WO2024206821A1 - Méthodes d'amélioration de l'immunogénicité de vaccins cellulaires - Google Patents

Méthodes d'amélioration de l'immunogénicité de vaccins cellulaires Download PDF

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Publication number
WO2024206821A1
WO2024206821A1 PCT/US2024/022230 US2024022230W WO2024206821A1 WO 2024206821 A1 WO2024206821 A1 WO 2024206821A1 US 2024022230 W US2024022230 W US 2024022230W WO 2024206821 A1 WO2024206821 A1 WO 2024206821A1
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Prior art keywords
hla
cell
gene
cells
dendritic cell
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Inventor
William V. Williams
Miguel LOPEZ-LAGO
Jay Berzofsky
Purevdorj Olkhanud
Noriko SEISHIMA
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Briacell Therapeutics Corp USA
US Department of Health and Human Services
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Briacell Therapeutics Corp USA
US Department of Health and Human Services
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Priority to CN202480022688.5A priority Critical patent/CN121152635A/zh
Priority to AU2024246543A priority patent/AU2024246543A1/en
Priority to EP24720692.3A priority patent/EP4687956A1/fr
Publication of WO2024206821A1 publication Critical patent/WO2024206821A1/fr
Priority to IL323635A priority patent/IL323635A/en
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/19—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/24—Antigen-presenting cells [APC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/46—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/50—Cellular immunotherapy characterised by the use of allogeneic cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure provides an engineered mammalian dendritic cell comprising one or more exogenous alleles of at least one Major Histocompatibility Complex (MHC) class II gene.
  • MHC Major Histocompatibility Complex
  • the one or more exogenous alleles are introduced through homologous recombination or through transfection or transduction of one or more expression vectors into the cell.
  • the exogenous alleles comprise an exogenous allele of a first MHC class II gene and an exogenous allele of a second MHC class II gene. In other instances, the exogenous alleles comprise a first exogenous allele of an MHC class II gene and a second exogenous allele of the same MHC class II gene.
  • the engineered mammalian dendritic cell is an engineered human dendritic cell.
  • the MHC class II gene comprises an HLA class II alpha subunit gene, an HLA class II beta subunit gene, or a combination thereof.
  • the MHC class II gene comprises an HLA-DR gene, an HLA-DP gene, an HLA- DQ gene, an HLA-DM gene, an HLA-DO gene, or a combination thereof.
  • the HLA-DR gene comprises an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4 gene, an HLA-DRB5 gene, or a combination thereof.
  • the HLA-DP gene comprises an HLA-DPA1 gene, an HLA-DPB1 gene, or a combination thereof.
  • the HLA-DQ gene comprises an HLA-DQA1 gene, an HLA-DQB1 gene, or a combination thereof.
  • the engineered mammalian dendritic cell comprises an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof.
  • the cell is engineered from a cell line.
  • the cell line is HL-60, THP-1, K562, MUTZ3, or an immortalized dendritic cell.
  • the immortalized dendritic cell expresses HTLV-1 transactivator (Tax) protein, SV40 proteins, and/or hTERT.
  • the cell is engineered from a primary cell.
  • the primary cell is from a patient.
  • the patient has a cancer.
  • the present disclosure provides a composition comprising an engineered mammalian dendritic cell comprising one or more exogenous alleles of at least one Major Histocompatibility Complex (MHC) class II gene.
  • MHC Major Histocompatibility Complex
  • the present disclosure provides a pharmaceutical composition comprising a composition described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further comprises a cryoprotectant.
  • the present disclosure provides a method for semi-allogeneic dendritic cell-based immunotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.
  • the method prior to the administering, further comprises: (i) obtaining an MHC class II allele profile by genotyping a plurality of MHC class II genes in a biological sample from the subject; and (ii) selecting an engineered mammalian dendritic cell for administering to the subject, wherein the engineered mammalian dendritic cell comprises one or more mismatches to the MHC class II allele profile of the subject.
