dako), E1L3N (Cell Signaling Technology), 4059 (ProSci, Inc.), h5H1 (Advanced Cell Diagnostics), and 9A11. In some examples, the anti-PD-L1 antibody is SP142. In other examples, the anti-PD-Lantibody is SP263. In some examples, the anti-PD-L1 antibody is 22C3. In some examples, the anti-PD- L1 antibody is 28-8.In some examples, a tumor sample obtained from the individual has a detectable expression level of PD-L1 in less than 1% of the tumor cells in the tumor sample, in 1% or more of the tumor cells in the tumor sample, in from 1% to less than 5% of the tumor cells in the tumor sample, in 5% or more of the tumor cells in the tumor sample, in from 5% to less than 50% of the tumor cells in the tumor sample, or in 50% or more of the tumor cells in the tumor sample.In some examples, a tumor sample obtained from the individual has a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise less than 1% of the tumor sample, more than 1% of the tumor sample, from 1% to less than 5% of the tumor sample, more than 5% of the tumor sample, from 5% to less than 10% of the tumor sample, or more than 10% of the tumor sample.In some aspects, a tumor sample obtained from the individual has a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise 5%-19% of the tumor sample (e.g., TIC 5%- 19%); e.g., has a PD-L1 expression level that is PD-L1 low. In some aspects, a tumor sample obtained from the individual has a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise >20% of the tumor sample (e.g., TIC >20%); e.g., has a PD-L1 expression level that is PD-Lhigh. In some embodiments, tumor samples that have been determined to have a TIC of greater than, or equal to, 5% are comparable to a CPS of greater than, or equal to, 1.

WO 2024/186790 PCT/US2024/018472 In some examples, tumor samples may be scored for PD-L1 positivity in tumor-infiltrating immune cells and/or in tumor cells according to the criteria for diagnostic assessment shown in Table 1 and/or Table 2, respectively.

Table 1. Tumor-infiltrating immune cell (IC) IHC diagnostic criteria PD-L1 Diagnostic Assessment IC Score Absence of any discernible PD-L1 staining ORPresence of discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering <1% of tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peri-tumoral desmoplastic stroma ICO Presence of discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering >1% to <5% of tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peri-tumoral desmoplastic stroma IC1 Presence of discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering >5% to <10% of tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peri-tumoral desmoplastic stroma IC2 Presence of discernible PD-L1 staining of any intensity in tumor-infiltrating immune cells covering >10% of tumor area occupied by tumor cells, associated intratumoral stroma, and contiguous peri-tumoral desmoplastic stroma ICS Table 2. Tumor cell (TC) IHC diagnostic criteria PD-L1 Diagnostic Assessment TC Score Absence of any discernible PD-L1 staining ORPresence of discernible PD-L1 staining of any intensity in <1% of tumor cells TCO Presence of discernible PD-L1 staining of any intensity in >1% to <5% of tumor cells TC1Presence of discernible PD-L1 staining of any intensity in >5% to <50% of tumor cells TC2Presence of discernible PD-L1 staining of any intensity in >50% of tumor cells TC3 In some instances, in any of the methods, uses, or compositions for use described herein, the individual has a PD-L1 -selected tumor (e.g., a proportion of tumor area occupied by PD-L1 expressing tumor-infiltrating immune cells (ICs) is greater than or equal to 5% in the tumor sample as determined by an IHC with the SP142 antibody). In some instances, the PD-L1-selected tumor is a tumor that has been determined to have a proportion of tumor area occupied by PD-L1 expressing immune cells (ICs) greater than or equal to 5% by an immunohistochemical (IHC) assay. In some instances, the IHC assay uses the anti-PD-L1 antibody SP142, SP263, 22C3, or 28-8. In some instances, the IHC assay uses anti-PD-Lantibody SP142. In some instances, the IHC assay uses anti-PD-L1 antibody SP263. In some instances, the IHC assay uses anti-PD-L1 antibody 22C3. In some instances, the IHC assay uses anti-PD-Lantibody 22C3. In some instances, the IHC assay uses anti-PD-L1 antibody 28-8.In some instances, the IC score has been determined to be greater than, or equal to, 5% (e.g., as determined using the Ventana (SP142) PD-L1 IHC assay). In some instances, the IC score has been determined to be 2 or 3 (e.g., as determined using the Ventana (SP142) PD-L1 IHC assay). In some instances, the IC score has been determined to be greater than, or equal to, 1% (e.g., as determined using the Ventana (SP142) PD-L1 IHC assay). In some instances, the IC score has been determined to be greater than, or equal to, 10% (e.g., as determined using the Ventana (SP142) PD-L1 IHC assay). In some instances, the IC score has been determined to be greater than, or equal to, 1% and less than 50% 61 WO 2024/186790 PCT/US2024/018472 (e.g., as determined using the Ventana (SP142) PD-L1 IHC assay). In some instances, the IC score has been determined to be greater than, or equal to, 1% and less than 30% (e.g., as determined using the Ventana (SP142) PD-L1 IHC assay).In some instances, in any of the methods, uses, or compositions for use described herein, a tumor sample obtained from the individual has a detectable protein expression level of PD-L1. In some instances, the detectable protein expression level of PD-L1 has been determined by an IHC assay. In some instances, the IHC assay uses anti-PD-L1 antibody SP142. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise greater than, or equal to, 5% of the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise greater than, or equal to, 1% of the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise greater than, or equal to, 1% and less than 5% of the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor- infiltrating immune cells that comprise greater than, or equal to, 5% and less than 10% of the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise greater than, or equal to, 10% of the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than, or equal to, 1 % of the tumor cells in the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than, or equal to, 1% and less than 5% of the tumor cells in the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than, or equal to, 5% and less than 50% of the tumor cells in the tumor sample. In some instances, the tumor sample has been determined to have a detectable expression level of PD-L1 in greater than, or equal to, 50% of the tumor cells in the tumor sample.In some instances, in any of the methods, uses, or compositions for use described herein, the individual has a PD-L1-selected tumor (e.g., a PD-L1 "high"-selected tumor (e.g., a PD-L1 tumor proportion score (TPS) greater than or equal to 50% in a tumor sample as determined by an IHC with the SP263 antibody)). In some instances, the PD-L1-selected tumor is a PD-L1 "high"-selected tumor. In some instances, the PD-L1-selected tumor is a tumor that has been determined to have TPS greater than or equal to 50% by an immunohistochemical (IHC) assay. In some instances, the IHC assay uses the anti-PD-L1 antibody SP263, SP142, 22C3, or 28-8. In some instances, the IHC assay uses anti-PD-Lantibody SP263. In some instances, the IHC assay uses anti-PD-L1 antibody SP142. In some instances, the IHC assay uses anti-PD-L1 antibody 22C3. In some instances, the TPS has been determined to be greater than, or equal to, 50% (e.g., as determined using the Ventana (SP263) PD-L1 IHC assay). In some instances, the TPS has been determined to be less than 50% (e.g., as determined using the Ventana (SP263) PD-L1 IHC assay). In some instances, the TPS has been determined to be greater than, or equal to, 1% (e.g., as determined using the Ventana (SP263) PD-L1 IHC assay). In some instances, the TPS has been determined to be greater than, or equal to, 1% and less than 50% (e.g., as determined using the Ventana (SP263) PD-L1 IHC assay).

WO 2024/186790 PCT/US2024/018472 In some instances, in any of the methods, uses, or compositions for use described herein, a tumor sample obtained from the individual has a detectable protein expression level of PD-L1. In some instances, the detectable protein expression level of PD-L1 has been determined by an IHC assay. In some instances, the IHC assay uses anti-PD-L1 antibody SP263. In some instances, the tumor sample has been determined to have a PD-L1-positive tumor cell fraction greater than, or equal to, 50% of the tumor sample. In some instances, the tumor sample has been determined to have a PD-L1 -positive tumor cell fraction less than 50% of the tumor sample. In some instances, the tumor sample has been determined to have a PD-L1-positive tumor cell fraction greater than, or equal to, 1% and less than 50% of the tumor sample.In some instances, the IHC assay uses the anti-PD-L1 antibody 22C3. In some instances, the IHC assay is the pharmDx 22C3 IHC assay. In some instances, the PD-L1-positive tumor cell fraction is greater than, or equal to, 50% as determined by positive staining with the anti-PD-L1 antibody 22C3. In some embodiments, the tumor sample has been determined to have a combined positive score (CPS) of greater than, or equal to, 10 or a tumor proportion score (TPS) of greater than or equal to 1 % in the tumor sample, e.g., as determined using the anti-PD-L1 antibody 22C3 as part of the pharmDx 22C3 IHC assay. In some embodiments, the tumor sample has been determined to have a CPS of greater than, or equal to, 10 or a TPS of greater than or equal to 1 % and less than 50% in the tumor sample, e.g., as determined using the anti-PD-L1 antibody 22C3 as part of the pharmDx 22C3 IHC assay. In some embodiments, the tumor sample has been determined to have a CPS of greater than, or equal to, 20 or a TPS of greater than or equal to 50% in the tumor sample, e.g., as determined using the anti-PD-Lantibody 22C3 as part of the pharmDx 22C3 IHC assay. In some embodiments, tumor samples that have been determined to have a CPS of greater than, or equal to, 1 are comparable to a TIC of greater than, or equal to, 5%.In some instances, the IHC assay uses the anti-PD-L1 antibody 28-8. In some instances, the IHC assay is the pharmDx 28-8 IHC assay. In some instances, the PD-L1-positive tumor cell fraction is greater than, or equal to, 50% as determined by positive staining with the anti-PD-L1 antibody 28-8.In some instances, in any of the methods, uses, or compositions for use described herein, a tumor sample obtained from the individual has a detectable nucleic acid expression level of PD-L1. In some instances, the detectable nucleic acid expression level of PD-L1 has been determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof. In some instances, the sample is selected from the group consisting of a tissue sample, a whole blood sample, a serum sample, and a plasma sample. In some instances, the tissue sample is a tumor sample. In some instances, the tumor sample comprises tumor-infiltrating immune cells, tumor cells, stromal cells, and any combinations thereof.

III. TIRAGOLUMAB AND ATEZOLIZUMAB Tiragolumab (an anti-TIGIT antagonist antibody) and atezolizumab (an anti-PD-L1 antibody), which are useful for treating an individual (e.g., a human) having a non-small cell lung cancer (NSCLC) in accordance with the methods, uses, and compositions for use of the invention, are described herein.

WO 2024/186790 PCT/US2024/018472 A. Tiragolumab Tiragolumab (CAS Registry Number: 1918185-84-8) is a fully human IgG1/kappa MAb that binds TIGIT and comprises the heavy chain sequence of SEQ ID NO: 4 and the light chain sequence of SEQ ID NO: 5. Tiragolumab comprises two N-linked glycosylation sites (N306) in the Fc domain. Tiragolumab is described in WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Proposed INN: List 117, Vol. 31, No. 2, published June 9, 2017 (see page 343).Tiragolumab (Genentech) is also known as MTIG7192A, RG6058, or RO7092284. Tiragolumab is described in PCT Pub. Nos. WO2003072305A8, WO2004024068A3, WO2004024072A3, WO2009126688A2, WO2015009856A2, WO2016011264A1, WO2016109546A2, WO2017053748A2, and WO2019165434A1; US Pub. Nos. 2017/0044256, 2017/0037127, 2017/0145093, 2017/260594, 2017/0088613, 2018/0186875, 2019/0119376; and US Pat. Nos. US9873740B2, US10626174B2, US10611836B2, US9499596B2, US8431350B2, US10047158B2, and US10017572B2.

S. Atezolizumab Atezolizumab is an anti-PD-L1 antagonist monoclonal antibody (mAb) having the International Nonproprietary Names for Pharmaceutical Substances (INN) List 112 (WHO Drug Information, Vol. 28, No. 4, 2014, p. 488), or the CAS Registry Number 1380723-44-3.

C. Methods of delivery The compositions utilized in the methods described herein (e.g., tiragolumab and atezolizumab) can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, topically, transdermally, intravitreally (e.g., by intravitreal injection), by eye drop, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The compositions utilized in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In some aspects, tiragolumab and/or atezolizumab is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.Tiragolumab and/or atezolizumab (and any additional therapeutic agent) may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical WO 2024/186790 PCT/US2024/018472 condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. Tiragolumab and/or atezolizumab need not be, but are optionally formulated with and/or administered concurrently with one or more agents currently used to prevent or treat the disorder in question, e.g., one or more of the agents provided herein. The effective amount of such other agents depends on the amount of tiragolumab or atezolizumab present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.For the treatment of a NSCLC, the appropriate dosage of tiragolumab and atezolizumab, or any combination thereof, described herein (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the tiragolumab and/or atezolizumab is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the tiragolumab and/or atezolizumab, and the discretion of the attending physician. Tiragolumab and/or atezolizumab is suitably administered to the patient at one time or over a series of treatments. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. Such doses may be administered intermittently, e.g., every week or every three weeks (e.g., such that the patient receives, for example, from about two to about twenty, or e.g., about six doses of the tiragolumab and/or atezolizumab). An initial higher loading dose, followed by one or more lower doses, may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

D. Dosing i. Dosing of tiragolumab As a general proposition, the therapeutically effective amount of tiragolumab administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight, whether by one or more administrations. In some embodiments, the therapeutically effective amount of tiragolumab administered to a human is in the range of 0.01 to 50 mg/kg of patient body weight, whether by one or more administrations.In some exemplary embodiments, tiragolumab is administered in a dose of about 0.01 to about mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, weekly, every two weeks, every three weeks, or every four weeks, for example. In exemplary embodiments, tiragolumab is administered in a dose of 0.01 to 45 mg/kg, 0.01 to 40 mg/kg, 0.01 to 35 mg/kg, 0.01 to mg/kg, 0.01 to 25 mg/kg, 0.01 to 20 mg/kg, 0.01 to 15 mg/kg, 0.01 to 10 mg/kg, 0.01 to 5 mg/kg, or 0.01 to mg/kg administered daily, weekly, every two weeks, every three weeks, or every four weeks, for example.

WO 2024/186790 PCT/US2024/018472 In some instances, tiragolumab is administered on about Day 1 (e.g., Day -3, Day -2, Day -1, Day 1, Day 2, or Day 3) of a dosing cycle.In some instances, tiragolumab is administered (e.g., every three weeks) in a tiered dosing regimen (e.g., dosing based on body weight (BW) or body surface area (BSA) of a subject). Such dosing regimens can be utilized in treatments for subjects having relatively low body weight (e.g., 40 kg or less (e.g., from 5 kg to 40 kg, from 15 kg to 40 kg, or from 5 kg to 15 kg)) and have been developed through biosimulation studies based on extrapolations of pharmacokinetic parameters estimated from adult data.In some instances, the effective amount of tiragolumab to treat a subject having a cancer is a tiered dose based on a subject’s body weight. In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 400 mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between about 30 mg to about 1200 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between about 250 mg to about 350 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between about 350 mg to about 450 mg every three weeks (e.g., about 4mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between about 550 mg to about 650 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of about 300 mg every three weeks; (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of about 400 mg every three weeks; or (c) greater than 40 kg, and tiragolumab is administered at a dose of about 600 mg every three weeks. In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 400 mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between 30 mg to 1200 mg every three weeks (e.g., 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between 250 mg to 350 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between 350 mg to 450 mg every three weeks (e.g., 400 mg every three weeks); or (c) greater than kg, and tiragolumab is administered at a dose of between 550 mg to 650 mg every three weeks (e.g., 6mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on WO 2024/186790 PCT/US2024/018472 a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of 300 mg every three weeks; (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of 400 mg every three weeks; or (c) greater than 40 kg, and tiragolumab is administered at a dose of 600 mg every three weeks.In some instances, the effective amount of tiragolumab is a dose of between about 30 mg to about 1200 mg (e.g., between about 30 mg to about 1100 mg, e.g., between about 60 mg to about 10mg, e.g., between about 100 mg to about 900 mg, e.g., between about 200 mg to about 800 mg, e.g., between about 300 mg to about 800 mg, e.g., between about 400 mg to about 800 mg, e.g., between about 400 mg to about 750 mg, e.g., between about 450 mg to about 750 mg, e.g., between about 5mg to about 700 mg, e.g., between about 550 mg to about 650 mg, e.g., 600 mg ± 10 mg, e.g., 600 ± mg, e.g., 600 ± 5 mg, e.g., 600 ± 3 mg, e.g., 600 ± 1 mg, e.g., 600 ± 0.5 mg, e.g., 600 mg) every three weeks (Q3W) for subject with a body weight greater than 40 kg (e.g., 40.5 kg, 41 kg, 42 kg, 43 kg, 44 kg, kg, 46 kg, 47 kg, 48 kg, 49 kg, 50 kg, 51 kg, 52 kg, 53 kg, 54 kg, 55 kg, 56 kg, 57 kg, 58 kg, 59 kg, kg, 61 kg, 62 kg, 63 kg, 64 kg, 65 kg, 66 kg, 67 kg, 68 kg, 69 kg, 70 kg, 75 kg, 80 kg, 85 kg, 90 kg, 95 kg, 100 kg, 110 kg, 120 kg, 130 kg, 140 kg, 150 kg or more). In some instances, the effective amount of tiragolumab is a dose of about 600 mg every three weeks for subject with a body weight greater than kg. In some instances, the effective amount of tiragolumab is a dose of between 30 mg to 1200 mg (e.g., between 30 mg to 1100 mg, e.g., between 60 mg to 1000 mg, e.g., between 100 mg to 900 mg, e.g.,between 200 mg to 800 mg, e.g., between 300 mg to 800 mg, e.g., between 400 mg to 800 mg, e.g.,between 400 mg to 750 mg, e.g., between 450 mg to 750 mg, e.g., between 500 mg to 700 mg, e.g.,between 550 mg to 650 mg, e.g., 600 mg ± 10 mg, e.g., 600 ± 6 mg, e.g., 600 ± 5 mg, e.g., 600 ± 3 mg,e.g., 600 ± 1 mg, e.g., 600 ± 0.5 mg, e.g., 600 mg) every three weeks (Q3W) for subject with a body weight greater than 40 kg (e.g., 40.5 kg, 41 kg, 42 kg, 43 kg, 44 kg, 45 kg, 46 kg, 47 kg, 48 kg, 49 kg, kg, 51 kg, 52 kg, 53 kg, 54 kg, 55 kg, 56 kg, 57 kg, 58 kg, 59 kg, 60 kg, 61 kg, 62 kg, 63 kg, 64 kg, 65 kg, kg, 67 kg, 68 kg, 69 kg, 70 kg, 75 kg, 80 kg, 85 kg, 90 kg, 95 kg, 100 kg, 110 kg, 120 kg, 130 kg, 1kg, 150 kg or more). In some instances, the effective amount of tiragolumab is a dose of 600 mg every three weeks for subject with a body weight greater than 40 kg.In some instances, the effective amount of tiragolumab is a dose of between about 10 mg to about 1000 mg (e.g., between about 20 mg to about 1000 mg, e.g., between about 50 mg to about 9mg, e.g., between about 100 mg to about 850 mg, e.g., between about 200 mg to about 700 mg, e.g., between about 250 mg to about 600 mg, e.g., between about 300 mg to about 500 mg, e.g., between about 350 mg to about 450 mg, e.g., between about 390 mg to about 410 mg, e.g., about 400 mg) every three weeks (Q3W) for subject with a body weight greater than 15 kg and less than or equal to 40 kg (e.g., 15.1 kg, 15.2 kg, 15.3 kg, 15.4 kg, 15.5 kg, 16 kg, 17 kg, 18 kg, 19 kg, 20 kg, 21 kg, 22 kg, 23 kg, kg, 25 kg, 26 kg, 27 kg, 28 kg, 29 kg, 30 kg, 31 kg, 32 kg, 33 kg, 34 kg, 35 kg, 36 kg, 37 kg, 38 kg, kg, or 39.5 kg). In some instances, the effective amount of tiragolumab is a dose of about 400 mg every three weeks (e.g., 400 mg ± 10 mg, e.g., 400 ± 6 mg, e.g., 400 ± 5 mg, e.g., 400 ± 3 mg, e.g., 400 ± mg, e.g., 400 ± 0.5 mg, e.g., 400 mg every three weeks) for subject with a body weight greater than 15 kg and less than or equal to 40 kg. In some instances, the effective amount of tiragolumab is a dose of between 10 mg to 1000 mg (e.g., between 20 mg to 1000 mg, e.g., between 50 mg to 900 mg, e.g., WO 2024/186790 PCT/US2024/018472 between 100 mg to 850 mg, e.g., between 200 mg to 700 mg, e.g., between 250 mg to 600 mg, e.g., between 300 mg to 500 mg, e.g., between 350 mg to 450 mg, e.g., between 390 mg to 410 mg, e.g., 4mg) every three weeks (Q3W) for subject with a body weight greater than 15 kg and less than or equal to kg (e.g., 15.1 kg, 15.2 kg, 15.3 kg, 15.4 kg, 15.5 kg, 16 kg, 17 kg, 18 kg, 19 kg, 20 kg, 21 kg, 22 kg, kg, 24 kg, 25 kg, 26 kg, 27 kg, 28 kg, 29 kg, 30 kg, 31 kg, 32 kg, 33 kg, 34 kg, 35 kg, 36 kg, 37 kg, 38 kg, kg, or 39.5 kg). In some instances, the effective amount of tiragolumab is a dose of 400 mg every three weeks (e.g., 400 mg ± 10 mg, e.g., 400 ± 6 mg, e.g., 400 ± 5 mg, e.g., 400 ± 3 mg, e.g., 400 ± mg, e.g., 400 ± 0.5 mg, e.g., 400 mg every three weeks) for subject with a body weight greater than 15 kg and less than or equal to 40 kg.In some instances, the effective amount of tiragolumab is a dose of between about 10 mg to about 1000 mg (e.g., between about 10 mg to about 900 mg, e.g., between about 50 mg to about 9mg, e.g., between about 100 mg to about 750 mg, e.g., between about 100 mg to about 600 mg, e.g., between about 150 mg to about 500 mg, e.g., between about 200 mg to about 400 mg, e.g., between about 250 mg to about 350 mg, e.g., between about 290 mg to about 310 mg, e.g., about 300 mg) every three weeks (Q3W) for subject with a body weight less than or equal to 15 kg (e.g., 0.5 kg, 1 kg, 1.5 kg, 2.0 kg, 2.5 kg, 3.0 kg, 3.5 kg, 4.0 kg, 4.5 kg, 5.0 kg, 5.5 kg, 6.0 kg, 6.5 kg, 7.0 kg, 7.5 kg, 8.0 kg, 8.5 kg, 9.0 kg, 9.5 kg, 10.0 kg, 10.5 kg, 11.0 kg, 11.5 kg, 12.0 kg, 12.5 kg, 13.0 kg, 13.5 kg, 14.0 kg, 14.5 kg, or 15.0 kg). In some instances, the effective amount of tiragolumab is a dose of about 300 mg every three weeks (e.g., 300 mg ± 10 mg, e.g., 300 ± 6 mg, e.g., 300 ± 5 mg, e.g., 300 ± 3 mg, e.g., 300 ± 1 mg, e.g., 300 ± 0.5 mg, e.g., 300 mg every three weeks) for subject with a body weight less than or equal to 15 kg. In some instances, the effective amount of tiragolumab is a dose of between 10 mg to 1000 mg (e.g., between 10 mg to 900 mg, e.g., between 50 mg to 900 mg, e.g., between 100 mg to 750 mg, e.g., between 100 mg to 600 mg, e.g., between 150 mg to 500 mg, e.g., between 200 mg to 400 mg, e.g., between 250 mg to 350 mg, e.g., between 290 mg to 310 mg, e.g., 300 mg) every three weeks (Q3W) for subject with a body weight less than or equal to 15 kg (e.g., 0.5 kg, 1 kg, 1.5 kg, 2.0 kg, 2.5 kg, 3.0 kg, 3.kg, 4.0 kg, 4.5 kg, 5.0 kg, 5.5 kg, 6.0 kg, 6.5 kg, 7.0 kg, 7.5 kg, 8.0 kg, 8.5 kg, 9.0 kg, 9.5 kg, 10.0 kg, 10.kg, 11.0 kg, 11.5 kg, 12.0 kg, 12.5 kg, 13.0 kg, 13.5 kg, 14.0 kg, 14.5 kg, or 15.0 kg). In some instances, the effective amount of tiragolumab is a dose of 300 mg every three weeks (e.g., 300 mg ± 10 mg, e.g., 300 ± 6 mg, e.g., 300 ± 5 mg, e.g., 300 ± 3 mg, e.g., 300 ± 1 mg, e.g., 300 ± 0.5 mg, e.g., 300 mg every three weeks) for subject with a body weight less than or equal to 15 kg.In some instances, the effective amount of tiragolumab to treat a subject having a cancer is a tiered dose based on a subject’s body surface area. In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 350 mg every three weeks); (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between about 30 mg to about 1200 mg every WO 2024/186790 PCT/US2024/018472 three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between about 250 mg to about 350 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between about 300 mg to about 400 mg every three weeks (e.g., about 350 mg every three weeks); or (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between about 400 mg to about 500 mg every three weeks (e.g., about 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between about 550 mg to about 650 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of about 3mg every three weeks; (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of about 400 mg every three weeks; (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of 450 mg every three weeks; or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of about 600 mg every three weeks. In some instances, the effective amount of tiragolumab to treat a subject having a cancer is a tiered dose based on a subject’s body surface area. In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 350 mg every three weeks); (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between 30 mg to 12mg every three weeks (e.g., 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between 250 mg to 350 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between 300 mg to 400 mg every three weeks (e.g., 350 mg every three weeks); or (c) greater than 0.75 m2 and less than or equal to 1.m2, and tiragolumab is administered at a dose of between 400 mg to 500 mg every three weeks (e.g., 4mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between 550 mg to 650 mg every three weeks (e.g., 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of 3mg every three weeks; (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of 400 mg every three weeks; (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of 450 mg every three weeks; or (d) greater than 1.m2, and tiragolumab is administered at a dose of 600 mg every three weeks.

WO 2024/186790 PCT/US2024/018472 In some instances, the effective amount of tiragolumab is a dose of between about 30 mg to about 1200 mg (e.g., between about 30 mg to about 1100 mg, e.g., between about 60 mg to about 10mg, e.g., between about 100 mg to about 900 mg, e.g., between about 200 mg to about 800 mg, e.g., between about 300 mg to about 800 mg, e.g., between about 400 mg to about 800 mg, e.g., between about 400 mg to about 750 mg, e.g., between about 450 mg to about 750 mg, e.g., between about 5mg to about 700 mg, e.g., between about 550 mg to about 650 mg, e.g., 600 mg ± 10 mg, e.g., 600 ± mg, e.g., 600 ± 5 mg, e.g., 600 ± 3 mg, e.g., 600 ± 1 mg, e.g., 600 ± 0.5 mg, e.g., 600 mg) every three weeks (Q3W) for subject with a body surface area greater than 1.25 m2 (e.g., 1.25 m2, 1.35 m2, 1.45 m2, 1.50 m2, 1.55 m2, 1.60 m2, 1.65 m2, 1.70 m2, 1.75 m2, 1.80 m2, 1.85 m2, 1.90 m2, 1.95 m2, 2.0 m2, 2.1 m2, 2.2 m2, 2.3 m2, 2.4 m2, 2.5 m2, 2.6 m2, 2.7 m2, 2.8 m2, 2.9 m2, 3.0 m2 or more). In some instances, the effective amount of tiragolumab is a dose of about 600 mg every three weeks for subject with a body surface area greater than 1.25 m2. In some instances, the effective amount of tiragolumab is a dose of between 30 mg to 1200 mg (e.g., between 30 mg to 1100 mg, e.g., between 60 mg to 1000 mg, e.g.,between 100 mg to 900 mg, e.g., between 200 mg to 800 mg, e.g., between 300 mg to 800 mg, e.g.,between 400 mg to 800 mg, e.g., between 400 mg to 750 mg, e.g., between 450 mg to 750 mg, e.g.,between 500 mg to 700 mg, e.g., between 550 mg to 650 mg, e.g., 600 mg ± 10 mg, e.g., 600 ± 6 mg,e.g., 600 ± 5 mg, e.g., 600 ± 3 mg, e.g., 600 ± 1 mg, e.g., 600 ± 0.5 mg, e.g., 600 mg) every three weeks (Q3W) for subject with a body surface area greater than 1.25 m2 (e.g., 1.25 m2, 1.35 m2, 1.45 m2, 1.m2, 1.55 m2, 1.60 m2, 1.65 m2, 1.70 m2, 1.75 m2, 1.80 m2, 1.85 m2, 1.90 m2, 1.95 m2, 2.0 m2, 2.1 m2, 2.m2, 2.3 m2, 2.4 m2, 2.5 m2, 2.6 m2, 2.7 m2, 2.8 m2, 2.9 m2, 3.0 m2 or more). In some instances, the effective amount of tiragolumab is a dose of 600 mg every three weeks for subject with a body surface area greater than 1.25 m2.In some instances, the effective amount of tiragolumab is a dose of between about 10 mg to about 1000 mg (e.g., between about 20 mg to about 1000 mg, e.g., between about 50 mg to about 9mg, e.g., between about 100 mg to about 850 mg, e.g., between about 200 mg to about 700 mg, e.g., between about 250 mg to about 600 mg, e.g., between about 300 mg to about 500 mg, e.g., between about 400 mg to about 500 mg, e.g., between about 440 mg to about 460 mg, e.g., about 450 mg) every three weeks (Q3W) for subject with a body surface area greater than 0.75 m2 and less than or equal to 1.25 m2 (e.g., 0.76 m2, 0.77 m2, 0.78 m2, 0.79 m2, 0.80 m2, 0.82 m2, 0.84 m2, 0.86 m2, 0.88 m2, 0.90 m2, 0.95 m2, 1.0 m2, 1.05 m2, 1.10 m2, 1.15 m2, 1.20 m2, or 1.25 m2). In some instances, the effective amount of tiragolumab is a dose of about 450 mg every three weeks (e.g., 450 mg ± 10 mg, e.g., 450 ± 6 mg, e.g., 450 ± 5 mg, e.g., 450 ± 3 mg, e.g., 450 ± 1 mg, e.g., 450 ± 0.5 mg, e.g., 450 mg every three weeks) for subject with a body surface area greater than 0.75 m2 and less than or equal to 1.25 m2.In some instances, the effective amount of tiragolumab is a dose of between about 10 mg to about 1000 mg (e.g., between about 20 mg to about 1000 mg, e.g., between about 50 mg to about 9mg, e.g., between about 100 mg to about 850 mg, e.g., between about 200 mg to about 700 mg, e.g., between about 250 mg to about 600 mg, e.g., between about 300 mg to about 500 mg, e.g., between about 300 mg to about 400 mg, e.g., between about 340 mg to about 360 mg, e.g., about 350 mg) every three weeks (Q3W) for subject with a body surface area greater than 0.5 m2 and less than or equal to 0.75 m2 (e.g., 0.51 m2, 0.52 m2, 0.53 m2, 0.54 m2, 0.55 m2, 0.56 m2, 0.57 m2, 0.58 m2, 0.59 m2, 0.60 m2, WO 2024/186790 PCT/US2024/018472 0.72 , 2 ודו 0.71 , 2 ודו 0.70 , 2 ודו 0.69 , 2 ודו 0.68 , 2 ודו 0.67 , 2 ודו 0.66 , 2 ודו 0.65 , 2 ודו 0.64 , 2 ודו 0.63 , 2 ודו 0.62 , 2 ודו 0.61ודו 0.74 , 2 ודו 0.73 , 2 ודו , or 0.75 2ודו). In some instances, the effective amount of tiragolumab is a dose of about 350 mg every three weeks (e.g., 350 mg ± 10 mg, e.g., 350 ± 6 mg, e.g., 350 ± 5 mg, e.g., 350 ± mg, e.g., 350 ± 1 mg, e.g., 350 ± 0.5 mg, e.g., 350 mg every three weeks) for subject with a body surface area greater than 0.5 m2 and less than or equal to 0.75 m2.In some instances, the effective amount of tiragolumab is a dose of between about 10 mg to about 1000 mg (e.g., between about 10 mg to about 900 mg, e.g., between about 50 mg to about 9mg, e.g., between about 100 mg to about 750 mg, e.g., between about 100 mg to about 600 mg, e.g., between about 150 mg to about 500 mg, e.g., between about 200 mg to about 400 mg, e.g., between about 250 mg to about 350 mg, e.g., between about 290 mg to about 310 mg, e.g., about 300 mg) every three weeks (Q3W) for subject with a body surface area less than or equal to 0.5 m2 (e.g., 0.02 m2, 0.m2, 0.06 m2, 0.08 m2, 0.1 m2, 0.15 m2, 0.20 m2, 0.25 m2, 0.30 m2, 0.35 m2, 0.40 m2, 0.45 m2, or 0.50 m2). In some instances, the effective amount of tiragolumab is a dose of about 300 mg every three weeks (e.g., 300 mg ± 10 mg, e.g., 300 ± 6 mg, e.g., 300 ± 5 mg, e.g., 300 ± 3 mg, e.g., 300 ± 1 mg, e.g., 300 ± 0.5 mg, e.g., 300 mg every three weeks) for subject with a body surface area less than or equal to 0.5 m2.In some instances, the effective amount of tiragolumab is a dose (e.g., a fixed dose) of between about 10 mg to about 1000 mg (e.g., between about 20 mg to about 1000 mg, e.g., between about 50 mg to about 900 mg, e.g., between about 100 mg to about 850 mg, e.g., between about 200 mg to about 8mg, e.g., between about 300 mg to about 600 mg, e.g., between about 400 mg to about 500 mg, e.g., between about 405 mg to about 450 mg, e.g., between about 410 mg to about 430 mg, e.g., about 4mg) every two weeks (Q2W). In some instances, the effective amount of tiragolumab is a dose of about 420 mg every two weeks (e.g., 420 mg ± 10 mg, e.g., 420 ± 6 mg, e.g., 420 ± 5 mg, e.g., 420 ± 3 mg, e.g., 420 ± 1 mg, e.g., 420 ± 0.5 mg, e.g., 420 mg every two weeks). In some instances, the method comprises administering to the subject or population of subjects tiragolumab at a dose of about 300 mg to about 600 mg every two weeks. In some instances, the method comprises administering to the subject or population of subjects tiragolumab at a dose of 300 mg to 600 mg every two weeks. In some instances, the method comprises administering to the subject or population of subjects tiragolumab at a dose of about 420 every two weeks. In some instances, the method comprises administering to the subject or population of subjects tiragolumab at a dose of 420 every two weeks. In some instances, the dose of tiragolumab is a fixed dose.In some instances, the effective amount of tiragolumab is a dose (e.g., a fixed dose) of between about 30 mg to about 1200 mg (e.g., between about 30 mg to about 1100 mg, e.g., between about 60 mg to about 1000 mg, e.g., between about 100 mg to about 900 mg, e.g., between about 200 mg to about 800 mg, e.g., between about 300 mg to about 800 mg, e.g., between about 400 mg to about 800 mg, e.g., between about 400 mg to about 750 mg, e.g., between about 450 mg to about 750 mg, e.g., between about 500 mg to about 700 mg, e.g., between about 550 mg to about 650 mg, e.g., 600 mg ± 10 mg, e.g., 600 ± 6 mg, e.g., 600 ± 5 mg, e.g., 600 ± 3 mg, e.g., 600 ± 1 mg, e.g., 600 ± 0.5 mg, e.g., 600 mg) every three weeks (Q3W). In some instances, the effective amount of tiragolumab is a dose of about 600 mg every three weeks. In some instances, the method comprises administering to the subject or population of subjects tiragolumab at a dose of about 600 every three weeks. In some instances, the method WO 2024/186790 PCT/US2024/018472 comprises administering to the subject or population of subjects tiragolumab at a dose of 600 mg every three weeks. In some instances, the dose of tiragolumab is a fixed dose.In some instances, the effective amount of tiragolumab is a dose of between about 200 mg to about 2000 mg (e.g., between about 200 mg to about 2000 mg, e.g., between about 400 mg to about 1900 mg, e.g., between about 500 mg to about 1800 mg, e.g., between about 600 mg to about 1700 mg, e.g., between about 700 mg to about 1400 mg, e.g., between about 800 mg to about 1600 mg, e.g., between about 900 mg to about 1500 mg, e.g., between about 1000 mg to about 1400 mg, e.g., between about 1050 mg to about 1350 mg, e.g., between about 1100 mg to about 1300 mg, e.g., between about 1150 mg to about 1250 mg, e.g., between about 1175 mg to about 1225 mg, e.g., between about 11mg to about 1210 mg, e.g., about 1200 mg, e.g., 1200 mg ± 10 mg, e.g., 1200 ± 6 mg, e.g., 1200 ± 5 mg, e.g., 1200 ± 3 mg, e.g., 1200 ± 1 mg, e.g., 1200 ± 0.5 mg, e.g., 1200 mg) every three weeks (Q3W). In some instances, the effective amount of tiragolumab is a dose of about 600 mg every three weeks. In some instances, the effective amount of tiragolumab is a dose of 600 mg every three weeks.In some instances, the effective amount of tiragolumab is a dose of between about 200 mg to about 2000 mg (e.g., between about 200-300 mg, between about 300-400 mg, between about 400-5mg, between about 500-600 mg, between about 600-700 mg, between about 700-800 mg, between about 800-900 mg, between about 900-1000 mg, between about 1000-1100 mg, between about 1100-1200 mg, between about 1200-1300 mg, between about 1300-1400 mg, between about 1400-1500 mg, between about 1500-1600 mg, between about 1600-1700 mg, between about 1700-1800 mg, between about 1800-1900 mg, or between about 1900-2000 mg, e.g., between about 200 mg to about 1600 mg, e.g., between about 250 mg to about 1600 mg, e.g., between about 300 mg to about 1600 mg, e.g., between about 400 mg to about 1500 mg, e.g., between about 500 mg to about 1400 mg, e.g., between about 6mg to about 1200 mg, e.g., between about 700 mg to about 1100 mg, e.g., between about 800 mg to about 1000 mg, e.g., between about 800 mg to about 900 mg, e.g., about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, about 1500, about 1550, about 16mg, about 1650 mg, about 1700 mg, about 1750 mg, about 1800 mg, about 1850 mg, about 1900 mg, about 1950 mg, or about 2000 mg, e.g., about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, or about 900 mg) every four weeks (Q4W). In some instances, the effective amount of tiragolumab is about 700 mg to about 1000 mg every four weeks. In some instances, the effective amount of tiragolumab is 700 mg to 1000 mg every four weeks. In some instances, the effective amount of tiragolumab is about 840 mg every four weeks. In some instances, the effective amount of tiragolumab is 840 mg every four weeks. The 840 mg Q4W dosing regimen is supported by results from PK modeling and simulation and exposure-safety analyses. Briefly, the average concentration following the 840 mg Q4W dosing regimen is similar to that of the 600 mg every weeks dosing regimen, which was evaluated in previous studies. The Cmax of the 840 mg Q4W dosing regimen was simulated to be 28% higher at steady state, relative to the Cmax for the 600 mg every weeks dosing regimen, but falls within the range of observed exposure of the highest administered dose in the clinic (1200 mg every 3 weeks). A preliminary analysis of the tiragolumab exposure-safety WO 2024/186790 PCT/US2024/018472 relationship based on previous observations (tiragolumab doses of 2-1200 mg every 3 weeks administered as monotherapy or in combination with atezolizumab 1200 mg every 3 weeks) suggest that tiragolumab exhibits a flat exposure-safety relationship. In summary, the 840 mg Q4W dosing regimen can provide comparable safety and efficacy as the 600 mg every-3-weeks dosing regimen, given that the predicted exposure is within the range of observed efficacious exposures and tiragolumab exhibits a flat exposure-safety relationship.In some instances, the effective amount of tiragolumab is a dose of between about 200 mg to about 2000 mg (e.g., between about 200 mg to about 2000 mg, e.g., between about 400 mg to about 1900 mg, e.g., between about 500 mg to about 1800 mg, e.g., between about 600 mg to about 1700 mg, e.g., between about 700 mg to about 1400 mg, e.g., between about 800 mg to about 1600 mg, e.g., between about 900 mg to about 1500 mg, e.g., between about 1000 mg to about 1400 mg, e.g., between about 1050 mg to about 1350 mg, e.g., between about 1100 mg to about 1300 mg, e.g., between about 1150 mg to about 1250 mg, e.g., between about 1175 mg to about 1225 mg, e.g., between about 11mg to about 1210 mg (e.g., between 200 mg to 2000 mg, e.g., between 400 mg to 1900 mg, e.g., between 500 mg to 1800 mg, e.g., between 600 mg to 1700 mg, e.g., between 700 mg to 1400 mg, e.g., between 800 mg to 1600 mg, e.g., between 900 mg to 1500 mg, e.g., between 1000 mg to 1400 mg, e.g., between 1050 mg to 1350 mg, e.g., between 1100 mg to 1300 mg, e.g., between 1150 mg to 1250 mg, e.g., between 1175 mg to 1225 mg, e.g., between 1190 mg to 1210 mg), e.g., about 1200 mg, e.g., 12mg ± 10 mg, e.g., 1200 ± 6 mg, e.g., 1200 ± 5 mg, e.g., 1200 ± 3 mg, e.g., 1200 ± 1 mg, e.g., 1200 ± 0.mg, e.g., 1200 mg) every four weeks (Q4W). In some instances, the effective amount of tiragolumab is a dose of about 840 mg every four weeks (e.g., 840 mg ± 10 mg, e.g., 840 ± 6 mg, e.g., 840 ± 5 mg, e.g., 840 ± 3 mg, e.g., 840 ± 1 mg, e.g., 840 ± 0.5 mg, e.g., 840 mg every four weeks). In some instances, the effective amount of tiragolumab is a dose of 840 mg every four weeks. In some instances, the dose of tiragolumab is a fixed dose.In some instances, the effective amount of tiragolumab is a dose of about 1200 mg every four weeks.In some instances, the dose of tiragolumab administered in a combination therapy (e.g., a combination treatment with atezolizumab) may be reduced as compared to a standard dose of tiragolumab administered as a monotherapy.In some instances, tiragolumab is administered intravenously. Alternatively, in some embodiments, tiragolumab is administered subcutaneously. In some instances, tiragolumab is administered to the patient intravenously at a dose of about 420 mg every 2 weeks, about 600 mg every weeks, or about 840 mg of every 4 weeks. In some instances, tiragolumab is administered to the patient intravenously at a dose of 420 mg every 2 weeks, 600 mg every 3 weeks, or 840 mg of every 4 weeks.In some instances, a subject is administered a total of 1 to 20 doses of tiragolumab, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses. In some instances, a subject is administered a total of 1 to 50 doses of tiragolumab, e.g., 1 to 50 doses, 1 to 45 doses, 1 to 40 doses, to 35 doses, 1 to 30 doses, 1 to 25 doses, 1 to 20 doses, 1 to 15 doses, 1 to 10 doses, 1 to 5 doses, 2 to doses, 2 to 45 doses, 2 to 40 doses, 2 to 35 doses, 2 to 30 doses, 2 to 25 doses, 2 to 20 doses, 2 to doses, 2 to 10 doses, 2 to 5 doses, 3 to 50 doses, 3 to 45 doses, 3 to 40 doses, 3 to 35 doses, 3 to 30 WO 2024/186790 PCT/US2024/018472 doses, 3 to 25 doses, 3 to 20 doses, 3 to 15 doses, 3 to 10 doses, 3 to 5 doses, 4 to 50 doses, 4 to 45doses, 4 to 40 doses, 4 to 35 doses, 4 to 30 doses, 4 to 25 doses, 4 to 20 doses, 4 to 15 doses, 4 to 10doses, 4 to 5 doses, 5 to 50 doses, 5 to 45 doses, 5 to 40 doses, 5 to 35 doses, 5 to 30 doses, 5 to doses, 5 to 20 doses, 5 to 15 doses, 5 to 10 doses, 10 to 50 doses, 10 to 45 doses, 10 to 40 doses, 10 todoses, 10 to 30 doses, 10 to 25 doses, 10 to 20 doses, 10 to 15 doses, 15 to 50 doses, 15 to doses, 15 to 40 doses, 15 to 35 doses, 15 to 30 doses, 15 to 25 doses, 15 to 20 doses, 20 to 50 doses, to 45 doses, 20 to 40 doses, 20 to 35 doses, 20 to 30 doses, 20 to 25 doses, 25 to 50 doses, 25 to doses, 25 to 40 doses, 25 to 35 doses, 25 to 30 doses, 30 to 50 doses, 30 to 45 doses, 30 to 40 doses, to 35 doses, 35 to 50 doses, 35 to 45 doses, 35 to 40 doses, 40 to 50 doses, 40 to 45 doses, or 45 to doses. In particular instances, the doses may be administered intravenously. ii. Dosing of atezolizumab As a general proposition, the therapeutically effective amount of atezolizumab to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight, whether by one or more administrations.In some exemplary embodiments, atezolizumab is administered in a dose of about 0.01 to about mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, weekly, every two weeks, every three weeks, or every four weeks, for example. In some exemplary embodiments, atezolizumab is administered in a dose of 0.01 to 45 mg/kg, 0.01 to 40 mg/kg, 0.01 to mg/kg, 0.01 to 30 mg/kg, 0.01 to 25 mg/kg, 0.01 to 20 mg/kg, 0.01 to 15 mg/kg, 0.01 to 10 mg/kg, 0.01 to mg/kg, or 0.01 to 1 mg/kg administered daily, weekly, every two weeks, every three weeks, or every four weeks, for example.In some instances, atezolizumab is administered on about Day 1 (e.g., Day -3, Day -2, Day -1, Day 1, Day 2, or Day 3) of a dosing cycle.In some instances, the effective amount of atezolizumab is a dose (e.g., a fixed dose) of between about 20 mg to about 1600 mg (e.g., between about 40 mg to about 1500 mg, e.g., between about 2mg to about 1400 mg, e.g., between about 300 mg to about 1400 mg, e.g., between about 400 mg to about 1400 mg, e.g., between about 500 mg to about 1300 mg, e.g., between about 600 mg to about 1200 mg, e.g., between about 700 mg to about 1100 mg, e.g., between about 800 mg to about 1000 mg, e.g., between about 800 mg to about 900 mg, e.g., about 800, about 810, about 820, about 830, about 840, about 850, about 860, about 870, about 880, about 890, or about 900 mg) every two weeks (Q2W). In some instances, the effective amount of atezolizumab is a dose (e.g., a fixed dose) of between 20 mg to 1600 mg (e.g., between 40 mg to 1500 mg, e.g., between 200 mg to 1400 mg, e.g., between 300 mg to 1400 mg, e.g., between 400 mg to 1400 mg, e.g., between 500 mg to 1300 mg, e.g., between 600 mg to 1200 mg, e.g., between 700 mg to 1100 mg, e.g., between 800 mg to 1000 mg, e.g., between 800 mg to 900 mg, e.g., 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, or 900 mg) every two weeks (Q2W). In some instances, the effective amount of atezolizumab is a dose of about 840 mg every two weeks (e.g., 840 mg ± 10 mg, e.g., 840 ± 6 mg, e.g., 840 ± 5 mg, e.g., 840 ± 3 mg, e.g., 840 ± 1 mg, e.g., 840 ± 0.