  • the method further comprises administering to the subject a regulatory T cell inhibitory agent (Treg agent).
  • Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA (siRNA), an siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof.
  • the Treg agent is administered after administering the pharmaceutical composition.
  • the subject is a human.
  • the human has a cancer, wherein the engineered dendritic cell comprises a tumor-specific antigen or a fragment thereof.
  • the present disclosure provides a method for autologous dendritic cell-based immunotherapy in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein, wherein the engineered mammalian dendritic cell is from a primary immune cell of the subject.
  • the method further comprises: (i) obtaining the primary immune cell or a plurality thereof from the subject; (ii) genotyping a plurality of MHC class II genes of the primary immune cell to determine an endogenous MHC class II allele profile; and (iii) engineering the primary immune cell into an engineered mammalian dendritic cell by: (a)introducing into the primary immune cell one or more exogenous MHC class II alleles comprising at least one mismatch to the endogenous MHC class II allele profile of the subject; and (b) introducing into the primary immune cell an antigen of a pathogen, a tumor- associated antigen, a neo-antigen, an allergen, an antigen that is the target of an autoimmune response, or a fragment thereof.
  • (iii) further comprises: (c) incubating the primary immune cell with Lipopolysaccharide (LPS), interferon-gamma (IFN-y), or a combination thereof.
  • the method further comprises administering to the subject a Treg agent.
  • the Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA (siRNA), an siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof.
  • the Treg agent is administered after administering the pharmaceutical composition.
  • FIG. 1 depicts the three mouse strains used in the study.
  • C57BL/6J mouse is wild type (WT) with MHC haplotype H2b
  • B 6.C-H2-K bml /ByJ (bml) mouse is MHC class I mutant with haplotype H2bml.
  • the 'Q6.C-H2-K bml /ByJ mice differ from the C57BL/6J Kb allele by 7 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide binding groove in positions 152, 155, and 156 of the a2 domain.
  • B6(C)-J/2- nZ>7 fem72 /KhEgJ (bm!2) mouse is MHC class II mutant with haplotype H2bml2.
  • the B6(C)- //2-4A/ / ’"' / -7l ⁇ hEgJ mice differ from the C57BL/6J lAb allele by 3 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide binding groove in positions 67, 70, and 71 of the pi domain.
  • FIG. 2 depicts schematic drawings of antigen presenting cells (APCs) with MHC alloantigens H2-D b , H2-K b , and H2-IA b and the genetic loci of the murine H-2 complex.
  • the E7 peptide is a peptide from human papilloma virus (HPV) protein E7, and only binds to MHC class I, H-2D b .
  • HPV human papilloma virus
  • APC WT contains wild type MHC alloantigens H2-D b , H2-K b , and H2- IA b
  • H2-D b presents the E7 peptide.
  • APC mutant bml contains wild type H2-D b which presents the E7 peptide, and wild type H2-IA b , and mutant H2-K bml which stimulates MHC class I allogeneic help.
  • APC mutant bml2 contains wild type H2-D b which presents the E7 peptide, and wild type H2-K' and mutant H2-IA bm12 which stimulates MHC class II allogeneic help.
  • D indicates the genetic loci of the murine H-2 complex, also known as the murine major histocompatibility complex (MHC).
  • Classical MHC class I comprises H-2D, H- 2K and H-2L subclasses in the K region and D region
  • non-classical MHC class lb comprises H-2Q
  • Classical MHC class II comprises H- 2A(IA) and H-2E(IE)
  • non-classical MHC-IIb comprises H-2P (P), H-2M (DM) and H-2O (DO) subclasses. All MHC class II subclasses are located in the I region.
  • MHC class III is located in the S region.
  • FIG. 3 depicts the expression of MHC class II (IAIE), CDl lc, CD40, CD80 and CD86 in immature and mature BMDCs as measured by flow cytometry.