WO 2024/186790 PCT/US2024/018472 mg, e.g., 840 mg every two weeks). In some instances, the effective amount of atezolizumab is a dose of about 840 mg every two weeks.In some instances, the effective amount of atezolizumab is a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 5 mg/kg to about 15 mg/kg, e.g., between about 7.5 mg/kg to about 12.5 mg/kg, e.g., about 10 ± 2 mg/kg, about 10 ± 1 mg/kg, about 10 ± 0.5 mg/kg, about 10 ± 0.2 mg/kg, or about 10 ± 0.mg/kg, e.g., about 10 mg/kg) every two weeks. In some instances, the effective amount of atezolizumab is a dose of between about 0.01 mg/kg to about 10 mg/kg of the subject’s body weight (e.g., between about 0.1 mg/kg to about 10 mg/kg, e.g., between about 0.5 mg/kg to about 10 mg/kg, e.g., between about 1 mg/kg to about 10 mg/kg, e.g., between about 2.5 mg/kg to about 10 mg/kg, e.g., between about mg/kg to about 10 mg/kg, e.g., between about 7.5 mg/kg to about 10 mg/kg, e.g., between about mg/kg to about 10 mg/kg, e.g., between about 9 mg/kg to about 10 mg/kg, e.g., between about 9.5 mg/kg to about 10 mg/kg, e.g., about 10 ± 1 mg/kg, e.g., about 10 ± 0.5 mg/kg, e.g., about 10 ± 0.2 mg/kg, e.g., about 10 ± 0.1 mg/kg, e.g., about 10 mg/kg) every two weeks. In some instances, the effective amount of atezolizumab is a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 5 mg/kg to 15 mg/kg, e.g., between 7.5 mg/kg to 12.5 mg/kg, e.g., 10 ± 2 mg/kg, 10 ± 1 mg/kg, 10 ± 0.5 mg/kg, 10 ± 0.2 mg/kg, or 10 ± 0.1 mg/kg, e.g., 10 mg/kg) every two weeks. In some instances, the effective amount of atezolizumab is a dose of between 0.01 mg/kg to 10 mg/kg of the subject’s body weight (e.g., between 0.1 mg/kg to 10 mg/kg, e.g., between 0.5 mg/kg to 10 mg/kg, e.g., between 1 mg/kg to 10 mg/kg, e.g., between 2.5 mg/kg to 10 mg/kg, e.g., between 5 mg/kg to 10 mg/kg, e.g., between 7.5 mg/kg to mg/kg, e.g., between 8 mg/kg to 10 mg/kg, e.g., between 9 mg/kg to 10 mg/kg, e.g., between 9.5 mg/kg to 10 mg/kg, e.g., 10 ± 1 mg/kg, e.g., 10 ± 0.5 mg/kg, e.g., 10 ± 0.2 mg/kg, e.g., 10 ± 0.1 mg/kg, e.g., mg/kg) every two weeks. In some instances, the effective amount of atezolizumab is a dose of about mg/kg every two weeks. In some instances, the effective amount of atezolizumab is a dose of 10 mg/kg every two weeks.In some instances, the effective amount of atezolizumab to treat a subject having a cancer is a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.mg/kg, or about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) every three weeks. In some instances, the effective amount of atezolizumab is a dose of between about 0.01 mg/kg to about 15 mg/kg of the subject’s body weight (e.g., between about 0.1 mg/kg to about 15 mg/kg, e.g., between about 0.5 mg/kg to about 15 mg/kg, e.g., between about 1 mg/kg to about 15 mg/kg, e.g., between about 2.5 mg/kg to about 15 mg/kg, e.g., between about 5 mg/kg to about 15 mg/kg, e.g., between about 7.5 mg/kg to about WO 2024/186790 PCT/US2024/018472 mg/kg, e.g., between about 10 mg/kg to about 15 mg/kg, e.g., between about 12.5 mg/kg to about mg/kg, e.g., between about 14 mg/kg to about 15 mg/kg, e.g., about 15 ± 1 mg/kg, e.g., about 15 ± 0.mg/kg, e.g., about 15 ± 0.2 mg/kg, e.g., about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) every three weeks. In some instances, the effective amount of atezolizumab to treat a subject having a cancer is a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.mg/kg to 15 mg/kg, e.g., 15 ± 2 mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) every three weeks. In some instances, the effective amount of atezolizumab is a dose of between 0.01 mg/kg to 15 mg/kg of the subject’s body weight (e.g., between 0.1 mg/kg to 15 mg/kg, e.g., between 0.5 mg/kg to 15 mg/kg, e.g., between 1 mg/kg to 15 mg/kg, e.g., between 2.5 mg/kg to mg/kg, e.g., between 5 mg/kg to 15 mg/kg, e.g., between 7.5 mg/kg to 15 mg/kg, e.g., between 10 mg/kg to 15 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., between 14 mg/kg to 15 mg/kg, e.g., 15 ± mg/kg, e.g., 15 ± 0.5 mg/kg, e.g., 15 ± 0.2 mg/kg, e.g., 15 ± 0.1 mg/kg, e.g., 15 mg/kg) every three weeks. In some instances, the effective amount of atezolizumab is a dose of about 15 mg/kg administered every three weeks. In some instances, the effective amount of atezolizumab is a dose of about 15 mg/kg administered every three weeks with a maximum dose of 1200 mg every three weeks. In some instances, the dose of atezolizumab administered in a combination therapy (e.g., a combination treatment with tiragolumab) may be reduced as compared to a standard dose of atezolizumab administered as a monotherapy. In some embodiments, atezolizumab is administered at a maximum dose of 1200 mg every three weeks.In some instances, the effective amount of atezolizumab is a dose of between about 80 mg to about 2000 mg (e.g., between about 100 mg to about 1600 mg, e.g., between about 200 mg to about 1600 mg, e.g., between about 300 mg to about 1600 mg, e.g., between about 400 mg to about 1600 mg, e.g., between about 500 mg to about 1600 mg, e.g., between about 600 mg to about 1600 mg, e.g., between about 700 mg to about 1600 mg, e.g., between about 800 mg to about 1600 mg, e.g., between about 900 mg to about 1500 mg, e.g., between about 1000 mg to about 1400 mg, e.g., between about 1050 mg to about 1350 mg, e.g., between about 1100 mg to about 1300 mg, e.g., between about 11mg to about 1250 mg, e.g., between about 1175 mg to about 1225 mg, e.g., between about 1190 mg to about 1210 mg, e.g., 1200 mg ± 5 mg, e.g., 1200 ± 2.5 mg, e.g., 1200 ± 1.0 mg, e.g., 1200 ± 0.5 mg, e.g., 1200 mg) every three weeks (Q3W). In some instances, the effective amount of atezolizumab is a dose of about 1200 mg every three weeks (e.g., 1200 mg ± 10 mg, e.g., 1200 ± 6 mg, e.g., 1200 ± 5 mg, e.g., 1200 ± 3 mg, e.g., 1200 ± 1 mg, e.g., 1200 ± 0.5 mg, e.g., 1200 mg every three weeks). In some instances, the effective amount of atezolizumab is a dose of 1200 mg every three weeks.In some instances, the effective amount of atezolizumab is a dose of between about 10 mg and about 800 mg (e.g., between about 10 mg and about 800 mg, e.g., between about 20 mg and about 7mg, e.g., between about 50 mg and about 600 mg, e.g., between about 75 mg and about 500 mg, e.g., between about 100 mg and about 400 mg, e.g., between about 100 mg and about 300 mg, e.g., between about 125 mg and about 275 mg, e.g., between about 150 mg and about 250 mg, e.g., between about 175 mg and about 225 mg, e.g., between about 190 mg and about 210 mg, e.g., about 200 mg ± 10 mg, WO 2024/186790 PCT/US2024/018472 e.g., 200 mg ± 7.5 mg, e.g., 200 mg ± 5 mg, e.g., 200 ± 2.5 mg, e.g., 200 ± 1.0 mg, e.g., 200 ± 0.5 mg, e.g., 200 mg) every three weeks (Q3W). In some instances, the effective amount of atezolizumab is a dose of about 200 mg every three weeks (e.g., 200 mg ± 10 mg, e.g., 200 ± 6 mg, e.g., 200 ± 5 mg, e.g., 200 ± 3 mg, e.g., 200 ± 1 mg, e.g., 200 ± 0.5 mg, e.g., 200 mg every three weeks). In some instances, the effective amount of atezolizumab is a dose of about 200 mg every three weeks (e.g., 200 mg ± mg, e.g., 200 ± 6 mg, e.g., 200 ± 5 mg, e.g., 200 ± 3 mg, e.g., 200 ± 1 mg, e.g., 200 ± 0.5 mg, e.g., 2mg every three weeks). In some instances, the effective amount of atezolizumab is a dose of between mg and 800 mg (e.g., between 10 mg and 800 mg, e.g., between 20 mg and 700 mg, e.g., between mg and 600 mg, e.g., between 75 mg and 500 mg, e.g., between 100 mg and 400 mg, e.g., between 1mg and 300 mg, e.g., between 125 mg and 275 mg, e.g., between 150 mg and 250 mg, e.g., between 175 mg and 225 mg, e.g., between 190 mg and 210 mg, e.g., 200 mg ± 10 mg, e.g., 200 mg ± 7.5 mg, e.g., 200 mg ± 5 mg, e.g., 200 ± 2.5 mg, e.g., 200 ± 1.0 mg, e.g., 200 ± 0.5 mg, e.g., 200 mg) every three weeks (Q3W). In some instances, the effective amount of atezolizumab is a dose of 200 mg every three weeks (e.g., 200 mg ± 10 mg, e.g., 200 ± 6 mg, e.g., 200 ± 5 mg, e.g., 200 ± 3 mg, e.g., 200 ± 1 mg, e.g., 200 ± 0.5 mg, e.g., 200 mg every three weeks). In some instances, the effective amount of atezolizumab is a dose of 200 mg every three weeks (e.g., 200 mg ± 10 mg, e.g., 200 ± 6 mg, e.g., 200 ± 5 mg, e.g., 200 ± 3 mg, e.g., 200 ± 1 mg, e.g., 200 ± 0.5 mg, e.g., 200 mg every three weeks). In some instances, the effective amount of atezolizumab is a dose of about 200 mg every three weeks. In some instances, the effective amount of atezolizumab is a dose of 200 mg every three weeks.In some instances, the effective amount of atezolizumab is a dose of between about 80 mg to about 3000 mg (e.g., between about 80-200 mg, between about 200-400 mg, between about 400-6mg, between about 600-800 mg, between about 800-1000 mg, between about 1000-1200 mg, between about 1200-1400 mg, between about 1400-1600 mg, between about 1600-1800 mg, between about 1800-2000 mg, between about 2200-2400 mg, between about 2400-2600 mg, between about 2600-28mg, or between about 2800-3000 mg, e.g., between about 100 mg and about 3000 mg, e.g., between about 200 mg and about 2900 mg, e.g., between about 500 mg to about 2800 mg, e.g., between about 600 mg to about 2700 mg, e.g., between about 650 mg to about 2600 mg, e.g., between about 700 mg to about 2500 mg, e.g., between about 1000 mg to about 2400 mg, e.g., between about 1100 mg to about 2300 mg, e.g., between about 1200 mg to about 2200 mg, e.g., between about 1300 mg to about 21mg, e.g., between about 1400 mg to about 2000 mg, e.g., between about 1500 mg to about 1900 mg, e.g., between about 1600 mg to about 1800 mg, e.g., between about 1620 mg to about 1700 mg, e.g., between about 1640 mg to about 1690 mg, e.g., between about 1660 mg to about 1680 mg, about 16mg, e.g., about 80 mg, about 200 mg, about 400 mg, about 600 mg, about 800 mg, about 1000 mg, about 1200 mg, about 1400 mg, about 1600 mg, about 1800 mg, about 2000 mg, about 2200 mg, about 24mg, about 2600 mg, about 2800 mg, or about 3000 mg, e.g., about 1600 mg, about 1610 mg, about 16mg, about 1630 mg, about 1640 mg, about 1650 mg, about 1660 mg, about 1670 mg, about 1680 mg, about 1690 mg, or about 1700 mg) every four weeks (Q4W). In some instances, the effective amount of atezolizumab is a dose of between 500 mg to 3000 mg (e.g., between 500 mg to 2800 mg, e.g., between 600 mg to 2700 mg, e.g., between 650 mg to 2600 mg, e.g., between 700 mg to 2500 mg, e.g., between 1000 mg to 2400 mg, e.g., between 1100 mg to 2300 mg, e.g., between 1200 mg to 2200 mg, e.g., WO 2024/186790 PCT/US2024/018472 between 1300 mg to 2100 mg, e.g., between 1400 mg to 2000 mg, e.g., between 1500 mg to 1900 mg, e.g., between 1600 mg to 1800 mg, e.g., between 1620 mg to 1700 mg, e.g., between 1640 mg to 16mg, e.g., between 1660 mg to 1680 mg, 1680 mg, e.g., 1600 mg, 1610 mg, 1620 mg, 1630 mg, 1640 mg, 1650 mg, 1660 mg, 1670 mg, 1680 mg, 1690 mg, or 1700 mg) every four weeks (Q4W). In some instances, the effective amount of atezolizumab is a dose of 1680 mg every four weeks (e.g., 1680 mg ± mg, e.g., 1680 ± 6 mg, e.g., 1680 ± 5 mg, e.g., 1680 ± 3 mg, e.g., 1680 ± 1 mg, e.g., 1680 ± 0.5 mg, e.g., 1680 mg every four weeks). In some instances, the effective amount of atezolizumab is a dose of about 1680 mg every four weeks. In some instances, the effective amount of atezolizumab is a dose of 1680 mg every four weeks.In some instances, the effective amount of atezolizumab is a dose of between about 50 mg to about 2000 mg (e.g., between about 50-100 mg, between about 100-250 mg, between about 250-5mg, between about 500-750 mg, between about 750-1000 mg, between about 1000-1250 mg, between about 1250-1500 mg, between about 1500-1750 mg, or between about 1750-2000 mg, e.g., between about 100 mg to about 1000 mg, between about 120 mg to about 900 mg, between about 150 mg to about 800 mg, between about 200 mg to about 700 mg, between about 250 mg to about 600 mg, between about 300 mg to about 500 mg, or between about 350 mg to about 450 mg, e.g., between about mg to about 100 mg, between about 100 mg to about 200 mg, between about 200 mg to about 3mg, between about 300 mg to about 400 mg, between about 400 mg to about 500 mg, between about 500 mg to about 600 mg, between about 600 mg to about 700 mg, between about 700 mg to about 8mg, or between about 800 mg to about 1000 mg, e.g., about 50 mg, about 100 mg, about 250 mg, about 500 mg, about 750 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 1750 mg, or about 20mg, e.g., about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg, e.g., 400 mg) every six weeks (Q6W). In some instances, the effective amount of atezolizumab is a dose of between 50 mg to 2000 mg (e.g., between 100 mg to 1000 mg, between 1mg to 900 mg, between 150 mg to 800 mg, between 200 mg to 700 mg, between 250 mg to 600 mg, between 300 mg to 500 mg, or between 350 mg to 450 mg, e.g., between 50 mg to 100 mg, between 1mg to 200 mg, between 200 mg to 300 mg, between 300 mg to 400 mg, between 400 mg to 500 mg, between 500 mg to 600 mg, between 600 mg to 700 mg, between 700 mg to 800 mg, or between 800 mg to 1000 mg, e.g., 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, 350 mg, 360 mg, 370 mg, 380 mg, 390 mg, 400 mg, 410 mg, 420 mg, 430 mg, 440 mg, 450 mg, 460 mg, 470 mg, 480 mg, 490 mg, or 500 mg, e.g., 400 mg) every six weeks (Q6W). In some instances, the effective amount of the anti-PD-1 antagonist antibody (e.g., pembrolizumab) is a dose of about 400 mg every six weeks (e.g., 400 mg ± 10 mg, e.g., 400 ± 6 mg, e.g., 400 ± 5 mg, e.g., 400 ± 3 mg, e.g., 400 ± 1 mg, e.g., 400 ± 0.5 mg, e.g., 400 mg every six weeks). In some instances, the dose of atezolizumab is a fixed dose. In some instances, the effective amount of atezolizumab is a dose of (e.g., a fixed dose) about 400 mg every six weeks. In some instances, the effective amount of atezolizumab is a dose (e.g., a fixed dose) of 400 mg every six weeks.In some instances, atezolizumab is administered intravenously. Alternatively, in some embodiments, atezolizumab is administered subcutaneously. In some instances, atezolizumab is WO 2024/186790 PCT/US2024/018472 administered to the patient intravenously at a dose of about 840 mg every 2 weeks, about 1200 mg every weeks, or about 1680 mg every 4 weeks. In some instances, atezolizumab is administered to the patient intravenously at a dose of 840 mg every 2 weeks, 1200 mg every 3 weeks, or 1680 mg every weeks.In some instances, a subject is administered a total of 1 to 20 doses of atezolizumab, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses. In some instances, a subject is administered a total of 1 to 50 doses of atezolizumab, e.g., 1 to 50 doses, 1 to 45 doses, 1 to 40 doses, to 35 doses, 1 to 30 doses, 1 to 25 doses, 1 to 20 doses, 1 to 15 doses, 1 to 10 doses, 1 to 5 doses, 2 to doses, 2 to 45 doses, 2 to 40 doses, 2 to 35 doses, 2 to 30 doses, 2 to 25 doses, 2 to 20 doses, 2 to doses, 2 to 10 doses, 2 to 5 doses, 3 to 50 doses, 3 to 45 doses, 3 to 40 doses, 3 to 35 doses, 3 to doses, 3 to 25 doses, 3 to 20 doses, 3 to 15 doses, 3 to 10 doses, 3 to 5 doses, 4 to 50 doses, 4 to doses, 4 to 40 doses, 4 to 35 doses, 4 to 30 doses, 4 to 25 doses, 4 to 20 doses, 4 to 15 doses, 4 to doses, 4 to 5 doses, 5 to 50 doses, 5 to 45 doses, 5 to 40 doses, 5 to 35 doses, 5 to 30 doses, 5 to doses, 5 to 20 doses, 5 to 15 doses, 5 to 10 doses, 10 to 50 doses, 10 to 45 doses, 10 to 40 doses, 10 to doses, 10 to 30 doses, 10 to 25 doses, 10 to 20 doses, 10 to 15 doses, 15 to 50 doses, 15 to doses, 15 to 40 doses, 15 to 35 doses, 15 to 30 doses, 15 to 25 doses, 15 to 20 doses, 20 to 50 doses, to 45 doses, 20 to 40 doses, 20 to 35 doses, 20 to 30 doses, 20 to 25 doses, 25 to 50 doses, 25 to doses, 25 to 40 doses, 25 to 35 doses, 25 to 30 doses, 30 to 50 doses, 30 to 45 doses, 30 to 40 doses, to 35 doses, 35 to 50 doses, 35 to 45 doses, 35 to 40 doses, 40 to 50 doses, 40 to 45 doses, or 45 to doses. In particular instances, the doses may be administered intravenously.

Hi. Dosing cycles for tiragolumab and atezolizumab In any of the methods and uses of the invention, tiragolumab and/or atezolizumab may be administered in one or more dosing cycles (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, or 50 or more dosing cycles). In some instances, the dosing cycles of tiragolumab and/or atezolizumab continue until there is a loss of clinical benefit (e.g., confirmed disease progression, drug resistance, death, or unacceptable toxicity). In some instances, the length of each dosing cycle is about to 42 days (e.g., 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, days, 41 days, 42 days). In some instances, the length of each dosing cycle is about 14 days. In some instances, the length of each dosing cycle is about 21 days. In some instances, the length of each dosing cycle is about 28 days. In some instances, the length of each dosing cycle is about 42 days. In some instances, the length of each dosing cycle is about 7 days. In some instances, tiragolumab is administered on about Day 1 (e.g., Day 1 ± 3 days) of each dosing cycle. In some instances, tiragolumab is administered on about Day 15 (e.g., Day 15 ± 3 days) of each dosing cycle. In some instances, tiragolumab is administered on about Day 22 (e.g., Day 22 ± 3 days) of each dosing cycle. In some instances, tiragolumab is administered on about Day 29 (e.g., Day 29 ± 3 days) of each dosing cycle. For example, tiragolumab may be administered intravenously at a dose (e.g., a fixed dose) of about 600 mg WO 2024/186790 PCT/US2024/018472 on Day 1 of each 21 -day cycle (i.eat a dose of about 600 mg every three weeks). In another example, tiragolumab is administered intravenously at a dose (e.g., a fixed dose) of about 600 mg on Day 1 and Day 15 of each 28-day cycle (i.e., at a dose of about 420 mg every two weeks). For example, tiragolumab may be administered intravenously at a dose (e.g., a fixed dose) of about 600 mg on Day 1, Day 15, and Day 29 of each 42-day cycle (i.e., at a dose of about 420 mg every two weeks). For example, tiragolumab may be administered intravenously at a dose (e.g., a fixed dose) of about 600 mg on Day 1 and Day 22 of each 42-day cycle (i.e., at a dose of about 600 mg every three weeks). In some instances, atezolizumab is administered on about Day 1 (e.g., Day 1 ± 3 days) of each dosing cycle. In some instances, atezolizumab is administered on about Day 15 (e.g., Day 15 ± 3 days) of each dosing cycle. For example, atezolizumab may be administered intravenously at a dose of about 1200 mg on Day of each 21-day cycle (i.e., at a dose of about 1200 mg every three weeks). For example, atezolizumab may be administered intravenously at a dose of about 1200 mg on Day 1 and Day 15 of each 28-day cycle (i.e., at a dose of about 840 mg every two weeks). In some examples, tiragolumab is administered intravenously at a dose (e.g., a fixed dose) of 600 mg on Day 1 of each 21-day cycle (i.e., at a dose of 600 mg every three weeks). In some instances, atezolizumab is administered on Day 1 (e.g., Day 1 ± days) of each dosing cycle. For example, atezolizumab may be administered intravenously at a dose of 1200 mg on Day 1 of each 21-day cycle (i.e., at a dose of 1200 mg every three weeks).In some instances, tiragolumab and atezolizumab are administered on about Day 1 (e.g., Day 1 ± days) of each dosing cycle.In some instances, tiragolumab is administered intravenously at a dose of about 600 mg on Day of each 21 -day cycle (i.e., at a dose of about 600 mg every three weeks) and atezolizumab is administered intravenously at a dose of about 1200 mg on Day 1 of each 21-day cycle (i.e., at a dose of about 1200 mg every three weeks). In some instances, tiragolumab is administered intravenously at a dose of 600 mg on Day 1 of each 21 -day cycle (i.e., at a dose of 600 mg every three weeks) and atezolizumab is administered intravenously at a dose of 1200 mg on Day 1 of each 21-day cycle (i.e., at a dose of 1200 mg every three weeks).In other instances, tiragolumab is administered intravenously at a dose of about 420 mg on Day of each 14-day cycle (i.e., at a dose of about 420 mg every two weeks) and atezolizumab is administered intravenously at a dose of about 840 mg on Day 1 of each 14-day cycle (i.e., at a dose of about 840 mg every two weeks). In some instances, tiragolumab is administered intravenously at a dose of 420 mg on Day 1 of each 14-day cycle (i.e., at a dose of about 420 mg every two weeks) and atezolizumab is administered intravenously at a dose of 840 mg on Day 1 of each 14-day cycle (i.e., at a dose of 840 mg every two weeks).In other instances, tiragolumab is administered intravenously at a dose of about 840 mg on Day of each 28-day cycle (i.e., at a dose of about 840 mg every four weeks) and atezolizumab is administered intravenously at a dose of about 1680 mg on Day 1 of each 28-day cycle (i.e., at a dose of about 1680 mg every four weeks). In some instances, tiragolumab is administered intravenously at a dose of 840 mg on Day 1 of each 28-day cycle (i.e., at a dose of 840 mg every four weeks) and atezolizumab is administered intravenously at a dose of 1680 mg on Day 1 of each 28-day cycle (i.e., at a dose of 1680 mg every four weeks).