  • DC Dendritic cells
  • BM bone marrow
  • GM-CSF murine granulocyte-macrophage colony-stimulating factor
  • the cells were incubated with or without 100 ng/ml lipopolysaccharide (LPS, Sigma-aldrich, Heidelberg, Germany) for overnight, and both matured and immature BMDCs were measured by flow cytometry.
  • LPS lipopolysaccharide
  • B-E indicate the maturation of BMDCs not only stimulate the expression of CD86, also increase the expression of MHC class II (IAIE), CD40, and CD80.
  • FIG. 4 depicts the maturation of BMDCs.
  • BMDCs from C57BL/6J (WT), B6.C-//2- 7f fe '" 7 /ByJ (bml), or B6(C)-T/2-4/7/ / ’"' / -7l ⁇ hEgJ (bml2) after 10 days culture with GM-CSF were pulsed with E743-77 (10 pg/ml) for 1-2 hours and maturated with LPS (100 ng/ml) overnight.
  • FIG. 5 depicts tumor volume growth curves of the mice in the first mouse study.
  • Female C57BL/6J mice 13 weeks old were inoculated subcutaneously with 1 x 10 5 TC-1 cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm in diameter.
  • mice were vaccinated intradermally with 2 x 10 6 (1.6 x 10 6 on Day 19) syngeneic E7 -pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi-allogeneic E7-pulsed BMDCs from 'Q6.C-H2-K bml /ByJ (E7-mBMDC bml), or semi-allogeneic E7-pulsed BMDCs from B6(C)-J/2MZ>7 6m72 /KhEgJ (E7-mBMDC bml2).
  • the control group was injected with phosphate-buffered saline (PBS).
  • vaccination with the E7-BMDC WT slowed tumor growth.
  • the E7-BMDC bml (MHC class I mutant) showed a similar effect as the E7-BMDC WT.
  • the E7-BMDC bml2 (MHC class II mutant) showed a greater effect in limiting tumor growth than either the E7-BMDCs WT or the E7-BMDC bml (MHC class I mutant).
  • B depicts tumor volume growth curves of individual mice. The tumor on one mouse had disappeared after the fifth vaccination with the E7-BMDC bml2 (MHC class II mutant).
  • FIG. 6 depicts tumor volume growth of the mice on different days in the first mouse study.
  • A depicts tumor volume growth on day 19;
  • B depicts tumor volume growth on day 21;
  • C depicts tumor volume growth on day 34;
  • D depicts tumor weight on day 35.
  • the PBS depicts the control group of the mice injected with PBS; E7-mBMDC WT depicts the mice vaccinated with syngeneic E7-pulsed BMDCs from C57BL/6J; E7-mBMDC bml depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from 6.C-H2-K bml /ByJ; and E7- mBMDC bml2 depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from B6(C)-//2-4/i/ / ’"' / -7l ⁇ hEgJ .
  • FIG. 7 depicts tumor volume growth of the mice in the second mouse study.
  • Female C57BL/6J mice (9 weeks old) were inoculated subcutaneously with 1 x 10 5 TC-1 cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm long diameter.
  • mice were vaccinated intradermally 2 x 10 6 (1.6 x 10 6 on Day 14) syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi-allogeneic E7-pulsed BMDCs from ⁇ 6.C-H2-K bml /ByJ (E7-mBMDC bml), or semi- allogeneic E7-pulsed BMDCs from B6(C)-7/2-HZ>7 fem72 /KhEgJ (E7-mBMDC bml2).
  • the control group was injected with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • vaccination with the E7-BMDC WT slowed tumor growth.
  • the E7-BMDC bml (MHC class I mutant) showed a similar effect as the E7-BMDC WT.
  • the E7-BMDC bml2 (MHC class II mutant) showed a greater effect than either the E7 -BMDCs WT or the E7-BMDC bml (MHC class I mutant).
  • (B) depicts the ratio of tumor/body weight of the mice on Day 28 after TC-1 cell inoculation.
  • C depicts the body weight of the mice on Day 28 after TC-1 cell inoculation.
  • D depicts the tumor weight of the mice on Day 28 after TC-1 cell inoculation.
  • PBS depicts the control group of the mice injected with PBS
  • E7-mBMDC WT depicts the mice vaccinated with syngeneic E7-pulsed BMDCs from C57BL/6J
  • E7-mBMDC bml depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from ⁇ 6.C-H2-K bml /ByJ
  • E7-mBMDC bml2 depicts the mice vaccinated with semi-allogeneic E7-pulsed BMDCs from B6(C)-772-HZ>7 fem72 /KhEgJ.
  • FIG. 8 depicts tumor volume growth curves of mice in a CD4 or CD8 T cell depletion study.
  • Female C57BL/6J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 5 TC-1 cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm in diameter.
  • Intraperitoneal injection of anti-CD4 antibody at an early stage of tumor growth (Early aCD4), or anti-CD8 antibody (aCD8) was started on day 6 (200 pg/mouse) after the inoculation and continued every 2-4 days (100 pg/mouse) to the end of the experiment.
  • Intraperitoneal injection of anti-CD4 antibody at a late stage of tumor growth was started on day 17 (200 pg/mouse) after the inoculation and continued every 2-4 days (100 pg/mouse) to the end of the experiment.
  • mice were vaccinated intradermally with 2 x 10 6 syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT) or semi-allogeneic E7-pulsed BMDCs from B6(C)-772- HZ>7 fem72 /KhEgJ (E7-mBMDC bml2).
  • the control group was injected with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • FIG. B depicts tumor volume growth curves of the mice in 4 groups either injected with phosphate-buffered saline (PBS) or vaccinated with the E7-BMDC WT with isotype control antibody (Isotype + the E7-BMDC WT), anti-CD4 antibody at an early stage of tumor growth (Early aCD4 + the E7-BMDC WT), or anti-CD8 antibody (aCD8 + the E7- BMDC WT).
  • the groups vaccinated with E7-BMDC WT with isotype control antibody or anti-CD4 antibody showed similar growth curves.
  • C depicts tumor volume growth curves of the mice in 5 groups either injected with phosphate-buffered saline (PBS) or vaccinated with the E7-BMDC bml2 with isotype control antibody (Isotype + E7-BMDC bml2), anti-CD4 antibody at an early stage of tumor growth (Early aCD4 + E7-BMDC bml2), anti-CD4 antibody at a late stage of tumor growth (late aCD4 + E7-BMDC bml2), or anti-CD8 antibody (aCD8 + E7-BMDC bml2).
  • PBS phosphate-buffered saline
  • Isotype + E7-BMDC bml2 isotype control antibody
  • anti-CD4 antibody at an early stage of tumor growth Early aCD4 + E7-BMDC bml2
  • anti-CD4 antibody at a late stage of tumor growth late aCD4 + E7-BMDC b
  • the group vaccinated with E7-BMDC bml2 with isotype control antibody (Isotype + E7-mBMDC bml2) showed a greater effect on limiting tumor growth.
  • Administration of anti-CD4 antibody to the vaccinated mice at an early stage of tumor growth (Early aCD4 + E7-mBMDC bml2) inhibited such effect until day 17.
  • Administration of anti- CD4 antibody to the vaccinated mice at a late stage of tumor growth (late aCD4 + E7-mBMDC bml2) showed the best effect on suppressing tumor growth after the 5 th vaccination dose.
  • C shows that CD4 depletion at a late stage of tumor growth had a greater effect on limiting tumor growth in mice vaccinated with E7-BMDC bml2.
  • FIG. 9 depicts tumor volume growth of the mice on different days of the CD4 or CD8 T cell depletion study shown in FIG. 8.
  • A depicts tumor volume growth on day 14;
  • B depicts tumor volume growth on day 20;
  • C depicts tumor volume growth on day 29.