WO 2024/186790 PCT/US2024/018472 In some instances, tiragolumab administered in a combination therapy (e.g., a combination treatment with atezolizumab) may be reduced as compared to a standard dose of tiragolumab administered as a monotherapy. In some instances, the dose of tiragolumab administered in a combination therapy (e.g., a combination treatment with atezolizumab) may be reduced as compared to a standard dose of tiragolumab administered as a monotherapy.In some instances, the dose of atezolizumab administered in a combination therapy (e.g., a combination treatment with tiragolumab) may be reduced as compared to a standard dose of atezolizumab administered as a monotherapy. In some instances, the dose of atezolizumab administered in a combination therapy (e.g., a combination treatment with tiragolumab) may be reduced as compared to a standard dose of atezolizumab administered as a monotherapy. iv. Intravenous infusion and subcutaneous administration of tiragolumab and atezolizumab In some instances, tiragolumab is administered intravenously. Alternatively, in some embodiments, tiragolumab is administered subcutaneously. In some instances, atezolizumab is administered intravenously. Alternatively, in some embodiments, atezolizumab is administered subcutaneously.In some instances, tiragolumab is administered to the subject or population of subjects by intravenous infusion over about 60 ± 15 minutes (e.g., about 45 minutes, about 46 minutes, about minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about minutes, about 59 minutes, about 60 minutes, about 61 minutes, about 62 minutes, about 63 minutes, about 64 minutes, about 65 minutes, about 66 minutes, about 67 minutes, about 68 minutes, about minutes, about 70 minutes, about 71 minutes, about 72 minutes, about 73 minutes, about 74 minutes, or about 75 minutes). In some instances, tiragolumab is administered to the subject or population of subjects by intravenous infusion over about 60 ± 10 minutes (e.g., about 50 minutes, about 51 minutes, about 52 minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 61 minutes, about 62 minutes,about 63 minutes, about 64 minutes, about 65 minutes, about 66 minutes, about 67 minutes, about 68minutes, about 69 minutes, or about 70 minutes). In some instances, atezolizumab is administered to thesubject by intravenous infusion over about 60 ± 15 minutes (e.g., about 45 minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49 minutes, about 50 minutes, about 51 minutes, about minutes, about 53 minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57 minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 61 minutes, about 62 minutes, about minutes, about 64 minutes, about 65 minutes, about 66 minutes, about 67 minutes, about 68 minutes, about 69 minutes, about 70 minutes, about 71 minutes, about 72 minutes, about 73 minutes, about minutes, or about 75 minutes).In some instances, tiragolumab is administered to the subject by intravenous infusion over about ± 10 minutes (e.g., about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, WO 2024/186790 PCT/US2024/018472 about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, or about 40 minutes). In some instances, atezolizumab is administered to the subject by intravenous infusion over about 30 ± minutes (e.g., about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, or about 40 minutes). v. Administration order and observation periods In some instances in which both tiragolumab and atezolizumab are administered to a subject or population of subjects, the tiragolumab is administered to the subject before the atezolizumab.In some instances, for example, following administration of tiragolumab and before administration of atezolizumab the method includes an intervening first observation period. In some instances, for example, following administration of tiragolumab, atezolizumab is administered to the subject. In some instances, tiragolumab is first administered to the subject and atezolizumab is administered to the subject following administration of tiragolumab.In some instances, the method further includes a second observation period following administration of atezolizumab.In some instances, the method includes both a first observation period following administration of tiragolumab and second observation period following administration of atezolizumab. In some instances, the first and second observation periods are each between about 30 minutes to about 60 minutes in length. In instances in which the first and second observation periods are each about 60 minutes in length, the method may include recording the subject’s vital signs (e.g., pulse rate, respiratory rate, blood pressure, and temperature) at about 30 ± 10 minutes after administration of tiragolumab or atezolizumab during the first or second observation periods. In instances in which the first and second observation periods are each about 30 minutes in length, the method may include recording the subject’s vital signs (e.g., pulse rate, respiratory rate, blood pressure, and temperature) at about 15 ± 10 minutes after administration of tiragolumab or atezolizumab during the first or second observation periods.In some instances, atezolizumab is administered to the subject or population of subjects before tiragolumab. In some instances, for example, following administration of atezolizumab)) and before administration of tiragolumab, the method includes an intervening first observation period.In some instances, the method further includes a second observation period following administration of tiragolumab.In some instances, the method includes both a first observation period following administration of atezolizumab and a second observation period following administration of tiragolumab. In some instances, the first and second observation periods are each between about 30 minutes to about minutes in length. In instances in which the first and second observation periods are each about minutes in length, the method may include recording the subject’s vital signs (e.g., pulse rate, respiratory rate, blood pressure, and temperature) at about 30 ± 10 minutes after administration of atezolizumab or tiragolumab during the first or second observation periods. In instances in which the first and second WO 2024/186790 PCT/US2024/018472 observation periods are each about 30 minutes in length, the method may include recording the subject’s vital signs (e.g., pulse rate, respiratory rate, blood pressure, and temperature) at about 15 ± 10 minutes after administration of atezolizumab or tiragolumab during the first or second observation periods. vi. Combination dosing of tiragolumab and atezolizumab In some instances, a dose of an effective amount of tiragolumab is administered with a dose of atezolizumab in a combination therapy (e.g., a combination treatment of tiragolumab with atezolizumab), e.g., for treatment of a subject having a NSCLC. In some instances, tiragolumab is administered every two weeks as described in Section IIl(D)(i) herein and atezolizumab is administered every two weeks as described in Section Ill(D)(ii) herein. In some instances, tiragolumab is administered every two weeks as described in Section 11l(D)(i) herein and atezolizumab is administered every three weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every two weeks as described in Section lll(D)(i) herein and atezolizumab is administered every four weeks as described in Section Ill(D)(ii) herein. In some instances, tiragolumab is administered every two weeks as described in Section 11l(D)(i) herein and atezolizumab is administered every six weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every three weeks as described in Section 11 l(D)(i) herein and atezolizumab is administered every two weeks as described in Section Ill(D)(ii) herein. In some instances, tiragolumab is administered every three weeks as described in Section 11 l(D)(i) herein and atezolizumab is administered every three weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every three weeks as described in Section 11 l(D)(i) herein and atezolizumab is administered every four weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every three weeks as described in Section 11 l(D)(i) herein and atezolizumab is administered every six weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every four weeks as described in Section 11 l(D)(i) herein and atezolizumab is administered every two weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every four weeks as described in Section 11l(D)(i) herein and atezolizumab is administered every three weeks as described in Section lll(D)(ii). In some instances, tiragolumab is administered every four weeks as described in Section 11l(D)(i) herein and atezolizumab is administered every four weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every four weeks as described in Section 11l(D)(i) herein and atezolizumab is administered every six weeks as described in Section lll(D)(ii) herein. In some instances, tiragolumab is administered every two, three, or four weeks as described in Section 111( D)(i) herein and atezolizumab is administered every two, three, four, or six weeks as described in Section IIl(D)(ii) herein.In some instances, the dose of tiragolumab is a dose of about 600 mg every three weeks. In some instances, the dose of tiragolumab is a dose of 600 mg every three weeks. In some instances, tiragolumab is administered (e.g., every three weeks) in a tiered dosing regimen (e.g., dosing based on body weight (BW) or body surface area (BSA) of a subject) and atezolizumab is administered at a dose from about 0.01 mg/kg to about 50 mg/kg (e.g., about 15 mg/kg) up to 1200 mg, e.g., every three weeks. In some instances, tiragolumab is administered (e.g., every three weeks) in a tiered dosing regimen (e.g., dosing based on body weight (BW) or body surface area (BSA) of a subject) and atezolizumab is WO 2024/186790 PCT/US2024/018472 administered at a dose from 0.01 mg/kg to 50 mg/kg (e.g., 15 mg/kg) up to 1200 mg, e.g., every three weeks. Such dosing regimens can be utilized in treatments for subjects having relatively low body weight (e.g., 40 kg or less (e.g., from 5 kg to 40 kg, from 15 kg to 40 kg, or from 5 kg to 15 kg)) and have been developed through biosimulation studies based on extrapolations of pharmacokinetic parameters estimated from adult data. In some instances, the dose of tiragolumab is a tiered dose based on a subject’s body weight (e.g., body weight (BW) > 40 kg: 600 mg, BW > 15 kg and < 40 kg: 400 mg, and BW < 15 kg: 300 mg). In some instances, the dose of atezolizumab is a dose based on a subject’s body weight (e.g., 15 mg/kg). In some instances, the dose of atezolizumab is a dose based on a subject’s body surface area (e.g., body surface area (BSA) > 1.25 m2: 600 mg, BSA > 0.75 m2 and < 1.25 m2: 4mg, BSA > 0.5 m2 and < 0.75 m2: 350 mg, and BSA < 0.5 m2: 300 mg). In some instances, the dose (e.g., about 600 mg) of tiragolumab is administered in combination with a dose of atezolizumab based on a subject’s body weight (e.g., 15 mg/kg) every three weeks. In some instances, the tiered dose (e.g., body weight (BW) > 40 kg: 600 mg, BW > 15 kg and < 40 kg: 400 mg, and BW < 15 kg: 300 mg) of tiragolumab is administered in combination with a dose of atezolizumab based on a subject’s body weight (e.g., 15 mg/kg) every three weeks. In some instances, the tiered dose (e.g., body weight (BW) > 40 kg: 600 mg, BW > 15 kg and < 40 kg: 400 mg, and BW < 15 kg: 300 mg) of tiragolumab is administered in combination with a dose of atezolizumab based on a subject’s body surface area (e.g., BSA > 1.25 m2: 600 mg, BSA > 0.75 m2 and < 1.25 m2: 450 mg, BSA > 0.5 m2 and < 0.75 m2: 350 mg, and BSA < 0.5 m2: 300 mg) every three weeks. In some embodiments, atezolizumab is administered at a maximum dose of 1200 mg every three weeks. In some instances, the combination therapy is administered with one or more chemotherapeutic agents (e.g., a platinum-based chemotherapeutic agent (e.g., carboplatin or cisplatin) and/or a non-platinum-based chemotherapeutic agent (e.g., an antimetabolite (e.g., pemetrexed or gemcitabine)).In some instances, the effective amount of tiragolumab to treat a subject having a cancer is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 400 mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between about 30 mg to about 1200 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between about 250 mg to about 350 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between about 350 mg to about 450 mg every three weeks (e.g., about 4mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between about 550 mg to about 650 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of about 300 mg every three weeks; (b) greater than 15 kg and less than or equal to 40 kg, and WO 2024/186790 PCT/US2024/018472 tiragolumab is administered at a dose of about 400 mg every three weeks; or (c) greater than 40 kg, and tiragolumab is administered at a dose of about 600 mg every three weeks. In some instances, a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.mg/kg, or about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) of atezolizumab is administered in combination with a tiered dose based on a subject’s body weight of tiragolumab, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between about mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 400 mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between about 30 mg to about 1200 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, a subject with a body weight of less than or equal to 15 kg is administered a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks) of tiragolumab and a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.2 mg/kg, or about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body weight of greater than 15 kg and less than or equal to 40 kg is administered a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 400 mg every three weeks) of tiragolumab and a dose of between about 0.mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.2 mg/kg, or about 15 ± 0.mg/kg, e.g., about 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body weight of greater than 40 kg is administered a dose of between about 30 mg to about 1200 mg every three weeks (e.g., about 600 mg every three weeks) of tiragolumab and a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.2 mg/kg, or about 15 ± 0.mg/kg, e.g., about 15 mg/kg) of atezolizumab every three weeks.

WO 2024/186790 PCT/US2024/018472 In some instances, the effective amount of tiragolumab to treat a subject having a cancer is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 400 mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between mg to 1200 mg every three weeks (e.g., 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between 250 mg to 350 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between 350 mg to 450 mg every three weeks (e.g., 400 mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between 550 mg to 650 mg every three weeks (e.g., 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body weight, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of 300 mg every three weeks; (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of 400 mg every three weeks; or (c) greater than 40 kg, and tiragolumab is administered at a dose of 600 mg every three weeks. In some instances, a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.mg/kg to 15 mg/kg, e.g., 15 ± 2 mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab is administered in combination with a tiered dose based on a subject’s body weight of tiragolumab, wherein the subject has a body weight of (a) less than or equal to 15 kg, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 15 kg and less than or equal to 40 kg, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 400 mg every three weeks); or (c) greater than 40 kg, and tiragolumab is administered at a dose of between 30 mg to 12mg every three weeks (e.g., 600 mg every three weeks). In some instances, a subject with a body weight of less than or equal to 15 kg is administered a dose of between 10 mg to 1000 mg every three weeks (e.g., 300 mg every three weeks) of tiragolumab and a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ±2 mg/kg, 15 ± mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body weight of greater than 15 kg and less than or equal to kg is administered a dose of between 10 mg to 1000 mg every three weeks (e.g., 400 mg every three weeks) of tiragolumab and a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg WO 2024/186790 PCT/US2024/018472 to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ± 2 mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body weight of greater than 40 kg is administered a dose of between 30 mg to 1200 mg every three weeks (e.g., 600 mg every three weeks) of tiragolumab and a dose of between 0.01 mg/kg to mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ± mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab every three weeks.In some instances, the effective amount of tiragolumab to treat a subject having a cancer is a tiered dose based on a subject’s body surface area. In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 350 mg every three weeks); (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between about 30 mg to about 1200 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between about 250 mg to about 350 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between about 300 mg to about 400 mg every three weeks (e.g., about 350 mg every three weeks); or (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between about 400 mg to about 500 mg every three weeks (e.g., about 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between about 550 mg to about 650 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of about 3mg every three weeks; (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of about 400 mg every three weeks; (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of 450 mg every three weeks; or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of about 600 mg every three weeks.In some instances, a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± WO 2024/186790 PCT/US2024/018472 0.5 mg/kg, about 15 ± 0.2 mg/kg, or about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) of atezolizumab is administered in combination with a tiered dose based on a subject’s body surface area of tiragolumab, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 350 mg every three weeks); (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between about 30 mg to about 1200 mg every three weeks (e.g., about 600 mg every three weeks). In some instances, a subject with a body surface area of less than or equal to 0.5 m2 is administered a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 300 mg every three weeks) of tiragolumab and a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.2 mg/kg, or about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body surface area of greater than 0.5 m2 and less than or equal to 0.75 m2 is administered a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 350 mg every three weeks) of tiragolumab and a dose of between about 0.01 mg/kg to about mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.2 mg/kg, or about 15 ± 0.1 mg/kg, e.g., about mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body surface area of greater than 0.75 m2 and less than or equal to 1.25 m2 is administered a dose of between about 10 mg to about 1000 mg every three weeks (e.g., about 450 mg every three weeks) of tiragolumab and a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about 5 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.5 mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.mg/kg, or about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body surface area of greater than 1.25 m2 is administered a dose of between about 30 mg to about 1200 mg every three weeks (e.g., about 600 mg every three weeks) of tiragolumab and a dose of between about 0.01 mg/kg to about 50 mg/kg of the subject’s body weight (e.g., between about 0.01 mg/kg to about 45 mg/kg, e.g., between about 0.1 mg/kg to about 40 mg/kg, e.g., between about 1 mg/kg to about 35 mg/kg, e.g., between about 2.5 mg/kg to about 30 mg/kg, e.g., between about WO 2024/186790 PCT/US2024/018472 mg/kg to about 25 mg/kg, e.g., between about 10 mg/kg to about 20 mg/kg, e.g., between about 12.mg/kg to about 15 mg/kg, e.g., about 15 ± 2 mg/kg, about 15 ± 1 mg/kg, about 15 ± 0.5 mg/kg, about 15 ± 0.2 mg/kg, or about 15 ± 0.1 mg/kg, e.g., about 15 mg/kg) of atezolizumab every three weeks.In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 350 mg every three weeks); (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between 30 mg to 1200 mg every three weeks (e.g., 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between 250 mg to 350 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between 300 mg to 400 mg every three weeks (e.g., 350 mg every three weeks); or (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between 400 mg to 500 mg every three weeks (e.g., 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between 550 mg to 6mg every three weeks (e.g., 600 mg every three weeks). In some instances, the effective amount of tiragolumab is a tiered dose based on a subject’s body surface area, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of 300 mg every three weeks; (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of 400 mg every three weeks; (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of 450 mg every three weeks; or (d) greater than 1.m2, and tiragolumab is administered at a dose of 600 mg every three weeks.In some instances, a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ± 2 mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab is administered in combination with a tiered dose based on a subject’s body surface area of tiragolumab, wherein the subject has a body surface area of (a) less than or equal to 0.5 m2, and tiragolumab is administered at a dose of between mg to 1000 mg every three weeks (e.g., 300 mg every three weeks); (b) greater than 0.5 m2 and less than or equal to 0.75 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 350 mg every three weeks); (c) greater than 0.75 m2 and less than or equal to 1.25 m2, and tiragolumab is administered at a dose of between 10 mg to 1000 mg every three weeks (e.g., 450 mg every three weeks); or (d) greater than 1.25 m2, and tiragolumab is administered at a dose of between mg to 1200 mg every three weeks (e.g., 600 mg every three weeks). In some instances, a subject with a body surface area of less than or equal to 0.5 m2 is administered a dose of between 10 mg to 1000 mg WO 2024/186790 PCT/US2024/018472 every three weeks (e.g., 300 mg every three weeks) of tiragolumab and a dose of between 0.01 mg/kg to mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ± mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body surface area of greater than 0.5 m2 and less than or equal to 0.75 m2 is administered a dose of between 10 mg to 1000 mg every three weeks (e.g., 350 mg every three weeks) of tiragolumab and a dose of between 0.01 mg/kg to mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ± mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body surface area of greater than 0.75 m2 and less than or equal to 1.25 m2 is administered a dose of between 10 mg to 1000 mg every three weeks (e.g., 450 mg every three weeks) of tiragolumab and a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to 45 mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.5 mg/kg to 30 mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ±2 mg/kg, 15 ± mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or 15 ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab every three weeks. In some instances, a subject with a body surface area of greater than 1.25 m2 is administered a dose of between 30 mg to 1200 mg every three weeks (e.g., 600 mg every three weeks) of tiragolumab and a dose of between 0.01 mg/kg to 50 mg/kg of the subject’s body weight (e.g., between 0.01 mg/kg to mg/kg, e.g., between 0.1 mg/kg to 40 mg/kg, e.g., between 1 mg/kg to 35 mg/kg, e.g., between 2.mg/kg to 30 mg/kg, e.g., between 5 mg/kg to 25 mg/kg, e.g., between 10 mg/kg to 20 mg/kg, e.g., between 12.5 mg/kg to 15 mg/kg, e.g., 15 ± 2 mg/kg, 15 ± 1 mg/kg, 15 ± 0.5 mg/kg, 15 ± 0.2 mg/kg, or ± 0.1 mg/kg, e.g., 15 mg/kg) of atezolizumab every three weeks.

IV. PHARMACEUTICAL COMPOSITIONS, FORMULATIONS, AND KITS In another aspect of the invention, an article of manufacture or kit containing materials useful for the prognostic assessment and/or treatment of individuals is provided.In some instances, such articles of manufacture or kits can be used to identify an individual having a NSCLC who may benefit from treatment with (a) atezolizumab or (b) atezolizumab and tiragolumab. Such articles of manufacture or kits may include (a) reagents for determining the expression level of one or more genes and (b) instructions for using the reagents to identify an individual having a NSCLC who may benefit from a treatment comprising (a) atezolizumab or (b) atezolizumab and tiragolumab.Any of the articles of manufacture or kits described may include a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. Where the article of manufacture or kit utilizes nucleic acid hybridization to detect the target WO 2024/186790 PCT/US2024/018472 nucleic acid, the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as an enzymatic, fluorescent, or radioisotope label.In some aspects, the article of manufacture or kit includes the container described above and one or more other containers including materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. A label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use, such as those described above. For example, the article of manufacture or kit may further include a container including a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution, and dextrose solution.The articles of manufacture or kits described herein may have a number of aspects. In one aspect, the article of manufacture or kit includes a container, a label on said container, and a composition contained within said container, wherein the composition includes one or more polynucleotides that hybridize to a complement of a locus described herein under stringent conditions, and the label on said container indicates that the composition can be used to evaluate the presence of a gene listed herein (e.g., CCL5, CXCR3, CCR7, or CXCR6, or a member of any one of the Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, and Cytotox.4 gene signatures) in a sample, and wherein the kit includes instructions for using the polynucleotide(s) for evaluating the presence of the gene RNA or DNA in a particular sample type.For oligonucleotide-based articles of manufacture or kits, the article of manufacture or kit can include, for example: (1) an oligonucleotide, e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a protein or (2) a pair of primers useful for amplifying a nucleic acid molecule. The article of manufacture or kit can also include, e.g., a buffering agent, a preservative, or a protein stabilizing agent. The article of manufacture or kit can further include components necessary for detecting the detectable label (e.g., an enzyme or a substrate). The article of manufacture or kit can further include components necessary for analyzing the sequence of a sample (e.g., a restriction enzyme or a buffer). The article of manufacture or kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the article of manufacture or kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

V. EXAMPLES The following are examples of the methods of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1. TIGIT and PD-L1 co-blockade promotes clonal expansion of non-exhausted anti-tumor CD8+ T cells WO 2024/186790 PCT/US2024/018472 A. Background Co-blockade of the immune checkpoints PD-1 and TIGIT has demonstrated activity in mouse tumor models and human cancer patients (Chiang et al., J Immunother Cancer, 10: 6004711,2022). Although these coinhibitory receptors restrict costimulatory receptor signaling by CD28 and CD226 in CD8+ T cells (Banta et al., Immunity, 55: 512-526, 2022), how combination blockade of PD-1 and TIGIT enhances T cell function remains a key unknown. This Example shows that combination efficacy in mouse tumor models involved leukocytes trafficking between the draining lymph nodes (dLN) and tumor. Compared to monotherapy treatment with anti-PD-L1 or anti-TIGIT, combination blockade elicited tumor antigen-specific CD8+ T cell clonal dual expansion in both dLN and tumor. Qualitatively, these clones possessed properties of early effector or memory cells that emerge from a pool of stem-like memory or progenitor exhausted cells (Hudson et al., Immunity, 51: 1043-1058, 2019; Huang et al., Cell, 185: 1-18, 2022) and exhibited a marked decrease in the expression of the Tox transcription factor (Scott et al., Nature, 571: 270-274, 2019; Khan et al., Nature, 571: 211-218, 2019). Together, the effects of combination treatment suggest that PD-1 and TIGIT shape and maintain the repertoire of effector and memory CD8+ T cells that are critical for anti-tumor immunity. Interestingly, patients harboring a similar dual-expanded CD8+ T cell profile were associated with favorable clinical outcomes in a randomized Phase 2 trial evaluating tiragolumab (anti-TIGIT monoclonal antibody; mAb) plus atezolizumab (anti-PD- L1 mAb) in non-small cell lung carcinoma. It is therefore proposed that PD-L1 and TIGIT co-blockade promotes expansion of antigen-specific CD8+ T effector and memory cells while restricting their differentiation along the exhaustion pathway, thereby contributing to enhanced anti-tumor responses.Clonotypically expanded effector-like CD8+ T cells are often found in tumors, normal adjacent tissue, and peripheral blood of patients with various types of cancer (Wu et al., Nature, 579: 274-278, 2020). The presence of these peripherally expanded T cell clones likely correlates with an active anti- tumor immune response since patients bearing transcriptional signatures indicative of clonal expansion are associated with favorable progression-free survival when treated with the anti-PD-L1 monoclonal antibody (mAb) atezolizumab in various clinical trials (Wu et al., Nature, 579: 274-278, 2020). T cells in the blood do not exhibit the features of exhausted T cells (Tex) and are not believed to be derived from tumor-infiltrating lymphocytes (TILs), suggesting that expansion may not reflect a reversal of intratumoral Tex exhaustion by PD-1 blockade (Scott et al., Nature, 571: 270-274, 2019; Khan et al., Nature, 571:211- 218, 2019; Wherry etal., Nat Rev Immunol, 15: 486-499, 2015; Sade-Feldman etal., Cell, 175:998- 1013.61020, 2018).Indeed, mounting evidence indicates that inhibitory effects of PD-1 signaling in T cells are mediated via binding to PD-L1 expressed by dendritic cells (DCs) found in tumor draining lymph node (dLN), tertiary lymphoid structures (TLS) or intratumoral lymphoid aggregates (Fransen et al., JCI Insight, 3: 6124507, 2018; Yost et al., Nat Med, 25: 1251-1259, 2019; Dammeijer et al., Cancer Cell, 38: 685-700, 2020; Oh et al., Nature Cancer, 1: 681-691,2020). Temporal analysis of tumor-specific T cells reveals that T cells primed in the dLN acquire PD-1 expression and are subsequently detected as PD-expressing T cells after migrating to the tumor site (Dammeijer et al., Cancer Cell, 38: 685-700, 2020). PD-1 expression thus appears to more accurately reflect T cell activation status rather than indicating the presence of exhaustion. Indeed, PD-1 is expressed on "stem cell-like memory (Tscm)" or "resource" WO 2024/186790 PCT/US2024/018472 CD8+ T cells that are believed to be the primary targets of PD-1/PD-L1 blockade (Siddiqui et al., Immunity, 50: 195-211 e110, 2019). Blocking PD-1 signaling thus may result in the differentiation of these progenitors into T cells with cytolytic effector activity against tumor cells, perhaps via a recently described transient population of T precursor exhausted cells (Tpex) (Huang et al., Cell, 185: 1-18, 2022; Dammeijer et al., Cancer Cell, 38: 685-700, 2020; Kallies et al., Nat Rev Immunol, 20: 128-136, 2020; Connolly et al., Sci Immunol, 6: eabg7836, 2021). Recently, tumor-specific CD8+ T cells have been shown to adopt a stem-like phenotype in response to priming in tumor dLN, maintain their stern-like state as they migrate to the tumor, and then acquire an effector program following co-stimulation in the tumor microenvironment (Prokhnevska et al., Immunity, 56: 107-124, 2023).TIGIT (T cell immunoreceptor with ig and immunoreceptor tyrosine-based inhibitory domains) is another immune checkpoint receptor that has garnered widespread attention due to the efficacy observed in early clinical trials using a combination of blocking antibodies to TIGIT and PD-L1 (Chiang et al., J Immunother Cancer, 10: 6004711,2022). In CD8+ TILs, TIGIT and PD-1 expression are highly correlated (Johnston et al., Cancer Cell, 26: 923-937, 2014). Co-expression may also define distinct populations of Tscm cells, with those co-expressing TIGIT and PD-1 reflecting cells more likely to differentiate into dysfunctional exhausted CD8+ T cells as opposed to functional effector or memory cells. TIGIT and PD-together regulate the function of CD226 (Banta et al., Immunity, 55: 512-526, 2022), whose signaling activates the PI3K/AKT pathway leading to inactivation of FOXO1, a widely expressed transcription factor with important roles in regulating T cell differentiation, including the effector and memory pathways (Luo et al., Cancer Biol, 50: 13-20, 2018; Du et al., Proc Natl Acad Sci USA, 115: E11731 -E11740, 2018; Hedrick et al., Nat Rev Immunol, 12: 649-661,2012). Despite its potential value as a target for immunotherapy, key unknowns remain concerning the effects of TIGIT on T cell function and whether TIGIT blockade acts on T cells in lymphoid organs or in tumors.The present study investigated whether the addition of TIGIT blockade to PD-1/PD-L1 blockade affected T cell function primarily in the dLN, as has been reported for PD-1/PD-L1 blockade alone, and how combination blockade impacted T cell function and differentiation. Trafficking of T cells from dLN to the tumor was found to be necessary to elicit the full effect of dual blockade. However, once tumors were seeded with competent T effectors, these previously trafficked T cells were sufficient to control tumor growth in response to PD-1/TIGIT inhibition. In both cases, dual checkpoint blockade elicited a marked expansion of anti-tumor T cell clonotypes lacking canonical features of T cell exhaustion. These outcomes were dependent on the expression of CD226, a target of negative regulation by both TIGIT and PD-1 (Banta et al., Immunity, 55: 512-526, 2022). It is therefore proposed that dual blockade restricts entry of T cells into the exhaustion pathway, yielding expanded clonotypes that preferentially expand and differentiate into T effector and memory cells with a consequent potential for therapeutic benefit.