  • FIG. 10 depicts tumor volume growth of the mice in a Treg cell depletion study.
  • Six groups of female B6.129(Cg)-Foxp3tm3(Hbegf/GFP)Ayr/J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 5 TC-1 cells. Seven days after the inoculation, all tumors on the mice reached around 5 mm in diameter.
  • mice The first group of mice was injected with phosphate-buffered saline (PBS) only as a control group.
  • PBS phosphate-buffered saline
  • Two groups of mice (2 doses of E7- mBMDC bml2) were vaccinated intradermally 2 x 10 6 semi-allogeneic E7-pulsed BMDCs from B6(C)-J/2-24Z>7 fem72 /KhEgJ (E7-mBMDC bml2) on day 8 and 13 after the TC-1 cell inoculation.
  • Three groups of mice (5 doses of E7-mBMDC bml2) received the same vaccine on day 8, 13, 18, 23, and 28.
  • mice express the human diphtheria toxin (DT) receptor, and they are depleted for Treg cells when they are injected with DT.
  • DT diphtheria toxin
  • FIG. 11 depicts IFNy production in TC-1 bearing mouse CD4 or CD8 T cells cocultured with mBMDC WT, E7-mBMDCs WT, mBMDC bml2, or E7-mBMDC bml2.
  • CD4 and CD8 T cells were isolated from spleens of TC-1 bearing mice 8 days after TC-1 inoculation and incubated at a concentration of 2 x 10 5 cells per well, alone or co-cultured with BMDCs (1 x 10 5 cells per well, with or without E7-pulsing), in a 96-well round plate for 24 hrs or 72 hrs.
  • the isolated CD8 T cells either alone (CD8) or co-cultured with mBMDC WT (WT CD8), E7-pulsed mBMDCs WT (E7-WT CD8), mBMDC bml2 (bml2 CD8), or E7-pulsed mBMDC bml2 (E7-bml2 CD8), were tested for IFNy production via intracellular staining and flow cytometry (A) and for IFNy production in the supernatant by ELISA (C).
  • the isolated CD4 T cells either alone (CD4) or co-cultured with mBMDC WT (WT CD4), E7-pulsed mBMDCs WT (E7-WT CD4), mBMDC bml2 (bml2 CD4), or E7-pulsed mBMDC bml2 (E7-bml2 CD4), were tested for IFNy production via intracellular staining and flow cytometry (B) and for IFNy production in the supernatant by ELISA (C).
  • CD8 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8) generated significantly higher levels of IFNy in comparison to a mix of CD8 and CD4 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8+CD4).
  • CD8 T cells co-cultured with E7-mBMDC bml2 (E7- bml2 CD8) and a mix of CD8 and CD4 T cells co-cultured with E7-mBMDC bml2 (E7-bml2 CD8+CD4) showed similar high IFNy production compared to CD8 T cells co-cultured with E7-mBMDC WT (E7-WT CD8).
  • FIG. 1 depicts IFNy production of CD4 T cells by via intracellular staining and flow cytometry 24hrs after co-culture.
  • CD4 T cells co-cultured with E7-mBMDC bml2 (E7-BM12 CD4) generated significantly higher levels of IFNy in comparison to CD4 T cells co-cultured with E7-mBMDC WT (E7-WT CD4).
  • C depicts IFNy production in the supernatant of CD4 and/or CD8 T cell cultures by ELISA 72hrs after co-culture.
  • CD4 T cells co-cultured with E7-mBMDC bml2 showed a good amount of IFNy release, significantly higher than CD8 T cells co-cultured with E7-mBMDC bml2 (E7-bml2 CD8).
  • Robust IFNy production was observed in the supernatant of the mix of CD8 and CD4 T cells co-cultured with E7-mBMDC bml2 (E7-bml2 CD8+CD4).