B. Combination treatment requires trafficking of lymphocytes from draining lymph nodes to tumor The observation that PD-1 and TIGIT regulate costimulatory signals in T cells suggests that both receptors act at the same steps and anatomical sites in T cell activation (Chiang et al., J Immunother Cancer, 10: 6004711,2022). Since this issue had not yet been explored experimentally, the present WO 2024/186790 PCT/US2024/018472 examples evaluated the role of dLN in TIGIT blockade by restricting the trafficking of T cells with FTY720, a sphingosine 1-phosphate (S1P) receptor inhibitor that prevents egress of T cells from lymphoid organs (Pinschewer et al., J Immunol, 164: 5761-5770, 2000). Tumor growth was monitored in mice with established CT26 tumors following treatment with anti PD-L1 and TIGIT mAbs, singly and in combination, with and without FTY720. Consistent with previous observations (Johnston et al., Cancer Cell, 26: 923- 937, 2014), anti-PD-L1 monotherapy did not significantly impact tumor growth and anti-TIGIT had limited single-agent activity, whereas the combination demonstrated clear therapeutic efficacy (Figs. 1A and 7A). However, the presence of FTY720 limited both single-agent activity of anti-TIGIT and the effects of TIGIT/PD-L1 co-blockade (Fig. 1A). Similar results were observed in the EO771 tumor model (Fig. 7B).Treatment with anti-PD-L1 and/or anti-TIGIT did not affect total CD8+ T cell, CD4+ T cell, or regulatory T cell (Treg) numbers in dLN or tumor, either with or without FTY720 treatment (Fig. 7C). To determine if the effects of immune checkpoint blockade and FTY720 were restricted to tumor-specific CD8+ T cells, a gp70 tetramer was used to detect T cell receptors (TCRs) specific for gp70, a tumor- associated, immunodominant retroviral antigen expressed by CT26 cells (Huang et al., Proc Natl Acad Sci U SA, 93: 9730-9735, 1996). Within the dLN, anti-TIGIT, especially in combination with anti-PD-L1, significantly increased the fraction of gp70+CD8+ T cells; anti-PD-L1 alone had little effect (Fig. 1B, left panel and Fig. 7D). The addition of FTY720 to anti-TIGIT, or the combination treatment, further increased the frequency of gp70+CD8+ T cells in dLN, suggesting their accumulation in dLN by preventing T cell egress. The effectiveness of FTY720 treatment was further confirmed as gp70+CD8+ T cell numbers were significantly increased in blood with anti-TIGIT or combination treatment, but not in FTY720 treated animals (Fig. 1B, middle panel and Fig. 7D). In tumors, only the combination of anti-TIGIT and anti-PD- L1 significantly increased the fraction of gp70+CD8+ T cells, with a similar trend noted in tumors from FTY720-treated mice (Fig. 1B, right panel and Fig. 7D). Since trafficking via blood had been blocked, the expansion of intratumoral T cells was likely to have occurred locally. Treatment with combination checkpoint blockade also increased the fraction of intratumoral CD8+ T cells producing the proinflammatory cytokines IFN-g and TNF-a, with FTY720 eliminating this effect, suggesting that T cells derived from the periphery might possess superior effector function (Figs. 1C, 7E, and 8A).It was next investigated whether the anti-tumor efficacy of combination checkpoint blockade relied on the continuous recruitment of newly generated T cells or could be sustained once trafficked T cells that were previously reshaped by combination therapy in the dLN had infiltrated into the tumor. FTY720 was injected either at the time of combination checkpoint blockade or 7 days later to arrest continued T cell recruitment to the tumor. Although early administration of FYT720 blocked combination efficacy, delaying the blockade of T cell trafficking until 7 days after combination treatment resulted in only slight impairment in anti-tumor efficacy (Fig. 1D).Taken together, these results indicate that efficacy with combination checkpoint blockade depends on the induction of tumor-specific CD8+ T cells in dLN that then trafficked to and infiltrated tumors via the circulation. Once tumors become seeded with competent T effector cells, however, they appear sufficient to sustain therapeutic benefit in response to anti-TIGIT plus anti-PD-L1.

WO 2024/186790 PCT/US2024/018472 C. TIGIT and PD-L1 co-blockade promotes and expands different CD8+ T cell states in dLN and tumor compared to either single agent alone It was next examined how co-blockade affects the generation and phenotype of tumor-specific T cells. Single-cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing (scTCR-seq) were performed on T cells isolated from tumor, dLN, and blood from 31 mice. Further, antibody-derived tag sequencing (ADT-seq) was conducted with tetramers against gp70 or control antigens and cellular indexing of transcriptomes and epitopes (CITE-seq) against a panel of 18 proteins. Gene expression profiles of a large dataset of 305,908 T cells yielded 22 distinct clusters (Fig. 9A; top row, far left). Effector status as indicated by granzyme B expression was confined primarily to CD8+/gp70-tetramer+ T cells, which showed clonal expansion and high ADT counts, a measure of the number of tetramers bound and therefore the avidity for gp70 tumor antigen (Fig. 9A). CITE-seq provided a complementary characterization of T cell differentiation, effector, and memory states based on surface marker expression (Fig. 9B).Greater resolution of CD8+ T cell phenotypes was obtained by re-analyzing the 174,514 T cells belonging to the initial clusters with high CD8a expression. This secondary analysis yielded 16 distinct CD8+ clusters (Figs. 2A and 2B; see Table 3 for genes defining each cluster) exhibiting various phenotypes: (a) two naive-like clusters (Ribo.1-2) characterized by high expression of ribosomal proteins such as Rps20 and Rps24; (b) three Ccr7clusters (Ccr7.1-3) distinguished by Ccr7, a marker expressed by naive, Tscm and central memory (Tom) cells but low or absent in cytotoxic CD8+ T effector (Teff) and memory (Tern) cells (Mahnke et al., Eur J Immunol, 43: 2797-2809, 2013), as well as genes associated with Tscm-like cells such as Sell, Left, and Tcf7(Siddiqui et al., Immunity, 50: 195-211 e110, 2019); (c) an Egr cluster that shared similarities with the Ccr7 clusters but was characterized by expression of the early activation marker Cd69 as well as immediate-early response genes Ier2 and Ier5; (d) an ifng cluster marked by expression of Ifng, Tnf and Cc/4; checkpoint inhibitor receptors including Pdcdl and Tigit; as well as transcription factors belonging to the orphan nuclear receptor family, Nr4a1 and Nr4a3, which are involved in CD8+ T cell memory transcriptional programming (Odajiu et al., Front Indocrin, 11: 624122, 2021); (e) an Ifit cluster with hallmarks of interferon response genes; (f) a Mitotic cluster exclusively expressing genes associated with mitosis such as Ptma, Mcm3 and Mcm5; (g) a Cxcr3 cluster identified by the expression of Cxcr3, possibly associated with tuning CD8+ T cell differentiation toward Tscm or Teff cell pathways (Duckworth et al., Nat Immunol, 22: 434-448, 2021; Rahimi et al., Adv Immunol, 138: 71-98, 2018), as well as the highest level of Cd226, and also expression of Zfp683, which may correlate with establishing residency in tumors (Mackay et al., Science, 352: 459-463, 2016); (h) two Ccl5 clusters (Ccl5.1-2) with Ccl5.2 expressing higher levels of Ccl5, a chemokine that can exert chemotactic effects on T cells and is associated with CD8+ T cell infiltration into tumors (Dangaj et al., Cancer Cell, 35: 885-900, 2019); and (i) four Cytotox clusters (Cytotox.1-4) that exhibited graded expression of genes associated with exhaustion such as Tox and checkpoint inhibitory checkpoint receptors with Cytotox.4 expressing the highest levels of these markers and Cytotox.2 and Cytotox.3 appearing to be highly mitotic. The Cclclusters shared expression of a number of genes associated with the four Cytotox clusters. CITE-seq analysis using protein expression of various surface markers corroborated the categorization by gene expression (Figs. 10A and 10B).

WO 2024/186790 PCT/US2024/018472 Table 3. Markers of mouse CD8+ T cell clusters Iog2fc Ribo.1 Iog2fc Ribo.2 Iog2fc Ccr7.1 Iog2fc Ccr7.2 0.56 Rps20 0.66 Rps20 0.44 Ccr7 0.56 Stall0.54 Rps24 0.64 Rps24 0.43 Rflnb 0.45 Lef10.52 Rps3a1 0.62 Sell 0.43 Lef1 0.45 Ifi470.5 Rpl13 0.59 Rps27 0.36 Tsc22d3 0.44igtp0.5 Rps8 0.59 Rps29 0.36 Malat 0.44 Ccr70.48 Rps5 0.59 Rpl13 0.35 Smc6 0.42 Sell0.44 Rps29 0.58 Rps5 0.32 Gm42418 0.42 Rps240.43 Rplp1 0.58 Rplp1 0.31 Tdrp 0.41 Rps270.42 Rps4x 0.57 Rps3a1 0.3 Klf2 0.4 Gbp20.41 Rps27 0.57 Rps8 0.29 Acp5 0.39 Rps290.4 Rpsa 0.51 Rps4x 0.28 Cd8b1 0.38 Gm89950.36 RpIpO 0.45 Rpl32 0.27 Sell 0.38 Rps3a10.33 Rpl32 0.45 Ifngri 0.27 Dgka 0.37 Rps200.32 Rps15a 0.45 Rpsa 0.26 Btg1 0.37 Klf20.31 Rps11 0.45 Arl4c 0.25 Actn1 0.35 Rplp10.28 Rpl41 0.43 Sidtl 0.25 Rps27 0.35 Rpl130.27 Eef1a1 0.42 H7r 0.25 S1pr1 0.34 Dapll0.24 Tmsb10 0.42 RpIpO 0.25 Dapll 0.34 Smc60.24 Rps12 0.42 Rps15a 0.24 mt-Cytb 0.33 Rflnb0.23 Klf2 0.41 Nsg2 0.24 mt-Rnr1 0.32 Rps4xIog2fc Ccr7.3 Iog2fc Egr Iog2fc Ifng Iog2fc Ifit 0.67 Dapll 1.11 Nfkbia 2.18 Ccl4 1.62 lsg150.66 Ccr7 0.88 Egr1 2.05 Xcl1 1.21 Ifitl0.62 Smc4 0.82 Zfp36 1.85 Nr4a1 1.14 IfitS0.58 Rgcc 0.77 Junb 1.43 Egr1 1.09 lsg200.51 Mxd4 0.75 Cd69 1.42 Srgn 1.07 Ifi27l2a0.48 Cd8a 0.69 Ier2 1.34 Nr4a3 1.06 Samhdi0.48 Tcf7 0.67 Nr4a1 1.17 Tagap 1.03 Zbp10.39 Itgae 0.67 Zfp36l1 1.13 Myc 1.03 Slfn50.39 2410006H16Rik 0.62 Tagap 1.08 Zfp36l1 1.01 Irf70.39 Rgs10 0.57 Dusp2 1.07 Nfkbia 0.98 Rtp40.38 Znrfl 0.55 Ier5 1 Tnf 0.93 Usp180.37 Chd3 0.55 Myc 1 Rilpl2 0.9 Phf11b0.35 Cd52 0.54 Srgn 0.92 Ifng 0.89 Lgals3bp0.35 mt-Rnr2 0.47 Icaml 0.91 Dusp2 0.88 Bst20.35 Ddit4 0.45 Btg1 0.9 Gd69 0.86 Gbp20.35 Lef1 0.44 Relb 0.9 Egr2 0.83 Ifitm30.34 Izumol r 0.43 Cd5 0.88 Tnfrsf9 0.82 Ly6a WO 2024/186790 PCT/US2024/018472 0.33 Inpp4b 0.42 Kdm6b 0.85 Nr4a2 0.81 Statl0.33 Rflnb 0.41 Egr2 0.84 Hspa5 0.77 lfi2030.31 Malat 0.38 Batf 0.83 Rei 0.76 Ifihl Iog2fc Mitotic Iog2fc Cxcr3 Iog2fc Ccl5.1 Iog2fc Ccl5.2 1.46 Hsp90ab1 1.03 Cxcr6 0.83 Ccl5 2.58 Ccl51.19 Ptma 0.79 Ckb 0.49 Malatl 1.69 S100a61.13 Xcl1 0.77 GimapS 0.41 Itgbl 1.2 Lgals30.78 Odd 0.73 Id2 0.4 mt-Rnr2 1.18 Lgalsl0.68 Tnfrsf9 0.67 Ltb 0.4 Btg1 1.02 Cxcr60.65 Itm2a 0.64 Fgl2 0.4 Gzmk 0.99 Id20.61 Myc 0.63 Rampl 0.39 Ltb 0.96 Ahnak0.54 Ybx3 0.6 Lyst 0.38 H7r 0.95 Klrd0.54 Hspa5 0.57 Asb2 0.38 Zfp36l2 0.94 AW1120100.52 Gpx1 0.56 Il2rb 0.33 Itga4 0.94 S100a100.5 Rps12 0.55 Cxcr3 0.32 Txnip 0.92 Vim0.49 Tubalb 0.54 AW112010 0.3 Slamf6 0.9 Nkg70.48 Mcm6 0.54 SerincS 0.29 Host 0.81 Ly6c10.47 Rpi41 0.52 Inpp4b 0.27 mt-Rnr1 0.8 Ccr20.46 TubbS 0.52 Anxal 0.27 Ets1 0.78 Gripl0.45 Mcm3 0.5 Xcl1 0.26 AW112010 0.77 Ctsd0.41 Tpi1 0.5 Socs2 0.26 Cxcr3 0.76 Gzmk0.4 Mcm5 0.5 Cd82 0.25 Ms4a4b 0.75 Gzmb0.36 Dut 0.49 Cd4 0.25 Dgka 0.7 Itgbl0.33 Ftn 0.48 Gimap7 0.25 Ypel3 0.67 Cd52 Iog2fc Cytotox.1 Iog2fc Cytotox.2 Iog2fc Cytotox.3 Iog2fc Cytotox.4 2.4 Hmgb2 2.8 Hist1h1b 1.18 Mcm6 0.89 S100a62.2 Lgalsl 2.4 Hmgb2 1.17 Mcm3 0.59 Nrn11.9 Vim 2.4 Hist1 h2ae 1.07 Mcm5 0.58 Cxcr61.7 Ly6e 2.3 Hist1h1e 1.07 Dut 0.57 Klrd1.6 Ccnb2 2.2 Top2a 0.97 Ptma 0.54 Pdcdl1.6 Birc5 2.2 Mki67 0.77 Lig1 0.5 Lgalsl1.6 H2afz 2.2 Rrm2 0.72 Uhrfl 0.5 Lag31.5 Anxa2 2.1 Pclaf 0.7 Tk1 0.5 AW1120101.5 Cenpa 2 TubbS 0.68 Hsp90ab1 0.49 Itgbl1.5 Hmgn2 2 H2afx 0.67 Lgalsl 0.47 Id21.5 Pttgl 1.8 H2afz 0.66 Gzmb 0.47 Lgals31.4 Mki67 1.8 Birc5 0.64 Stmn1 0.46 Nrgn1.4 Lgals3 1.7 Lmnbi 0.62 Tpi1 0.44 Ctla2a1.4 S100a10 1.6 Ube2c 0.6 Pclaf 0.43 Cst71.3 Lmnbi 1.6 Ptma 0.58 H2afz 0.43 Nkg71.3 Cdc20 1.6 Stmn1 0.58 Tubalb 0.41 S100a10 WO 2024/186790 PCT/US2024/018472 1.3 Ube2c 1.5 Hmgn2 0.58 Lmnb1 0.41 Tnfrsf91.3 Cmtm7 1.5 Tubalb 0.55 Hmgn2 0.4 Cd521.2 S100a6 1.5 Ly6e 0.55 Tyms 0.4 Vim1.2 Pclaf 1.3 Dut 0.54 Ybx3 0.4 Ifitm2 This table presents the results of a procedure used to identify markers specific to each T cell cluster found by unsupervised cluster analysis. The FindMarkers procedure from the Seurat software package in R was applied to each cluster against all other clusters. Results are provided as the average log2 fold change (log2fc) for each gene.

Cross-labelling of the cells against reference gene signatures from published datasets (Huang et al., Cell, 185: 1-18, 2022; Daniel etal., Nat Immunol, 23: 1614-1627, 2022; Deaketal., Nature, 610: 173- 181,2022) showed concordance of the clusters with many reported signatures as well as providing more granularity, with several of the clusters matching to existing categories of T cell phenotypes (Fig. 11 A). Of particular relevance to the present study, recent papers have shown that the combination of anti-PD- L1 therapy with IL2 agonists promotes the creation of "better effectors", or T cells contributing to enhanced antitumor responses, which are most similar to the cluster Ccl5.2 (Deak et al., Nature, 610: 161-172, 2022; Hashimoto et al., Nature, 610: 173-181,2022).It was then assessed how each therapeutic treatment impacted the clonal behavior of CD8+ T cell clusters within the dLN, blood, and tumor. To this end, scTCR-seq was used to measure clonal expansion and the distribution of all clones, not just tumor antigen-specific clones, across cell clusters. Cells in dLN were predominantly singletons (having only one cell expressing a given TCR clonotype) with the exception of the two Ccl5 clusters, which showed evidence of clonal expansion following combination treatment, with and without FTY720. In contrast, cells in blood and tumor were almost exclusively expanded clones (Figs. 2C and 2D; Fig. 11B).At day 7, activated cells were present in the blood of treated mice and consisted primarily of clonally expanded Ccl5.2 cells, especially after anti-TIGIT or combination treatment; only a slight increase was observed over controls with anti-PD-L1 alone (Figs. 2C and 2D). FTY720 largely ablated these effects, as expected since the expanded Ccl5.2 cells in the blood likely originated in dLN.Tumors had relatively large proportions of clonally expanded gp70־ and gp70+ TILs, comprised of Ccl5, Cytotox, Ifng, and Ifit clusters (Figs. 20 and 2D). These cells were observed in the control group and with FTY720 treatment, however, indicating that many of them were pre-existing in tumor and may expand and differentiate intratumorally. Combination treatment showed larger proportions of Ccl5.2 cells that were tetramer gp70+ versus gp70־, reflecting their probable infiltration from blood.

D. Combination treatment focuses the TCR clonal diversity of tumor antigen-specific CD8+ T cells Next, the quantitative relationship between clonal expansion and antigen specificity of CD8+ T cells was examined. Using ADT count as a measure of CD8+ T cell specificity for the gp70 tumor antigen, the phenotypes of T cells with high antigen binding matched those with high clonal expansion, predominantly the Ccl5 and Cytotox clusters (Figs. 3A and 3B). Each clone was characterized by its majority ADT barcode type, with gp70+ as an indicator of tumor reactivity and gp70- as non-tumor reactivity (or reactivity to some other tumor antigen). T cells that bound the gp70 tetramer nearly WO 2024/186790 PCT/US2024/018472 exclusively had high avidity (> 100 sequenced ADT barcodes) and were detected widely in all clusters, whereas T cells that bound non-gp70 antigens were more likely to have high avidity in Ribo and Ccrclusters, and low avidity in Ifng, Ccl5.2 and Cytotox clusters (Fig. 3C).To see how clonal expansion was related across tissues, the degree of clonal expansion in dLN, blood, and tumor was compared at day 7 post-treatment, characterizing each clone by its majority cluster at each site (Figs. 3D and 12A). Combination treatment showed a striking phenomenon of coordinated clonal expansion, wherein 5 clones exhibited relatively large expansion in both dLN and tumor, as well as in the blood at day 7 (Fig. 3D). These clones were composed primarily of Ccl5.2 or Cytotox.1 cells in the dLN, and Ccl5.2 or Cytotox.4 cells in tumor. Interestingly, no clones were observed to have expanded in both dLN and tumor in the control or anti-PD-L1 single-agent treatment groups. Anti-TIGIT monotherapy promoted limited dual expansion of cluster Cytotox.1 and Ccl5.2 clones in dLN and tumor, respectively, but not nearly to the extent of combination treatment. When FTY720 was added to the combination treatment, many clones were observed with dual expansion in dLN and tumor, but relatively little expansion in blood. Since FTY720 prevents T cell egress from lymphoid tissue, one interpretation is that these dual-expanded clones represent clonal expansion occurring independently in dLN and tumor, with the clones in tumor having been seeded prior to FTY720 treatment (Fig. 1D). In contrast, the largest clones observed with combination treatment represent T cells that have expanded in dLN, trafficked into blood, and infiltrated the tumor (Fig. 12A), with possible additional expansion subsequently in tumor.Dual-expanded clones were either high-avidity tumor-reactive or low-avidity non-tumor reactive types (Fig. 3E). Although both anti-PD-L1 and anti-TIGIT single-agent treatments expanded several high- avidity tumor-specific clones, these expanded in either dLN or tumor but not both, and were not highly expanded in blood. Most of the dual-expanded clones in single-agent treatments were typically low- avidity non-tumor reactive, which may be consistent with "bystander" non-tumor reactive T clones that have been found to be prevalent in tumors (Meier et al., Nat Cancer, 3: 143-155, 2022; Simoni et al., Nature, 557: 575-579, 2018).In contrast, combination treatment induced a predominantly tumor-reactive response with four of the five most triple-expanded (dLN, blood, and tumor) clones being high-avidity tumor-reactive, but also several other less-expanded clones being of the low-avidity non-tumor-reactive bystander type. In the presence of FTY720, dual-expanded clones were almost all of the high-avidity tumor-reactive type, supporting the hypothesis that these represent pre-existing clones already present in dLN and tumors prior to treatment, and further indicating that such in situ clonal expansion occurs predominantly in T cells that are tumor-reactive, whereas bystander clones infiltrate predominantly from blood.Since these scatterplots indicate only the primary cluster type for each clone, to study the composition of individual clones across clusters, the most expanded clones were profiled in tumor, and were matched in dLN and blood (Fig. 3F). It was found that the largest clones in tumor had counterparts in dLN under combination but not single-agent treatment, and that these clones in dLN consisted predominantly of the Ccl5.2 phenotype. In blood, the same expanded clones were virtually all of the Ccl5.2 phenotype. In tumor, these clones had a more diverse mixture of clusters, indicating that expansion of Ccl5.2 cells occurs in dLN, these cells egress into blood, and subsequent differentiation occurs in tumors, predominantly into IFN-g producing and cytotoxic phenotypes. In contrast, the most WO 2024/186790 PCT/US2024/018472 expanded clones in tumors of control or anti-TIGIT and anti-PD-L1 single-agent groups had little or no representation in dLN or blood, indicating that these clones were likely pre-existing.By examining the cluster composition of the top 50 clones from each site independently, it was found that each treatment had distinct effects on T cell differentiation (Figs. 12B and 12C). In dLN, anti- TIGIT, but not anti-PD-L1, caused expansion of Ccl5.2 T cells and, to a lesser extent, Cytotox cells. This expansion was also seen in combination treatment, and FTY720 treatment shifted the composition to almost exclusively Ccl5.2, suggesting that these cells accumulated in dLN instead of egressing into blood. In tumor, anti-PD-L1 showed higher numbers of the Cytotox.4 group when compared with other treatments, suggesting that anti-PD-L1 has an effect on T cell differentiation in tumor. FTY720 treatment did not appear to change the differentiation patterns in tumor, supporting the notion that in situT cell differentiation in tumors occurred subsequent to infiltration.Taken together, the application of scRNA-seq, scTCR-seq, and ADT-seq to a large cell cohort reveals that combination treatment drives dual expansion and trafficking of a limited subset of highly avid tumor-reactive clonal populations preferentially belonging to cluster Ccl5.2, acting to focus the TCR clonal diversity and differentiation of CD8+ T cells in both dLN and tumors.

E. Anti-PD-L 1 and anti-TIGIT differentially reshape migration and differentiation patterns of CD8+ T cells in dLN and tumor Next, better understanding of the differentiation relationships across CD8+ T cell clusters in various treatments was sought. Although several methods exist for inferring differentiation trajectories using scRNA-seq data and similarities based on gene expression, none exploit the valuable information provided by scTCR-seq, which unambiguously identifies lineages of T cells. For this purpose, a computational method was developed to infer trafficking and differentiation pathways based on the co- occurrence of both T cell phenotypes and tissue sites within clones. This method computes a minimum spanning tree from pairwise co-occurrence frequencies, weighted by clone size, and allows the effects of different treatments across and within tissue-specific UMAP plots to be depicted, with line widths that reflect the degree of co-occurrence (Figs. 4A-4E and 13A). For all treatments, co-occurrences of the same clusters were detectable across dLN, blood, and tumor, but particularly for the Ccl5.2 cluster, supporting the migration of these cells from dLN to blood to tumor. Trafficking was most prevalent for anti-TIGIT and the combination treatment, with relatively little migration induced by anti-PD-L1 alone from dLN to blood or blood to tumor. Co-occurrences within each tissue indicate differentiation pathways, with a similar pattern observed within each unique clonotypic group of Ccl5.2 T cells differentiating into Cytotox.4 T cells, and subsequently developing into other cytotoxic phenotypes and IFN-g-producing T cells. Combination treatment, both with and without FTY720 treatment, showed a greater tendency than other treatments for Ccl5.2 phenotypes to co-occur with Ccl5.1 and Ifit phenotypes.Recently, in vivo trafficking of T cells into and out of tumors has been elegantly visualized using photoactivation, revealing a relationship between CD8+ T cell phenotypes and their tumor residency, and the recirculation of stem-like TCF1+ T cells from tumor back to dLN (Li et al., J Exp Med, 219: 620210749, 2022). Because the co-occurrence analysis did not allow such directionality to be determined, the dataset was assigned to their gene expression signatures, concordance was found between the two 100 WO 2024/186790 PCT/US2024/018472 classifications (Fig. 13B), supporting that Ccl5.1 and Ccl5.2 clusters are associated with recent infiltration, and that tumor-retained T cells are most cytotoxic. The phenomenon of recirculation also means that the presence of some clusters in dLN may not be due to differentiation within the dLN following activation but rather recirculation from the tumor.The clonal trajectory analysis shows that the Ccl5.2 cluster is a key phenotype involved in both the expansion and trafficking induced by the combination of anti-TIGIT and anti-PD-L1.

F. CD226 involvement in tumor-specific CD8+ T cell differentiation in dLN To validate the transcriptomic analyses highlighting the prominence of the Ccl5.2 and Cytotox.clusters, tumor-specific CD8+ T cells were immunophenotyped, focusing on markers associated with Tscm/Tpex/transitory states and exhaustion. It has previously been reported that human TILs in non- small cell lung cancer (NSCLC) differentially express CD226 and CD28 in various CD8+ T cell clusters and may thereby necessitate combination treatment to elicit optimal activity of the entire repertoire of potentially tumor-reactive TILs (Banta et al., Immunity, 55: 512-526, 2022). CD226 gene and protein expression overlayed with many of the clusters responsive to treatment based on trajectory analysis, while CD28 showed some overlapping expression but also marked distinct clusters as well, consistent with previous findings for human NSCLC TILs (Banta et al., Immunity, 55: 512-526, 2022) (see Figs. 2B, 10A, and 10B).CD226 signaling can lead to reduced FOXO1 expression, allowing CD226 to impact CD8+ T cell differentiation decisions (Du et al., Proc Natl Acad Sci USA, 115: E11731-E11740, 2018). To determine whether CD226 was required for the promotion of Tscm/Tpex cells rather than progression towards exhaustion, tumor-specific gp70+ CD8+ T cells were segregated based on CD226 expression. Anti-TIGIT alone or in combination with anti-PD-L1 increased the frequency of gp70+CD8+ T cells expressing CD2in both dLN and tumor, with FTY720 treatment having minimal impact (Figs. 5A and 5H). In response to checkpoint blockade, gp70+CD8+ T cells expressing CD226 were significantly more proliferative, as measured using Ki67 as a marker, but this was observed only in dLN and not tumor (Figs. 5B and 51). Few CD226+gp70+CD8+ T cells in dLN were naive as compared to the CD226 fraction (Fig. 5C), and combination treatment, but not either monotherapy, increased the frequency of CD226+gp70+CD8+ T cells with a Teff or Tem phenotype whereas no effects were observed in the CD226 population (Fig. 5D).Using PD-1 as a marker of activation or exhaustion, it was found that combination treatment appeared to increase the frequency of CD226+gp70+CD8+ T cells expressing PD-1 in dLN. In contrast, anti-TIGIT and combination treatment decreased the frequency of PD-1-expressing cells in the CD226- fraction (Fig. 5E). In addition, combination treatment as well as anti-TIGIT alone increased the frequencies of both CD226+ and CD226- gp70+CD8+ T cells in tumor with TCF-1 and Tim3, markers of Tscm/Tpex cells, whereas their effects in the dLN were limited to the CD226+ subset; FTY720 largely abolished these effects (Figs. 5F and 5J). The transcription factor Tox is a key regulator of the T cell exhaustion pathway (Scott et al., Nature, 571: 270-274, 2019; Seo et al., Proc Natl Acad Sci USA, 116: 12410-12415,2019). Strikingly, treatment with either anti-TIGIT alone or anti-TIGIT plus anti-PD-Lmarkedly decreased Tox expression in tumor antigen-specific CD226+, but not CD226, CD8+ T cells in dLN, while decreased Tox expression was seen in both CD226+ and CD226 fractions in tumor; FTY7 101 WO 2024/186790 PCT/US2024/018472 appeared to diminish the combination effect on Tox expression (Figs. 5G and 5K). Similar effects were seen in the EO771 model, with combination treatment increasing the frequency of CD8+ T cells in tumors, promoting CD226 expression on tumor CD8+ T cells, and increasing the TCF1 +Tim3+ phenotype while reducing Tox+ frequencies (Figs. 14A-14F).As anti-TIGIT plus anti-PD-L1 appeared to have more pronounced effects on CD8+ T cells expressing CD226, particularly in dLN, mice receiving the combination were concurrently treated with CD226 blocking mAb. Anti-CD226 mAb showed a trend towards impairing the ability of combination treatment to increase the frequency of TCF1+Tim3+ tumor-specific CD8+ T cells in dLN, but not in the tumor, consistent with the observation that tumor antigen-specific CD8+ T cells acquired stem-like phenotypes in the tumor dLN (Prokhnevska et al., Immunity, 56: 107-124, 2023) (Fig. 51). Anti-CD2mAb ablated the reduction in Tox-expressing cells mediated by combination therapy in dLN that was also maintained in tumor (Fig. 5M).Taken together, combination blockade elicited the generation of qualitatively better effector cells within dLN, with the unleashing of CD226 signaling likely playing a key role.

G. Discussion This study examined the mechanisms by which the combination of anti-TIGIT with anti-PD-Lgenerates superior anti-tumor activity compared to either Ab alone. Using a multi-omics approach in a mouse tumor model, several key findings were made (illustrated in Extended Data Fig. 10): (1) CD8+ T cell trafficking between dLN and tumor is strictly required for anti-tumor efficacy but only until sufficient tumor-specific CD8+ T cells have infiltrated the tumor, at which point additional trafficking is less important; (2) combination treatment drives expansion of tumor-specific CD8+ T cell clones in both the dLN and tumor; (3) dual-expanded clones have phenotypic characteristics of effector and memory cells derived from Tscm/Tpex cells that have reduced exhaustion programming likely reflecting the reduced induction of the Tox transcription factor; (4) combination treatment focuses dual-expanded clones towards a limited, but largely tumor antigen-specific, TOR repertoire; and (5) modification of the tumor-specific CD8+ T cell profile is associated with CD226 expression. In summary, the study shows how checkpoint blockade shapes the differentiation decisions of tumor-specific CD8+ T cells to elicit better and more productive anti-tumor responses.The study shows that both anti-TIGIT and anti-PD-L1 were individually capable of expanding tumor-specific CD8+ T cells clones in dLN or tumors, indicative of activity at both sites, with anti-TIGIT somewhat surprisingly exerting a more significant effect than anti-PD-L1 alone. However, only the combination blockade of both PD-L1 and TIGIT invoked coordinated dual expansion of a focused tumor- reactive CD8+ T cell clonal repertoire together with a less exhausted phenotype. Focusing of the TOR repertoire may reflect the enhancement of CD226 signaling in the Tscm/Tpex compartment to generate more targeted effector cells with anti-tumor activity. It was previously reported that TIGIT and PD-coinhibitory pathways converge to inactivate CD226, providing a mechanistic rationale for dual blockade of these two checkpoint inhibitor receptors (Banta et al., Immunity, 55: 512-526, 2022). Combination TIGIT/PD-1 blockade-enabled CD226 signaling would then contribute to the multitude of signals that determine CD8+ T cell differentiation fates following antigen-specific activation. It is possible that CD2 102 WO 2024/186790 PCT/US2024/018472 optimizes selection of clones with desired effector and/or memory properties, whereas ablation or dampening of CD226 signals by TIGIT and/or PD-1 tilts the balance towards differentiation states such as exhaustion that are less capable of controlling tumor growth. It is noteworthy that enhanced CDsignaling alone, resulting from PD-1/PD-L1 blockade, was relatively less effective at directing Tscm/Tpex cells towards the effector/memory pathway.While Tscm/Tpex cells have been proposed as targets for PD-1/PD-L1 targeted immunotherapies (Huang et al., Cell, 185: 1-18, 2022; Siddiqui et al., Immunity, 50: 195-211 e110, 2019; Kallies et al., Nat Rev Immunol, 20: 128-136, 2020; Connolly et al., Sci Immunol, 6: eabg7836, 2021), activation and differentiation of these cells may not be sufficient for driving robust efficacious activity in the cancer setting. Although anti-PD-(L)1 may act on Tscm/Tpex cells and expand tumor-specific CD8+ Teff cells, these Teff cells are often transitory and eventually accumulate as exhausted T cells (Deak et al., Nature, 610: 161-172, 2022), a phenomenon that was observed in the CT26 tumor model with anti-PD-Lmonotherapy. Anti-TIGIT monotherapy, in contrast, may also target Tscm/Tpex cells but exert a stronger effect on modification of differentiation programming, perhaps due to higher expression levels of TIGIT relative to PD-1 on Tscm/Tpex cells and its greater effect on CD226 activity. The differential monotherapy effects also highlight the nonredundant mechanisms of PD-1/PD-L1 and TIGIT targeted therapies. Nonetheless, dual blockade appears to be necessary to ensure full functionality of CD226, as well as CD28. It is therefore proposed that the TIGIT and PD-1 co-inhibitory molecules serve to direct the differentiation of activated T cells along the Tex pathway. By limiting Tox expression, their combined blockade may favor T effector and memory cell differentiation, perhaps by ensuring optimal co-stimulatory signaling via CD28 and CD226.CD226 signaling is an attractive mechanism to explain modulation of CD8+ T cell differentiation pathway decisions, through activation of AKT and subsequent degradation of FOXO1. Strength of FOXO1 transcriptional regulatory activity can have widely disparate effects on CD8+ T cell differentiation, with intense FOXO1 activity driving a pathway towards memory cell anergy/tolerance and minimal activity resulting in deletion of effector cells (Kim et al., Front Immunol, 4: 20, 2013). FOXO1 has been shown to be a transcriptional activator of PD-1, a requirement for sustainment of T cell memory and maintenance of stem-like properties, and a promoter of differentiation to terminal exhaustion (Staron et al., Immunity, 41: 802-814, 2014; Delpoux etal., J Exp Med, 215: 575-594, 2018; Delpoux etal., Cell Rep, 34: 108674, 2021; Utzschneider et al., Cell Rep, 22: 3454-3467, 2018). However, rather than exerting an all-or-none effect on FOXO1, CD226 is but one of many signals that are integrated by CD8+ T cells and determine the fate decisions following antigen-specific activation. CD226 may play a critical role in the dLN to promote TCF1+Tim3+ CD8+ T cells that are or become more competent effector cells, conferring protection from the exhaustion pathway. Once CD8+ T cells infiltrate tumors, however, CD226 may no longer be required to maintain this phenotype, suggestive of a CD226-mediated programming mechanism during early priming in dLN in response to tumor-specific antigens. Alternatively, the effect of combination treatment on the CD226- fraction in tumor may be attribute to downregulation of CD2following activation through tumor-expressed PVR (Braun et al., Immunity, 53: 805-823 6815, 2020; Weulersse et al., Immunity, 53: 824-839 6810, 2020). Thus, blockade of the immune checkpoints TIGIT 103 WO 2024/186790 PCT/US2024/018472 and PD-1/PD-L1 drives qualitatively advantageous differentiation of tumor-specific CD8+ T cells in dLN that can then traffic to and infiltrate into tumors where they maintain their qualitatively better effector state.It is also possible that other mechanisms, including the role of other immune cells or Fc format of anti-TIGIT mAb, may contribute to the shaping of the anti-tumor CD8+ T cell response. The finding that the CD8+ T cell clusters most profoundly affected by or responsive to the combination treatment of anti- TIGIT and anti-PD-L1 were those most highly expressing the chemokine receptors CXCR3 (Cxcr3, Ccl5.and Ccl5.2) and CXCR6 (Cxcr3, Ccl5.2, Cytotox.3 and Cytotox.4) raises implications as to whether the effects of combination treatment are acting solely on CD8+ T cells or if there may also be effects on CXCR3- or CXCR6-ligand producing antigen-presenting cells that also express PVR and PD-L1, the ligands for TIGIT and PD-1, respectively. As myeloid cells also express Fc gamma receptors (FegR), the contributions of anti-TIGIT Fc format and Fc-FcgR interactions should also be taken into account, particularly in light of the broad Fc formats used by the dozens of anti-TIGIT mAbs in clinical development (Chiang et al., J Immunother Cancer, 10: 6004711,2022).This study reveals that within a single clonal population of tumor-specific CD8+ T cells, the phenotypic composition is quite diverse and representative of varying stages of differentiation and developmental trajectories. Furthermore, the same clone may have different phenotypic composition in dLN, blood, and tumor. Clonal diversity and some degree of dual expansion was seen in all treatment groups in the study, yet efficacy was only observed with anti-TIGIT plus anti-PD-L1 combination where clones were highly expanded in dLN and tumor and present in high frequencies in blood. These findings provide the underpinnings for the generation of productive anti-tumor CD8+ T cell clonotypes and predictive biomarkers (e.g., predictive biomarkers for benefit from anti-TIGIT plus anti-PD-L1 combination treatment) (see Example 2).The effects of combined TIGIT and PD-1 blockade are comparable to recently described effects of combination therapy of anti-PD-L1 mAbs plus either wild-type IL-2 (Hashimoto et al., Nature, 610: 173- 181,2022), which can result in systemic immune responses, or a version that aims to avoid toxicity by targeting PD-1 instead of activating CD25 (Deak et al., Nature, 610: 161-172, 2022). Both IL-2 strategies altered the differentiation program of Tscm/Tpex cells to favor generation of highly functional Teff cells rather than exhausted cells. However, unlike the potential for potentially life-threatening reactions, such as capillary leak syndrome, associated with IL-2 therapies such as aldesleukin (Rosenberg et al., J Immunol, 192: 5451-5458, 2014) or possibly even targeted or modified IL2 variants (Deak et al., Nature, 610:161-172, 2022), tiragolumab plus atezolizumab has a favorable safety profile and is well-tolerated (Cho et al., Lancet Oncol, 23: 781-792, 2022; Rodriguez-Abreu et al., J Clin Oncol, 38: 9503, 2020). Thus, anti-TIGIT plus anti-PD-L1/PD-1 may represent an ideal cancer immunotherapy combination that could achieve the goal of generating desired tumor-specific clonotypes whose differentiation favors the production of memory and effector cells rather than Tex, and thus may be more effective for controlling tumor growth.

Example 2. Gene signatures derived from reference mouse CD8+ T cell clusters show association with response to tiragolumab plus atezolizumab in cancer patients The prominence of the Ccl5.2 cluster in dLN, blood and tumor of CT26 tumor-bearing mice treated with the combination of anti-TIGIT and anti-PD-L1, together with the co-occurrence depicting 104 WO 2024/186790 PCT/US2024/018472 differentiation pathways of individual clones (Example 1), prompted examination of scRNA-seq data of peripheral blood T cells from NSCLC patients treated with the combination of tiragolumab (anti-TIGIT mAb) plus atezolizumab (anti-PD-L1 mAb) (T+A) from a phase 1b study (Bendell et al., Cancer Res, AACR Annual Meeting 2020, Abstract CT302, 2020).A gene expression score for the clusters that are characteristic of dual-expanded clones, including a composite gene signature comprised of CCR7, CXCR3, CXCR6 and CCL5, was identified and showed association with clinical benefit in human NSCLC patients treated with tiragolumab plus atezolizumab, demonstrating translatability of the mouse findings to the human disease setting.Human CD8+ T cells were mapped onto the mouse reference CD8+ T cell UMAP (Fig. 15A), with a median prediction accuracy above 60% for human CD8+ T cells assigned to the mouse Ccl5.2 cluster (Fig. 15B). Patients with a clinical response, evaluated as either complete response (CR) or partial response (PR), trended with an increased frequency of CD8+ T cells mapping to the Ccl5.2 cluster compared to non-responders, evaluated as either stable disease (SD) or progressive disease (PD) (Fig. 15C). These human CD8+ T cells mapped to Ccl5.2 were also the most prevalent subset found in peripheral blood of responders, consistent with this cluster being the primary tumor-specific population trafficking from dLN to tumor based on the mouse tumor model data.It was next asked whether the gene signature of the Ccl5.2 cluster or gene signatures from the other mouse CD8+ T cell clusters were associated with improved overall survival (OS) benefit. To address this question, RNA-seq data from baseline tumor samples from patients in a separate, randomized phase 2 CITYSCAPE study which evaluated the efficacy of tiragolumab plus atezolizumab (T+A) versus placebo plus atezolizumab (P+A) in patients with PD-L1 positive NSCLC (Cho et al., Lancet Oncol, 23: 781-792, 2022) were analyzed.The top 20 differentially expressed signature genes for each mouse CD8+ T cell cluster were identified and the average expression of orthologous human genes was examined (average expression referred to as the gene signature ‘score’) in CITYSCAPE samples (Table 4). These CD8+ T cell signature gene scores were compared in CITYSCAPE patients treated with T+A or P+A, clinically evaluated as being responders (complete response or partial response (CRPR)) or non-responders (stable disease or progressive disease (SDPD)), where baseline tumor bulk RNA-seq data was available. Ccr7.3, Cxcr3, and Ccl5.1 gene signature scores were significantly higher in responders as compared to non-responders (Fig. 6A). While all CD8+ T cell cluster signatures trended with favorable OS hazard ratio (HR) in patients treated with T+A compared to P+A, high expression of Ccr7.2, Ifit, Mitotic and Cxcr3 gene scores were associated with significantly improved HR for OS, as were low expression of Cytotox.2 and Cytotox.(Fig. 15D). Dichotomization of patients on the basis of high or low cluster gene signature score and by treatment showed that high expression of the Ccr7.2, Ccr7.3, or Cxcr3 gene signatures trended with increased OS with T+A but not P+A (Fig. 15E). 105 WO 2024/186790 PCT/US2024/018472 Table 4. Gene sets derived from mouse CD8+ T cell reference clusters to define human gene signature scores Ribo.1 Ribo.2 Ccr7.1 Ccr7.2 Ccr7.3 Egr Ifng Ifit RPS20 RPS20 CCR7 STAT1 DAPL1 NFKBIA CCL4 ISG15RPS24 RPS24 RFLNB LEF1 CCR7 EGR1 XCL2 IFIT1BRPS3A SELL LEF1 IRGM SMC4 ZFP36 NR4A1 IFIT3RPL13 RPS27 TSC22D3 CCR7 RGCC JUNE EGR1 ISG20RPS8 RPS29 SMC6 SELL MXD4 CD69 SRGN IFI27L2RPS5 RPL13 TDRP RPS24 CD8A IER2 NR4A3 SAMHD1RPS29 RPS5 KLF2 RPS27 TCF7 NR4A1 TAGAP ZBP1RPLP1 RPLP1 ACP5 GBP2 ITGAE ZFP36L1 MYC SLFN5RPS4X RPS3A CD8B RPS29 RGS10 TAGAP ZFP36L1 IRF7RPS27 RPS8 SELL RPS3A ZNRF1 DUSP2 NFKBIA RTP4RPSA RPS4X DGKA RPS20 CHD3 IER5 TNF USP18RPLPO RPL32 BTG1 KLF2 CD52 MYC RILPL2 PHF11RPL32 IFNGR1 ACTN1 RPLP1 DDIT4 SRGN IFNG LGALS3BPRPS15A RPSA RPS27 RPL13 LEF1 ICAM1 DUSP2 BST2RPS11 ARL4C S1PR1 DAPL1 IZUMO1R BTG1 CD69 GBP2EEF1A1 SIDT1 DAPL1 SMC6 INPP4B RELB EGR2 IFITM3TMSB10 IL7R RFLNB RFLNB CDS TNFRSF9 STAT1RPS12 RPLPO RPS4X KDM6B NR4A2 IFH6KLF2 RPS15A EGR2 HSPA5 IFIH1BATF REL Mitotic Cxcr3 Ccl5.1 Ccl5.2 Cytotox.1 Cytotox.2 Cytotox.3 Cytotox.4 HSP90AB1 CXCR6 CCL5 CCL5 HMGB2 HMGB2 MCM6 S100A6PTMA CKB ITGB1 S100A6 LGALS1 TOP2A MCM3 NRN1XCL2 GIMAP5 BTG1 LGALS3 VIM MKI67 MCMS CXCR6ODC1 ID2 GZMK LGALS1 LY6E RRM2 DUT KLRC1TNFRSF9 LTB LTB CXCR6 CCNB2 PCLAF PTMA PDCD1ITM2A FGL2 IL7R ID2 BIRC5 TUBB LIG1 LGALS1MYC RAMP1 ZFP36L2 AHNAK ANXA2 BIRC5 UHRF1 LAGSYBX3 LYST ITGA4 KLRC1 CENPA LMNB1 TK1 ITGB1HSPA5 ASB2 TXNIP S100A10 HMGN2 UBE2C HSP90AB1 ID2GPX1 IL2RB SLAMF6 VIM PTTG1 PTMA LGALS1 LGALS3RPS12 CXCR3 HCST NKG7 MKI67 STMN1 GZMB NRGNTUBA1B SERINC3 ETS1 CCR2 LGALS3 HMGN2 STMN1 CST7MCM6 INPP4B CXCR3 CRIP1 S100A10 TUBA1B TPH NKG7TUBB ANXA1 MS4A4A CTSD LMNB1 LY6E PCLAF S100A10MCM3 XCL2 DGKA GZMK CDC20 DUT TUBA1B TNFRSF9TPH SOCS2 YPEL3 GZMB UBE2C LMNB1 CD52MCM5 CD82 ITGB1 CMTM7 HMGN2 VIMDUT CD4 CD52 S100A6 TYMS IFITM2FTL GIMAP7 PCLAF YBX3 106 WO 2024/186790 PCT/US2024/018472 This table presents the human genes converted from their mouse orthologs that were used to calculate the gene signature scores from CITYSCAPE data.

Collectively, these data suggest that gene signatures that are predominantly associated with response to T+A are characterized by high expression of chemokines or chemokine receptors that may have roles in tuning CD8+ T cell differentiation and supporting tumor infiltration, or are representative of a stem-like or early differentiation state. For these reasons, further investigation focused on CCR7, CXCR3, CXCR6 and CCL5, genes that were amongst the most highly expressed in each of the clusters (Table 3). High expression of each of these individual genes was associated with response in patients treated with T+A (Fig. 6B), and high expression of CCL5, CXCR3 or CCRZwas individually associated with favorable OS HR in T+A compared to P+A, outperforming CD8A (OS HR and 95% confidence interval (Cl) of 0.32 and 0.14-0.73; 0.41 and 0.18-0.94; 0.42 and 0.19-0.92; and 0.43 and 0.20-0.91, respectively) (Fig. 6C). CXCR3, CXCR6 and CCL5 were associated with improved OS for T+A, again outperforming CD8A (Fig. 6D).Next, a composite gene signature score comprised of the average expression of CCR7, CXCR3, CXCR6 and CCL5 was tested. Since CCR7 may also be expressed by naive cells, a gene signature excluding CCR7 was also tested. The gene signature score including CCR7was significantly higher (p=0.0098) in responder CITYSCAPE patients as compared to non-responders, and was similar to the gene score that excluded CCR7 (p=0.012) (Fig. 6E). A high gene signature score including CCR7was associated with favorable OS HR in patients treated with T+A (HR=0.37, 95% Cl: 0.17-0.81, p=0.016) compared to P+A, while a low signature score did not associate significantly with OS benefit (HR=0.78, 95% Cl: 0.40-1.52, p=0.467), and inclusion of CCR7outperformed the gene signature score excluding CCR7(HR=0.43, 95% Cl: 0.20-0.94, p=0.035) (Fig. 6F). Segregation of patients on the basis of high or low gene signature scores showed that those treated with T+A who had high expression of either gene signature score had improved OS compared to patients with a low gene signature (Fig. 6G). These gene signature scores were also associated with improved progression-free survival (PFS) and OS in patients with locally advanced or metastatic, previously treated NSCLC from the phase 3 OAK study who received atezolizumab monotherapy (Fig. 15F) (Rittmeyer et al., Lancet, 389: 255-265, 2017), suggesting that improved outcomes to checkpoint blockade may be associated with the CD8+ T cell clusters represented by the composite gene signature. Further, in a phase 3 NSCLC study of first-line atezolizumab versus chemotherapy (IMpowerl 10), patients with high expression of the CDS gene panel comprised of CCR7, CXCR3, CXCR6 and CCL5 demonstrated longer progression-free and overall survival in the PD-L1- positive subset (tumor proportion score (TPS) > 1%) in atezolizumab treated patients compared to patients with low expression of the gene panel (Fig. 17).Thus, this analysis of patients treated with T+A largely recapitulates the effects of anti-TIGIT plus anti-PD-L1 in the mouse tumor studies, providing translational evidence that the events occurring in dLN of tumor-bearing mice may also be detected in human blood and tumors. Furthermore, this analysis highlights that it is not just the presence of CD8+ T cells in tumors that may be predictive of response to immunotherapy (Tumeh et al., Nature, 515: 568-571,2014), but rather the quality of those CD8+ T cells at baseline that more strongly associates with clinical benefit. 107 WO 2024/186790 PCT/US2024/018472 While TCR diversity and clone frequencies can be measured by a number of different metrics, attempts to associate TCR diversity with clinical outcome to immunotherapies are constrained by a number of factors, including limited samples, lack of pre- and post-treatment and responder vs. non- responder samples and phenotypic diversity of clones (Kidman et al., Front Immunol, 11: 587014, 2020). Beyond these hurdles, the need for sampling multiple tissues (tumor, blood, and dLN) becomes paramount when considering that cancers in humans are detected well after they become established and therefore productive anti-tumor T cell responses may depend on both expansion of new clones in dLN as well as pre-existing T cells within the tumor. Despite these caveats, the preclinical identification of CD8+ T cell clusters important for response to anti-TIGIT plus anti-PD-L1 has been used to identify genes and gene signatures that are predictive biomarkers associated with clinical benefit in human NSCLC patients post checkpoint blockade.Furthermore, application of the gene signature to the NSCLC OAK and IMpowerl 10 studies indicates that the gene signature is applicable to treatment with atezolizumab alone, warranting further investigation in mouse tumor models that are amenable to anti-PD-L1 single agent activity, unlike the CT26 or EO771 models that are not responsive to anti-PD-L1 monotherapy.

Example 3. Materials and Methods The following materials and methods were used in the experiments described in Examples and 2.

Mice BALB/c or C57BL/6 mice were purchased from the Charles River Laboratories.

Cell Lines CT26 and EO771 cell lines were maintained at a dedicated internal cell line facility and tested to be mycoplasma-free. CT26 or EO771 cells were cultured in RPMI 1640 media supplemented with 10% FBS and 100 U/mL penicillin and 100 mg/mL streptomycin, and grown in a 37°C humidified, 5% COincubator.

Syngeneic tumor studies CT26 tumor studies were performed by inoculating age-matched 6-8 week old BALB/c female mice with a sub-cutaneous injection of 0.1 x 106 CT26 cells in 100 pL Hank’s balanced solution (HBSS) and MATRIGEL® (BD Biosciences, San Jose, CA). EO771 tumor studies were performed by inoculating age-matched 6-8 week old C57BL/6 female mice with an injection into the fifth mammary fat pad of 0.1 x 106 EO771 cells in 100 pL HBSS + MATRIGEL®. Once tumors achieved a mean volume of 150-2mm3, animals were apportioned into treatment groups and treated with isotype control (anti-gp1mlgG2a), 10 mg/kg; anti-PD-L1 .mlgG2a LALAPG mAb (clone 6E11), 10 mg/kg followed by 5 mg/kg; anti- TIGIT.mlgG2a mAb (clone 10A7), 10 mg/kg; or TIGIT.mlgG2a.LALAPG, 10 mg/kg, and administered intravenously for the first dose and subsequently intraperitoneally. For the tracking of tumor volume, doses were given three times a week for three weeks. For single-cell analyses, the mlgG2a version of 108 WO 2024/186790 PCT/US2024/018472 anti-TIGIT was used, and three doses were given over the course of one week. To inhibit trafficking, FTY720 (Cayman Chemical Company, 1 mg/kg) was administered by daily oral gavage starting day -before indicated treatment, or where indicated, day 7 after treatment, and continued until end of study. Tumor volumes were measured and calculated twice per week using the modified ellipsoid formula: 1/2 x (length x width2). For pharmacodynamic analyses, mice were euthanized at day 7 after initial treatment. Tumors were dissociated into single cell suspensions by using GENTLEMACS™ Dissociator (Miltenyi Biotec) and enzymatically digested in a buffer containing collagenase D (2 mg/mL) and DNAse (40 U/mL, Roche). Single cell suspensions of draining lymph nodes were obtained by mechanical dissociation through 40 pm cell strainers and performing red blood cell lysis as needed. Blood was obtained by terminal cardiac puncture and collected in lavender MICROTAINER™ Blood Collection Tubes (BD Biosciences, 365974) and subjected to red blood cell lysis. Animals bearing tumors exceeding 2,0mm3 or showing ulceration were euthanized following approved protocols.

Flow cytometry and FACS sorting Immune cell phenotyping by flow cytometry was performed on single cell suspensions from mouse draining lymph nodes, tumor, and blood obtained and described elsewhere. Briefly, dead cells were excluded by using a fixable viability dye. Cell surface markers were stained on ice after tetramer staining. The FoxP3 nuclear staining buffer set (INVITROGEN™) was then performed using recommended manufacturer’s instructions to detect intracellular or nuclear staining. For intracellular cytokine detection, cells were stimulated for 4 hours with Cell Stimulation Cocktail (INVITROGEN™, 00- 4970-93) at 37°C. After stimulation, cells were stained for surface markers and intracellular factors as described above. For obtaining cells for single cell analysis, tumors and dLNs were processed into single cell suspensions as described elsewhere, and subjected to first tetramer staining, then surface markers and CITE-seq antibodies together. Processing of blood samples at day 0 before any treatment or at day were first stained with hashed-tagged antibodies, then stained with surface markers. Cells were purified by fluorescence-activated cell sorting (FACS) on a Becton Dickinson FACSARIA™ Fusion cell sorter equipped with four lasers (405 nm, 488 nm, 561 nm and 638nm). A 70-pm nozzle running at 70 psi and 90 kHz was used as the setup for each sort session. FACSDIVA™ (v.8.0.1) and FlowJo (v.10) were used to collect and analyze the flow cytometry data. Before gating on fluorescence, single cells were gated using forward scatter (FSC-A) and side scatter (SSC-A) (for intact cells) and SSC-W/SSC-H and FSC-W/FSC-H (to ensure that only singlets were sorted). FACS gates were drawn to include only live single cells based on Calcein Blue AM+ and propidium iodide (Thermo Fisher Scientific). Antibodies and tetramers used for flow cytometry, cell sorting by FACS, or CITE-seq are shown in Tables 5 and 6. All samples were acquired on LSR-FORTESSA™, BD SYMPHONY™ Instruments (BD Biosciences) or CYTEK® Aurora and analyzed using FlowJo v10.5 or higher version software (Tree Star, Inc.). 109 WO 2024/186790 PCT/US2024/018472 Table 5. Antibodies and tetramers used for in vivo studies, flow cytometry, cell sorting by FACS, and CITE-seq Fluorophore / Type Target Catalog Number Supplier Clone Number Lot Number Species anti-PD-L1 n/a n/a 6E11 n/a n/aanti-TIGIT (lgG2a; LALAPG)n/a n/a 10A7 n/a n/a APC-Cy7 B220 103224 BioLegend RA3-6B2 B308558 RatPerCP-Cy5.5 B220 103235 BioLegend RA3-6B2 B308915 RatDye Calcein blue C1429 Invitrogen n/a 2326042 n/aAPC-Cy7 CD11b 561039 BioLegend DX5 7278811 RatPerCP-Cy5.5 CD11b 101227 BioLegend M1/70 B308467 RatAPC-Cy7 CD11c 117323 BioLegend N418 B197821Armenian HamsterPerCP-Cy5.5 CD226 133624 BioLegend TX42.1 B316769 RatBV785 CD226 133611 BioLegend TX42.1 B317733BUV737 CD4 612843 BD Biosciences RM4-5 1198910 RatQdot 605 CD450-113- 7562Invitrogen RM4-5 2366139 RatPE-Cy7 CD44 103030 BioLegend IM7 B308091 RatAF700 CD45 56-0441-82 Invitrogen IM7 1980496 RatBUV395 CD45 564279 BD Biosciences 30-F11 1145827 RatBV605 CD62L 104437 BioLegend MEL-14 B336181 RatBV421 CD69 104545 BioLegend H1.2F3 B291490Armenian HamsterAF700 CDS 100730 BioLegend 53-6.7 B285812 RatBUV737 CDS 564297 BD Biosciences 53.6-7 6294901 RatFITC CDS 100706 BioLegend 53.6-7 B318296 RatBV785 CD90.2 105331 BioLegend 30-H12 B289707 RatBUV395 CD90.2 565257 BD Biosciences 53-2.1 9311233 RatBUV805 CD90.2 741908 BD Biosciences 30-H12 2075373 RateFluor 780FixableViability Dye65-0856-14 Invitrogen n/a 2450571 n/aeFluor 506 FoxP3 69-5773-82 Invitrogen FJK-16s 2246975 RatPE H3K27Me 40724S Cell Signaling C36B11 1 RabbitBV650 IFNy 563854 BD Biosciences XMG1.2 7096621 RatBV711 Ki67 350516 BioLegend Ki-67 B345424 MouseBUV496 Ly108/SlamF6 750046 BD Biosciences 13G3 2126118 MouseBV605 Ly108/SlamF6 745250 BD Biosciences 13G3 1068515 MousePE-Cy7 PD-1 135216 BioLegend 29F.1A12 B355884 RatDyePropidiumIodide50-66211E Invitrogen n/a 9352710 n/aBV650 Streptavidin 405231 BioLegend n/a B196153 n/aPE TCF1/TCF7 14456S Cell Signaling C63D9 6147819 RabbitFITC TIGIT 11-9501-82 Invitrogen GIGD7 2318622 Rat110 WO 2024/186790 PCT/US2024/018472 APC TIMS 134008 BioLegend B8.2C12 B198857 RatBV421 TIMS 134019 BioLegend B8.2C12 B321967 RatBV711 TIMS 119727 BioLegend RMT3-23 B284683 RatBV421 TNFa 506328 BioLegendMP6-XT22B333754 Rat APC Tox130-118-474Miltenyi Biotec REA47352111049HumanTotal-Seq C CD4 100571 BioLegend RM4-5 B318116Total-Seq C CD8a 100785 BioLegend 53-6.7 B310956Total-Seq CCD122 (IL- 2Rp)105915 BioLegend 5H4 B315383 Total-Seq CCD127 (IL- 7Ra)135047 BioLegend A7R34 B300524Total-Seq C CD137 (41BB) 106119 BioLegend 17B5 B305895Total-Seq CCD1(CXCR3)126545 BioLegendCXCR3- 173B302524 Total-Seq CCD223 (LAG- 3)125237 BioLegend C9B7W B305125 Total-Seq CCD226(DNAM-1)128825 BioLegend 1.00E+06 B331517Total-Seq C CD279 (PD-1) 109127 BioLegend RMP1-30 B300527Total-Seq C CD28 102133 BioLegend 37.51 B304136Total-Seq CCD366 (Tim- 3)119739 BioLegend RMT3-23 B302525Total-Seq C CD38 102735 BioLegend 90 B299602Total-Seq C CD39 143815 BioLegend DUHA59 B297080Total-Seq C CD69 104551 BioLegend H1.2F3 B319428Total-Seq C CD73 127237 BioLegend TY/11.8 B310991Total-Seq C CX3CR1 149043 BioLegend SA011 F11 B296692Total-Seq C Ly108 134613 BioLegend 330-AJ B328193Total-Seq C TIGIT (VstmS) 142119 BioLegend IG9 B299596Total-Seq C mouse lgG1 400187 BioLegend MOPC-21 B333559Total-Seq C mouse lgG2 400293 BioLegendMOPC- 173B319350Total-Seq C rat lgG1 400467 BioLegend RTK2071 B313972Total-Seq C rat lgG2a 400577 BioLegend RTK2758 B307173Total-Seq C rat lgG2b 400677 BioLegend RTK4530 B320446Total-Seq C Arm Hamster 400977 BioLegend HTK888 B313973 HashtagsTOTALSEQ™-C0301 anti- mouse155861 BioLegendM1/42;30-F11B325958 HashtagsTOTALSEQ™-C0302 anti- mouse155863 BioLegendM1/42;30-F12B331515 HashtagsTOTALSEQ™-C0303 anti- mouse155865 BioLegendM1/42;30-F13B332386 HashtagsTOTALSEQ™-C0304 anti- mouse155867 BioLegendM1/42;30-F14B296942 111 WO 2024/186790 PCT/US2024/018472 HashtagsTOTALSEQ™-C0305 anti- mouse155869 BioLegendM1/42;30-F15B339941 HashtagsTOTALSEQ™-C0306 anti- mouse155871 BioLegendM1/42;30-F16B322559 HashtagsTOTALSEQ™-C0307 anti- mouse155873 BioLegendM1/42;30-F17B323978 HashtagsTOTALSEQ™-C0308 anti- mouse155875 BioLegendM1/42;30-F18B322555 HashtagsTOTALSEQ™-C0309 anti- mouse155877 BioLegendM1/42;30-F19B328421 HashtagsTOTALSEQ™-C0310 anti- mouse155879 BioLegendM1/42;30-F20B322104 Table 6. pMHC monomers Fluorophore Antigen Streptavidin BioLegend Catalog Number PE gp70 405265PE gp70 405267APC CT26 neoantigen 1 405283APC CT26 neoantigen 1 405285PE CT26 neoantigen 2 405269PE CT26 neoantigen 2 405155none CT26 neoantigen 2 405271PE UV irrelevant 405153PE UV irrelevant 405299none UV irrelevant 405271ABC UV irrelevant 405293BV650 p15E 405232 Single-cell RNA-seq and TCR V(D)J clonotype profiling Processing for single-cell expression (scRNA-seq) and T cell receptor V(D)J clonotypes (scTCR-seq) was done using the Chromium Single Cell 5’ Library and Gel Bead Kit (10x Genomics), following manufacturer’s instructions. Cell density and viability from each mouse tissue of FACS-sorted CD90+ T cells from tumor and blood, or CD90+CD44+T cells from draining lymph nodes, were determined by hemacytometer. Approximately 6,000-10,000 cells per sample were used for the reverse transcriptionmastermix. A total of 14 cycles of PCR amplification was performed to obtain sufficient cDNAs used for both RNA-seq library generation and TCR V(D)J targeted enrichment followed by V(D)J library generation after Gel Bead-in-Emulsion reverse transcription (GEM-RT) reaction and clean-up. TCR V(D)J enrichment was done per manufacturer’s user guide using Chromium Single Cell V(D) J Enrichment Kit, Human T cell (1 Ox Genomics). Libraries for RNA-seq and V(D)J were prepared following the 112 WO 2024/186790 PCT/US2024/018472 manufacturer’s user guide (1 Ox Genomics), then profiled using Bioanalyzer High Sensitivity DNA kit (Agilent Technologies) and quantified with Qubit (Thermo Fisher Scientific). scRNA-seq libraries were sequenced in one lane of HiSeq4000 (Illumina). scTCR V(D)J libraries were tagged with a sample barcode for multiplexed pooling with other libraries, sequenced in both lanes of a HiSeq2500 machine (Illumina) using Rapid Run mode, and then demultiplexed. All sequencing was done according to the manufacturer’s specification (10x Genomics).

Pre-processing of single-cell data Sequencing files from Illumina assays were run through CellRanger version 6.1.1 against a transcriptome derived from ENSEMBL version 2.2.0 for the mouse genome GRCm38. The combined matrix files from the filtered_feature_bc_matrix directory for the RNA and ADT libraries were divided into separate submatrices for each sample, based on 52,636 genes for expression, 12 tetramer barcodes for ADT counts, 24 antibody measurements for CITE-seq, and 10 barcodes for multiplexing of the blood samples. Measurements corresponding to various alleles of T cell receptor genes (e.g., Trbvl through Trbv31) were combined into a single gene measurement (Trbv). Since blood samples were pooled from several mice based on an encoding scheme that used two multiplex barcodes to identify each mouse, single cells were de-multiplexed using the two multiplex barcodes with highest counts. In cases of a tie for the second highest multiplex count (4.6% of cells), those single cells could not be assigned to a particular mouse using this method. TOR sequence data from the filtered_contig_annotations.csv files were processed using a custom script that identified clones across multiple tissues in each mouse, based on identical sets of alpha and beta sequences. To handle the blood cells that could not be assigned using the multiplex counts, blood cells with a TOR nucleotide sequence uniquely matching a cell from lymph node or tumor of a mouse in the pool were assigned to the corresponding mouse. ADT barcodes came from 12 distinct tetramers, of which 2 had gp70 antigens and the remaining 10 had a non-gpantigen (C28, UV, or C142). A cell was assigned to an antigen based on its ADT barcode with the highest count, and cells were not assigned in cases of ties.

Integration of single-cell expression data Analysis was performed in the statistical language R version 4.2.0 and with scripts written for Perl version 5.16.3. Single-cell expression matrices were processed using Seurat version 4.1.1 using the SCTransform function (unless specified otherwise, Seurat functions were run using default parameters). All samples were combined into a single Seurat object using a hierarchical process, first combining the tumor and lymph node samples from each mouse, and then combining these objects from all mice in each experimental group. The combinations were performed using the FindlntegrationAnchors function, using k.filter=200, and IntegrateData. The four pooled blood samples were obtained from two batches of mice at days 0 and 7, and these were combined from the same batch, and then together. Finally, the five group objects and single blood object were combined. This hierarchical process was required to avoid computational difficulties arising from otherwise trying to combine 52 separate samples and to ensure that biologically similar samples were matched first. 113 WO 2024/186790 PCT/US2024/018472 Isolation of CDS expression data The combination process above yielded expression data on 305,909 T cells. This data was clustered using the ScaleData, RunPCA, RunUMAP, FindNeighbor, and FindCluster functions from Seurat, yielding 22 clusters, of which 10 were identified as CD8+, 6 as CD4+, and 6 as contaminant clusters, with these identifications confirmed using the CITE-seq data. To obtain better resolution and a clustering that was not affected by the CD4+ T cells, the 174,515 single cells belonging to the 10 CD8+ clusters were isolated, then a clustering of that data was performed to yield 15 phenotypic clusters. Plotting of the CITE-seq data on the UMAR plots revealed a subregion within the Ccl5.1 cluster that was high in CD226 surface protein expression. A separate Cxcr3 cluster was created from cells in this region having CD226 expression above the 90th percentile.

Correspondences with clusters from external single-cell datasets Single-cell RNA-seq datasets generated or analyzed from five previous published datasets were obtained, using raw counts from NCBI GEO (Gene Expression Omnibus) unless specified otherwise: GSM5452712 and GSM5452714 from GSE180094 (Huang et al., Cell, 185: 1-18, 2022), GSE1227(Miller et al., Nat Immunol, 20(3): 326-336, 2019), GSM5530561 and GSM5530563 from GSE1825(processed data) (Connolly et al., Sol Immunol, 6: eabg7836, 2021), and GSM4618806 from GSE1526for the analysis by Huang et al. (Ce//, 185: 1 -18, 2022); the LCMV samples from GSE188666 (Daniel et al., Nat Immunol, 23: 1614-1627, 2022); E-MTAB-1 1773 from ArrayExpress (Deak et al., Nature, 610: 173-181,2022); GSE206739 (Hashimoto et al., Nature, 610: 173-181,2022); and GSE193654 (Li et al., J Exp Med, 219: 620210749, 2022). For the Daniel et al. study (Nat Immunol, 23: 1614-1627, 2022), metadata with cluster assignments of individual cell barcodes was obtained from the NCBI GEO repository; for all other studies, metadata was obtained by direct communication with the authors. The metadata were used to create centroids of each of the published clusters by normalizing each cell by its total count to yield a value in transcripts per million and adding 1 as a pseudocount (tpm); computing a trimmed mean of the tpm for each gene, rejecting 10% of measurements from each end of the range; and taking the logarithm base 2. These centroids were used as reference gene signatures to assign each cell from the dataset, where genes with zero expression across an entire sample were excluded, gene expression for each cell was converted to log2(tpm+1), and assignment was performed by the SingleR package in R, using default parameters. Assignments between the two clustering schemes were cross- tabulated, and normalized by the total counts for each of the clusters.

Clone-based migration and differentiation trajectories To compute trajectories of T cells within clonal lineages, a trajectory method that utilizes clone information from scTCR-seq data was developed. First, a co-occurrence matrix was computed, with rows and columns corresponding to each phenotypic cluster in each tissue type. Each clone was processed with ncells, where n > 1,by normalizing its distribution of cells across cluster/tissue groups as a vector x, whose sum is 1. Each clone contributes to the co-occurrence matrix with its weighted outer product nxxT, where the outer product represents the frequency of any two cluster/tissue pairs occurring in the same clone, and weighting by nemphasizes data from larger clones. The resulting co-occurrence matrix is 114 WO 2024/186790 PCT/US2024/018472 then analyzed for differentiation within each cluster by computing a minimum-spanning tree, where values from the co-occurrence matrix are considered as edges between the nodes represented on the rows and columns of the matrix. Migration between two tissues is computed as the edge weights between corresponding phenotypic clusters from the two tissues.

Projection of human CD8+ T cells from a Ph1b scRNAseq dataset to a mouse reference Human genes from the Ph1b scRNAseq data were first converted to their mouse orthologs using babelgene (version 22.9). Human genes without mouse orthologs or with mouse orthologs not present in the mouse scRNAseq dataset were left unmodified without renaming. Human CD8+ T cells were then separated by patient and normalized with SCTransform in Seurat (version 4.2) using the default parameters. These samples were then integrated using reference-based integration to overcome the memory limits of canonical correlation analysis (CCA) integration. T he second patient in the dataset was chosen at random as the integration reference. After integration, transfer anchors were identified between the query human CD8+ T cell dataset and the mouse CD8+ T cell reference. The MapQuery function in Seurat was used to transfer cell type labels, integrate embeddings, and to project the query data onto the reference UMAP.

Gene signature scores for CITYSCAPE The top 20 differentially expressed genes in each of the mouse CD8+ T cell clusters identified from scRNAseq were converted to their human orthologs using babelgene (version 22.9) in R (4.2.0). Mouse genes that did not have human orthologs or with human orthologs that were not present in the CITYSCAPE dataset were removed. The final curated table of signature genes used for analysis are in Table 4.

Analysis of CITYSCAPE and OAK clinical trial data CITYSCAPE (NCT01903993) is a phase 2 trial investigating tiragolumab with atezolizumab compared to placebo with atezolizumab in patients with locally advanced or metastatic NSCLC (Cho et al., Lancet Oncol, 23: 781 -792, 2022). Patients were treated until disease progression or loss of clinical benefit. Patient tumor samples were submitted for RNAseq and the average, log-normalized expression of the genes in Table 4 or selected genes as indicated in the text was used to define gene signature scores. Objective response was categorized according to RECIST (version 1.1). For Kaplan-Meier (KM) survival curves and hazard ratios, patients in the CITYSCAPE trial were separated by treatment group and further sub-divided by high or low expression of individual genes or gene signatures, where high or low is defined as at or above or below the global median expression, respectively, of that gene or gene signature score. The survminer package (version 0.4.9), survival package (version 3.4-0) and R (version 4.2.0) were used to generate the KM plot. A log-rank test was used for statistical testing on the survival data. A Cox proportional hazards regression model was fit on gene or gene signature score high or low data and the hazard ratio and 95% confidence interval for overall survival calculated and plotted for patients receiving tiragolumab with atezolizumab compared to patients receiving placebo with 115 WO 2024/186790 PCT/US2024/018472 atezolizumab. Similarly, KM survival curves for PFS and OS were generated for the phase 3 OAK study (NCT02008227) evaluating atezolizumab versus chemotherapy in PD-L1-positive previously treated patients with advanced or metastatic NSCLC.

Statistical Analysis Data were analyzed using GraphPad Prism software version 9 (GraphPad, San Diego, CA). Measures between two groups were performed with a Student’s t test (two-tailed). Groups of three or more were analyzed by one-way or two-way analysis of variance (ANOVA) followed by Tukey’s post- testing for multiple comparisons, as appropriate.Although the foregoing invention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference. 116

Claims (203)

WO 2024/186790 PCT/US2024/018472 What is claimed is: CLAIMS

1. A method of identifying an individual having a non-small cell lung cancer (NSCLC) who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual, wherein an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

2. A method for selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual, wherein an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

3. The method of claim 1 or 2, wherein the individual has an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab.

4. A method of treating an individual having a NSCLC, the method comprising:(a) detecting an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual, wherein the expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6 and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and(b) administering an effective amount of atezolizumab and tiragolumab to the individual.

5. A method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

6. The method of any one of claims 1 -5, wherein the benefit is a clinical response.

7. The method of claim 6, wherein the clinical response is a complete response (CR) or a partial response (PR). 117 WO 2024/186790 PCT/US2024/018472

8. The method of any one of claims 1 -7, wherein the individual has an expression level of one or more of CCL5, CXCR3, and CCR7 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CCR7 and the benefit is an increase in overall survival (OS) hazard ratio (HR).

9. The method of any one of claims 1 -7, wherein the individual has an expression level of one or more of CCL5, CXCR3, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CXCR6 and the benefit is an increase in overall survival (OS).

10. The method of any one of claims 1 -9, wherein the reference expression level of CCL5, CXCR3, or CXCR6 is a pre-assigned expression level.

11. The method of any one of claims 1 -10, wherein the reference expression level of CCL5, CXCR3, or CXCR6 is an expression level in a reference population.

12. The method of claim 11, wherein the expression level in the reference population is the median expression level of CCL5, CXCR3, or CXCR6 in the reference population.

13. The method of claim 11 or 12, wherein the reference population is a population of individuals having the NSCLC.

14. A method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

15. A method for selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

16. The method of claim 14 or 15, wherein the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab.

17. A method of treating an individual having a NSCLC, the method comprising:(a) detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or 118 WO 2024/186790 PCT/US2024/018472 above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and(b) administering an effective amount of atezolizumab and tiragolumab to the individual.

18. A method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCRdetected in a sample from the individual.

19. The method of any one of claims 14-18, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.

20. The method of any one of claims 14-17, wherein the method comprises further detecting the expression level of CCR7 in the sample from the individual.

21. The method of claim 20, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

22. The method of claim 18, wherein the expression level of CCR7 has been detected in the sample from the individual.

23. The method of claim 22, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

24. The method of any one of claims 14-23, wherein the benefit is a clinical response.

25. The method of claim 24, wherein the clinical response is a CR or a PR.

26. The method of any one of claims 14-25, wherein the benefit is an increase in OS HR.

27. The method of any one of claims 14-26, wherein the benefit is an increase in OS.

28. The method of any one of claims 14-27, wherein the reference gene signature score is a pre-assigned gene signature score.

29. The method of any one of claims 14-28, wherein the reference gene signature score is a gene signature score in a reference population. 119 WO 2024/186790 PCT/US2024/018472

30. The method of claim 29, wherein the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population.

31. The method of claim 29 or 30, wherein the reference population is a population of individuals having the NSCLC.

32. A method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab and tiragolumab, the method comprising:(a) detecting an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1 , RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual and determining a Ccr7.2 gene signature score therefrom;(b) detecting an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS1 0, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB in a sample from the individual and determining a Ccr7.3 gene signature score therefrom;(c) detecting an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual and determining a Cxcr3 gene signature score therefrom;(d) detecting an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual and determining a Ccl5.1 gene signature score therefrom;(e) detecting an expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1 in a sample from the individual and determining an Ifit gene signature score therefrom;(f) detecting an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual and determining a Mitotic gene signature score therefrom;(g) detecting an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT in a sample from the individual and determining a Cytotox.2 gene signature score therefrom; or(h) detecting an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual and determining a Cytotox.4 gene signature score therefrom;wherein (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab,and (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab. 120 WO 2024/186790 PCT/US2024/018472

33. A method for selecting a therapy for an individual having a NSCLC, the method comprising:(a) detecting an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1 , RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual and determining a Ocr7.2 gene signature score therefrom;(b) detecting an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS1 0, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB in a sample from the individual and determining a Ocr7.3 gene signature score therefrom;(c) detecting an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual and determining a Cxcr3 gene signature score therefrom;(d) detecting an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual and determining a Ccl5.1 gene signature score therefrom;(e) detecting an expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1 in a sample from the individual and determining an Ifit gene signature score therefrom;(f) detecting an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual and determining a Mitotic gene signature score therefrom;(g) detecting an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT in a sample from the individual and determining a Cytotox.2 gene signature score therefrom; or(h) detecting an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual and determining a Cytotox.4 gene signature score therefrom;wherein (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab,and (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

34. The method of claim 32 or 33, wherein the individual has (i) a Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab and tiragolumab. 121 WO 2024/186790 PCT/US2024/018472

35. A method of treating an individual having a NSCLC, the method comprising:(i)(a) detecting an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1 , RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual and determining a Ccr7.2 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(b) detecting an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS1 0, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1R, INPP4B, and RFLNB in a sample from the individual and determining a Ccr7.3 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(c) detecting an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual and determining a Cxcr3 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(d) detecting an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual and determining a Ccl5.1 gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(e) detecting an expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFI16, and IFIH1 in a sample from the individual and determining an Ifit gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(f) detecting an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual and determining a Mitotic gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab;(g) detecting an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT in a sample from the individual and determining a Cytotox.2 gene signature score therefrom, wherein the gene signature score is below a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; or(h) detecting an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual and determining a Cytotox.4 gene signature score therefrom, wherein the 122 WO 2024/186790 PCT/US2024/018472 gene signature score is below a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab; and (ii)administering an effective amount of atezolizumab and tiragolumab to the individual.

36. A method of treating an individual having a NSCLC, the method comprising administering atezolizumab and tiragolumab to the individual, wherein the individual has been determined to have:(a) a Ccr7.2 gene signature score based on an expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X in a sample from the individual that is at or above a reference Ccr7.2 gene signature score;(b) a Ccr7.3 gene signature based on an expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB in a sample from the individual that is at or above a reference Ccr7.3 gene signature score;(c) a Cxcr3 gene signature score based on an expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, SERINC3, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 in a sample from the individual that is at or above a reference Cxcr3 gene signature score;(d) a Ccl5.1 gene signature score based on an expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 in a sample from the individual that is at or above a reference Ccl5.1 gene signature score;(e) an Ifit gene signature score based on an expression level of each of ISG15, IFIT1B, I FITS, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 in a sample from the individual that is at or above a reference Ifit gene signature score;(f) a Mitotic gene signature score based on an expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL in a sample from the individual that is at or above a reference Mitotic gene signature score;(g) a Cytotox.2 gene signature score based on an expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1 B, LY6E, and DUT in a sample from the individual that is below a reference Cytotox.2 gene signature score; or(h) a Cytotox.4 gene signature score based on an expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 in a sample from the individual that is below a reference Cytotox.gene signature score;thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab. 123 WO 2024/186790 PCT/US2024/018472

37. The method of any one of claims 32-36, wherein the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.

38. The method of any one of claims 32-37, wherein the individual has a Ccr7.3, Cxcr3, or Ccl5.gene signature score in the sample that is at or above a reference gene signature score and the benefit is a clinical response.

39. The method of claim 38, wherein the clinical response is a CR or a PR.

40. The method of any one of claims 32-37, wherein the individual has (i) a Ccr7.2, Cxcr3, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score and the benefit is an increase in OS HR.

41. The method of any one of claims 32-37, wherein the individual has a Ccr7.2, Ccr7.3, or Cxcrgene signature score in the sample that is at or above a reference gene signature score and the benefit is an increase in OS.

42. The method of any one of claims 32-41, wherein the reference gene signature score is a pre- assigned gene signature score.

43. The method of any one of claims 32-42, wherein the reference gene signature score is a gene signature score in a reference population.

44. The method of claim 43, wherein the gene signature score in the reference population is the median Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population.

45. The method of claim 43 or 44, wherein the reference population is a population of individuals having the NSCLC.

46. A method of identifying an individual having a NSCLC who may benefit from a treatment comprising atezolizumab, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab. 124 WO 2024/186790 PCT/US2024/018472

47. A method for selecting a therapy for an individual having a NSCLC, the method comprising detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein a gene signature score that is at or above a reference gene signature score identifies the individual as one who may benefit from a treatment comprising atezolizumab.

48. The method of claim 46 or 47, wherein the individual has a gene signature score in the sample that is at or above a reference gene signature score, and the method further comprises administering to the individual an effective amount of atezolizumab.

49. A method of treating an individual having a NSCLC, the method comprising:(a) detecting an expression level of each of CCL5, CXCR3, and CXCR6 in a sample from the individual and determining a gene signature score therefrom, wherein the gene signature score is at or above a reference gene signature score and thereby identifies the individual as one who may benefit from a treatment comprising atezolizumab; and(b) administering an effective amount of atezolizumab to the individual.

50. A method of treating an individual having a NSCLC, the method comprising administering atezolizumab to the individual, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, and wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

51. The method of any one of claims 46-50, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.

52. The method of any one of claims 46-49, wherein the method comprises further detecting the expression level of CCR7 in the sample from the individual.

53. The method of claim 52, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

54. The method of claim 50, wherein the expression level of CCR7 has been detected in the sample from the individual.

55. The method of claim 54, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

56. The method of any one of claims 46-55, wherein the benefit is an increase in progression-free survival (PFS) or OS. 125 WO 2024/186790 PCT/US2024/018472

57. The method of any one of claims 46-56, wherein the reference gene signature score is a pre- assigned gene signature score.

58. The method of any one of claims 46-57, wherein the reference gene signature score is a gene signature score in a reference population.

59. The method of claim 58, wherein the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population.

60. The method of claim 58 or 59, wherein the reference population is a population of individuals having the NSCLC.

61. The method of any one of claims 46-60, wherein the treatment comprising atezolizumab is atezolizumab monotherapy.

62. The method of any one of claims 1 -61, wherein the expression level is a nucleic acid expression level or a protein expression level.

63. The method of claim 62, wherein the expression level is a nucleic acid expression level.

64. The method of claim 63, wherein the nucleic acid expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof.

65. The method of claim 63 or 64, wherein the nucleic acid expression level is an mRNA expression level.

66. The method of claim 65, wherein the mRNA expression level is determined by RNA-seq.

67. The method of claim 62, wherein the expression level is a protein expression level.

68. The method of claim 67, wherein the protein expression level is determined by massspectrometry.

69. The method of any one of claims 1 -68, wherein the sample is obtained from the individual prior to treatment with atezolizumab and/or tiragolumab. 126 WO 2024/186790 PCT/US2024/018472

70. The method of any one of claims 1 -69, wherein the sample is a tissue sample, a tumor sample, a blood sample, a plasma sample, a serum sample, or a combination thereof.

71. The method of claim 70, wherein the sample is a tissue sample.

72. The method of claim 71, wherein the tissue sample is a tumor tissue sample.

73. The method of claim 72, wherein the tumor tissue sample is a biopsy.

74. The method of claim 71, wherein the tissue sample is a tumor draining lymph node (dLN)sample.

75. The method of claim 70, wherein the sample is a blood sample.

76. The method of any one of claims 70-75, wherein the sample is an archival sample, a fresh sample, or a frozen sample.

77. The method of any one of claims 1 -76, wherein the individual has a PD-L1 -positive NSCLC.

78. The method of claim 77, wherein the PD-L1 -positive NSCLC has been determined to have a PD- L1 -positive tumor cell fraction by an immunohistochemical (IHC) assay.

79. The method of claim 78, wherein the PD-L1 -positive tumor cell fraction is determined by positive staining with an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is SP263, 22C3, SP142, or 28-8.

80. The method of any one of claims 1 -79, wherein the individual is a human.

81. The method of any one of claims 1 -80, wherein the individual has not previously been treated for NSCLC.

82. Use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

83. The use of claim 82, wherein the benefit is a clinical response.

84. The use of claim 83, wherein the clinical response is a complete response (CR) or a partial response (PR). 127 WO 2024/186790 PCT/US2024/018472

85. The use of any one of claims 82-84, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and CCR7 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CCR7 and the benefit is an increase in overall survival (OS) hazard ratio (HR).

86. The use of any one of claims 82-85, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CXCR6 and the benefit is an increase in overall survival (OS).

87. The use of any one of claims 82-86, wherein the reference expression level of CCL5, CXCR3, or CXCR6 is a pre-assigned expression level.

88. The use of any one of claims 82-87, wherein the reference expression level of CCL5, CXCR3, or CXCR6 is an expression level in a reference population.

89. The use of claim 88, wherein the expression level in the reference population is the median expression level of CCL5, CXCR3, or CXCR6 in the reference population.

90. The use of claim 88 or 89, wherein the reference population is a population of individuals having the NSCLC.

91. Use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

92. The use of claim 91, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.

93. The use of claim 91 or 92, wherein the expression level of CCR7 has been detected in the sample from the individual.

94. The use of claim 93, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

95. The use of any one of claims 91 -94, wherein the benefit is a clinical response. 128 WO 2024/186790 PCT/US2024/018472

96. The use of claim 95, wherein the clinical response is a CR or a PR.

97. The use of any one of claims 91 -96, wherein the benefit is an increase in OS HR.

98. The use of any one of claims 91 -97, wherein the benefit is an increase in OS.

99. The use of any one of claims 91 -98, wherein the reference gene signature score is a pre-assigned gene signature score.

100. The use of any one of claims 91 -99, wherein the reference gene signature score is a gene signature score in a reference population.

101. The use of claim 100, wherein the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population.

102. The use of claim 100 or 101, wherein the reference population is a population of individuals having the NSCLC.

103. Use of atezolizumab and/or tiragolumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have: (i)(a) a Ccr7.2 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X detected in a sample from the individual;(b) a Ccr7.3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB detected in a sample from the individual;(c) a Cxcr3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 detected in a sample from the individual;(d) a Ccl5.1 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and 129 WO 2024/186790 PCT/US2024/018472 tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HOST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 detected in a sample from the individual;(e) an Ifit gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 detected in a sample from the individual;(f) a Mitotic gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL detected in a sample from the individual;(g) a Cytotox.2 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT detected in a sample from the individual; or(h) a Cytotox.4 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 detected in a sample from the individual.

104. The use of claim 103, wherein the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.

105. The use of claim 103 or 104, wherein the individual has been determined to have a Ccr7.3, Cxcr3, or Ccl5.1 gene signature score in the sample that is at or above a reference gene signature score and the benefit is a clinical response.

106. The use of claim 105, wherein the clinical response is a CR or a PR.

107. The use of claim 103 or 104, wherein the individual has been determined to have (i) a Ccr7.2, Cxcr3, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score and the benefit is an increase in OS HR. 130 WO 2024/186790 PCT/US2024/018472

108. The use of claim 103 or 104, wherein the individual has been determined to have a Ccr7.2, Ccr7.3, or Cxcr3 gene signature score in the sample that is at or above a reference gene signature score and the benefit is an increase in OS.

109. The use of any one of claims 103-108, wherein the reference gene signature score is a pre- assigned gene signature score.

110. The use of any one of claims 103-109, wherein the reference gene signature score is a gene signature score in a reference population.

111. The use of claim 110, wherein the gene signature score in the reference population is the median Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.4 gene signature score in the reference population.

112. The use of claim 110 or 111, wherein the reference population is a population of individuals having the NSCLC.

113. Use of atezolizumab in the manufacture of a medicament for the treatment of an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

114. The use of claim 113, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.

115. The use of claim 113 or 114, wherein the expression level of CCR7 has been detected in the sample from the individual.

116. The use of claim 115, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

117. The use of any one of claims 113-116, wherein the benefit is an increase in progression-free survival (PFS) or OS.

118. The use of any one of claims 113-117, wherein the reference gene signature score is a pre- assigned gene signature score.

119. The use of any one of claims 113-118, wherein the reference gene signature score is a gene signature score in a reference population. 131 WO 2024/186790 PCT/US2024/018472

120. The use of claim 119, wherein the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population.

121. The use of claim 119 or 120, wherein the reference population is a population of individuals having the NSCLC.

122. The use of any one of claims 113-121, wherein the treatment comprising atezolizumab is atezolizumab monotherapy.

123. The use of any one of claims 82-122, wherein the expression level is a nucleic acid expression level or a protein expression level.

124. The use of claim 123, wherein the expression level is a nucleic acid expression level.

125. The use of claim 124, wherein the nucleic acid expression level is determined by RNA-seq, RT- qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof.

126. The use of claim 124 or 125, wherein the nucleic acid expression level is an mRNA expression level.

127. The use of claim 126, wherein the mRNA expression level is determined by RNA-seq.

128. The use of claim 123, wherein the expression level is a protein expression level.

129. The use of claim 128, wherein the protein expression level is determined by mass spectrometry.

130. The use of any one of claims 82-129, wherein the sample is obtained from the individual prior totreatment with atezolizumab and/or tiragolumab.

131. The use of any one of claims 82-130, wherein the sample is a tissue sample, a tumor sample, a blood sample, a plasma sample, a serum sample, or a combination thereof.

132. The use of claim 131, wherein the sample is a tissue sample.

133. The use of claim 132, wherein the tissue sample is a tumor tissue sample.

134. The use of claim 133, wherein the tumor tissue sample is a biopsy. 132 WO 2024/186790 PCT/US2024/018472

135. The use of claim 132, wherein the tissue sample is a tumor draining lymph node (dLN) sample.

136. The use of claim 131, wherein the sample is a blood sample.

137. The use of any one of claims 131-136, wherein the sample is an archival sample, a fresh sample, or a frozen sample.

138. The use of any one of claims 82-137, wherein the individual has a PD-L1-positive NSCLC.

139. The use of claim 138, wherein the PD-L1-positive NSCLC has been determined to have a PD- L1 -positive tumor cell fraction by an immunohistochemical (IHC) assay.

140. The use of claim 139, wherein the PD-L1 -positive tumor cell fraction is determined by positive staining with an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is SP263, 22C3, SP142, or 28-8.

141. The use of any one of claims 82-140, wherein the individual is a human.

142. The use of any one of claims 82-141, wherein the individual has not previously been treated for NSCLC.

143. Atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, CCR7, and CXCR6 in a sample from the individual that is at or above a reference expression level of CCL5, CXCR3, CCR7, or CXCR6, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab.

144. The atezolizumab and/or tiragolumab for use of claim 143, wherein the benefit is a clinical response.

145. The atezolizumab and/or tiragolumab for use of claim 144, wherein the clinical response is a complete response (CR) or a partial response (PR).

146. The atezolizumab and/or tiragolumab for use of any one of claims 143-145, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and CCRin the sample that is at or above a reference expression level of CCL5, CXCR3, or CCR7 and the benefit is an increase in overall survival (OS) hazard ratio (HR).

147. The atezolizumab and/or tiragolumab for use of any one of claims 143-146, wherein the individual has been determined to have an expression level of one or more of CCL5, CXCR3, and 133 WO 2024/186790 PCT/US2024/018472 CXCR6 in the sample that is at or above a reference expression level of CCL5, CXCR3, or CXCR6 and the benefit is an increase in overall survival (OS).

148. The atezolizumab and/or tiragolumab for use of any one of claims 143-147, wherein the reference expression level of CCL5, CXCR3, or CXCR6 is a pre-assigned expression level.

149. The atezolizumab and/or tiragolumab for use of any one of claims 143-148, wherein the reference expression level of CCL5, CXCR3, or CXCR6 is an expression level in a reference population.

150. The atezolizumab and/or tiragolumab for use of claim 149, wherein the expression level in the reference population is the median expression level of CCL5, CXCR3, or CXCR6 in the reference population.

151. The atezolizumab and/or tiragolumab for use of claim 149 or 150, wherein the reference population is a population of individuals having the NSCLC.

152. Atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

153. The atezolizumab and/or tiragolumab for use of claim 152, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.

154. The atezolizumab and/or tiragolumab for use of claim 152 or 153, wherein the expression level of CCR7 has been detected in the sample from the individual.

155. The atezolizumab and/or tiragolumab for use of claim 154, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

156. The atezolizumab and/or tiragolumab for use of any one of claims 152-155, wherein the benefit is a clinical response.

157. The atezolizumab and/or tiragolumab for use of claim 156, wherein the clinical response is a CR or a PR.

158. The atezolizumab and/or tiragolumab for use of any one of claims 152-157, wherein the benefit is an increase in OS HR. 134 WO 2024/186790 PCT/US2024/018472

159. The atezolizumab and/or tiragolumab for use of any one of claims 152-158, wherein the benefit is an increase in OS.

160. The atezolizumab and/or tiragolumab for use of any one of claims 152-159, wherein the reference gene signature score is a pre-assigned gene signature score.

161. The atezolizumab and/or tiragolumab for use of any one of claims 152-160, wherein the reference gene signature score is a gene signature score in a reference population.

162. The atezolizumab and/or tiragolumab for use of claim 161, wherein the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population.

163. The atezolizumab and/or tiragolumab for use of claim 161 or 162, wherein the reference population is a population of individuals having the NSCLC.

164. Atezolizumab and/or tiragolumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have:(i)(a) a Ccr7.2 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of STAT1, LEF1, IRGM, CCR7, SELL, RPS24, RPS27, GBP2, RPS29, RPS3A, RPS20, KLF2, RPLP1, RPL13, DAPL1, SMC6, RFLNB, and RPS4X detected in a sample from the individual;(b) a Ccr7.3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of DAPL1, CCR7, SMC4, RGCC, MXD4, CD8A, TCF7, ITGAE, RGS10, ZNRF1, CHD3, CD52, DDIT4, LEF1, IZUMO1 R, INPP4B, and RFLNB detected in a sample from the individual;(c) a Cxcr3 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CXCR6, CKB, GIMAP5, ID2, LTB, FGL2, RAMP1, LYST, ASB2, IL2RB, CXCR3, BERINGS, INPP4B, ANXA1, XCL2, SOCS2, CD82, CD4, and GIMAP7 detected in a sample from the individual;(d) a Ccl5.1 gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of CCL5, ITGB1, BTG1, GZMK, LTB, IL7R, ZFP36L2, ITGA4, TXNIP, SLAMF6, HCST, ETS1, CXCR3, MS4A4A, DGKA, and YPEL3 detected in a sample from the individual; 135 WO 2024/186790 PCT/US2024/018472 (e) an Ifit gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of ISG15, IFIT1B, IFIT3, ISG20, IFI27L2, SAMHD1, ZBP1, SLFN5, IRF7, RTP4, USP18, PHF11, LGALS3BP, BST2, GBP2, IFITM3, STAT1, IFH6, and IFIH1 detected in a sample from the individual;(f) a Mitotic gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HSP90AB1, PTMA, XCL2, ODC1, TNFRSF9, ITM2A, MYC, YBX3, HSPA5, GPX1, RPS12, TUBA1B, MCM6, TUBB, MCM3, TPI1, MCM5, DUT, and FTL detected in a sample from the individual;(g) a Cytotox.2 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of HMGB2, TOP2A, MKI67, RRM2, PCLAF, TUBB, BIRC5, LMNB1, UBE2C, PTMA, STMN1, HMGN2, TUBA1B, LY6E, and DUT detected in a sample from the individual; or(h) a Cytotox.4 gene signature score that is below a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab and tiragolumab, wherein the gene signature score is based on the expression level of each of S100A6, NRN1, CXCR6, KLRC1, PDCD1, LGALS1, LAGS, ITGB1, ID2, LGALS3, NRGN, CST7, NKG7, S100A10, TNFRSF9, CD52, VIM, and IFITM2 detected in a sample from the individual.

165. The atezolizumab and/or tiragolumab for use of claim 164, wherein the gene signature score is an average of the expression levels of the members of the gene signature in the sample from the individual.

166. The atezolizumab and/or tiragolumab for use of claim 164 or 165, wherein the individual has been determined to have a Ccr7.3, Cxcr3, or Ccl5.1 gene signature score in the sample that is at or above a reference gene signature score and the benefit is a clinical response.

167. The atezolizumab and/or tiragolumab for use of claim 166, wherein the clinical response is a CR or a PR.

168. The atezolizumab and/or tiragolumab for use of claim 164 or 165, wherein the individual has been determined to have (i) a Ccr7.2, Cxcr3, Ifit, or Mitotic gene signature score in the sample that is at or above a reference gene signature score or (ii) a Cytotox.2 or Cytotox.4 gene signature score that is below a reference gene signature score and the benefit is an increase in OS HR.

169. The atezolizumab and/or tiragolumab for use of claim 164 or 165, wherein the individual has been determined to have a Ccr7.2, Ccr7.3, or Cxcr3 gene signature score in the sample that is at or above a reference gene signature score and the benefit is an increase in OS. 136 WO 2024/186790 PCT/US2024/018472

170. The atezolizumab and/or tiragolumab for use of any one of claims 164-169, wherein the reference gene signature score is a pre-assigned gene signature score.

171. The atezolizumab and/or tiragolumab for use of any one of claims 164-170, wherein the reference gene signature score is a gene signature score in a reference population.

172. The atezolizumab and/or tiragolumab for use of claim 171, wherein the gene signature score in the reference population is the median Ccr7.2, Ccr7.3, Cxcr3, Ccl5.1, Ifit, Mitotic, Cytotox.2, or Cytotox.gene signature score in the reference population.

173. The atezolizumab and/or tiragolumab for use of claim 171 or 172, wherein the reference population is a population of individuals having the NSCLC.

174. Atezolizumab for use in treating an individual having a NSCLC, wherein the individual has been determined to have a gene signature score that is at or above a reference gene signature score, thereby identifying the individual as one who may benefit from a treatment comprising atezolizumab, wherein the gene signature score is based on the expression level of each of CCL5, CXCR3, and CXCR6 detected in a sample from the individual.

175. The atezolizumab for use of claim 174, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, and CXCR6 in the sample from the individual.

176. The atezolizumab for use of claim 174 or 175, wherein the expression level of CCR7 has been detected in the sample from the individual.

177. The atezolizumab for use of claim 176, wherein the gene signature score is an average of the expression levels of CCL5, CXCR3, CXCR6, and CCR7 in the sample from the individual.

178. The atezolizumab for use of any one of claims 174-177, wherein the benefit is an increase in progression-free survival (PFS) or OS.

179. The atezolizumab for use of any one of claims 174-178, wherein the reference gene signature score is a pre-assigned gene signature score.

180. The atezolizumab for use of any one of claims 174-179, wherein the reference gene signature score is a gene signature score in a reference population. 137 WO 2024/186790 PCT/US2024/018472

181. The atezolizumab for use of claim 180, wherein the gene signature score in the reference population is the median gene signature score based on the expression level of each of CCL5, CXCR3, and CXCR6 in the reference population.

182. The atezolizumab for use of claim 180 or 181, wherein the reference population is a population of individuals having the NSCLC.

183. The atezolizumab for use of any one of claims 174-182, wherein the treatment comprising atezolizumab is atezolizumab monotherapy.

184. The atezolizumab and/or tiragolumab for use of any one of claims 143-183, wherein the expression level is a nucleic acid expression level or a protein expression level.

185. The atezolizumab and/or tiragolumab for use of claim 184, wherein the expression level is a nucleic acid expression level.

186. The atezolizumab and/or tiragolumab for use of claim 185, wherein the nucleic acid expression level is determined by RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof.

187. The atezolizumab and/or tiragolumab for use of claim 185 or 186, wherein the nucleic acid expression level is an mRNA expression level.

188. The atezolizumab and/or tiragolumab for use of claim 187, wherein the mRNA expression level is determined by RNA-seq.

189. The atezolizumab and/or tiragolumab for use of claim 184, wherein the expression level is a protein expression level.

190. The atezolizumab and/or tiragolumab for use of claim 189, wherein the protein expression level is determined by mass spectrometry.

191. The atezolizumab and/or tiragolumab for use of any one of claims 143-190, wherein the sample is obtained from the individual prior to treatment with atezolizumab and/or tiragolumab.

192. The atezolizumab and/or tiragolumab for use of any one of claims 143-191, wherein the sample is a tissue sample, a tumor sample, a blood sample, a plasma sample, a serum sample, or a combination thereof. 138 WO 2024/186790 PCT/US2024/018472

193. The atezolizumab and/or tiragolumab for use of claim 192, wherein the sample is a tissue sample.

194. The atezolizumab and/or tiragolumab for use of claim 193, wherein the tissue sample is a tumor tissue sample.

195. The atezolizumab and/or tiragolumab for use of claim 194, wherein the tumor tissue sample is a biopsy.

196. The atezolizumab and/or tiragolumab for use of claim 193, wherein the tissue sample is a tumor draining lymph node (dLN) sample.

197. The atezolizumab and/or tiragolumab for use of claim 192, wherein the sample is a blood sample.

198. The atezolizumab and/or tiragolumab for use of any one of claims 192-197, wherein the sample is an archival sample, a fresh sample, or a frozen sample.

199. The atezolizumab and/or tiragolumab for use of any one of claims 143-198, wherein the individual has a PD-L1-positive NSCLC.

200. The atezolizumab and/or tiragolumab for use of claim 199, wherein the PD-L1 -positive NSCLC has been determined to have a PD-L1-positive tumor cell fraction by an immunohistochemical (IHC) assay.

201. The atezolizumab and/or tiragolumab for use of claim 200, wherein the PD-L1 -positive tumor cell fraction is determined by positive staining with an anti-PD-L1 antibody, wherein the anti-PD-Lantibody is SP263, 22C3, SP142, or 28-8.

202. The atezolizumab and/or tiragolumab for use of any one of claims 143-201, wherein the individual is a human.

203. The atezolizumab and/or tiragolumab for use of any one of claims 143-202, wherein the individual has not previously been treated for NSCLC. 139