  • the present disclosure is based, in part, on the inventors’ discovery that engineered mammalian dendritic cells (DCs) comprising semi-allogeneic Major Histocompatibility Complex (MHC) class II alleles can elicit a robust immune response in a subject.
  • DCs dendritic cells
  • MHC Major Histocompatibility Complex
  • the present disclosure relates to engineering mammalian DCs to express one or more exogenous MHC class II alleles.
  • the engineered mammalian DCs can be derived from a cell line and used as an “off-the-shelf’ semi-allogeneic cellular vaccine.
  • the engineered mammalian DCs can be derived from a subject and returned to the subject after modification for cancer and/or disease treatment as an autologous cellular immunotherapy.
  • the engineered mammalian DCs can be derived from one subject and used to treat other subject as a semi-allogeneic cellular immunotherapy.
  • BMDCs semi-allogeneic murine bone marrow dendritic cells
  • DCs are antigen- presenting cells expressing both MHC class I and class II molecules.
  • MHC class I molecules activate CD8+ T cells (killer T cells) to kill targeted cells
  • MHC class II molecules activate CD4+ T cells (helper T cells) to help other immune cell activities.
  • the expression of at least one exogenous allele of an MHC class II molecule provides additional help to the DC by enhancing the potency of antigen presentation and eliciting an allo-CD4+ Th response, therefore producing a more potent anti-cancer response.
  • the engineered mammalian DCs described herein can be used as either a semi-allogeneic or autologous DC-based vaccine.
  • compositions of engineered mammalian DCs expressing one or more exogenous MHC class II alleles and methods of using these cells for semi-allogeneic or autologous cellular immunotherapy of diseases such as cancer are also provided.
  • the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • the terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets.
  • Farm animals include, but are not limited to, cattle, goats, pigs, sheep, dogs, horses, and rabbits.
  • Sport animals include, but are not limited to, horses, bovines (calves, bulls, and steers), and dogs.
  • Pet animals include, but are not limited to, dogs, cats, rabbits, rats, pigs, horses, and guinea pigs. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • administering includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.
  • treating refers to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment.
  • Therapeutic benefit can also mean to effect a cure of one or more diseases, conditions, or symptoms under treatment.
  • the term “effective amount” or “sufficient amount” refers to the amount of an engineered mammalian cell or other composition that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the physical delivery system in which it is carried.
  • an effective amount is determined by such considerations as may be known in the art.
  • the amount must be effective to achieve the desired therapeutic effect in a subject suffering from cancer or disease.
  • the desired therapeutic effect may include, for example, amelioration of undesired symptoms associated with cancer or disease, prevention of the manifestation of such symptoms before they occur, slowing down the progression of symptoms associated with cancer or disease, slowing down or limiting any irreversible damage caused by cancer or disease, lessening the severity of or curing cancer or disease, or improving the survival rate or providing more rapid recovery from cancer or disease.
  • the effective amount depends, inter alia, on the type and severity of the disease to be treated and the treatment regime.
  • the effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount.
  • an effective amount depends on a variety of factors including the distribution profile of a therapeutic agent (e.g., a whole-cell cancer vaccine) or composition within the body, the relationship between a variety of pharmacological parameters (e.g., half-life in the body) and undesired side effects, and other factors such as age and gender, etc.
  • pharmaceutically acceptable carrier refers to a substance that aids the administration of an active agent to a cell, an organism, or a subject.
  • “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in the compositions of the disclosure and that causes no significant adverse toxicological effect on the subject.
  • Nonlimiting examples of pharmaceutically acceptable carriers include water, sodium chloride, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like.
  • the carrier may also be substances for providing the formulation with stability, sterility and isotonicity (e.g. antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g. antimicrobial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and the like) or for providing the formulation with an edible flavor etc.
  • the carrier is an agent that facilitates the delivery of an engineered mammalian cell to a target cell or tissue.
  • pharmaceutical carriers are useful in the present disclosure.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof.
  • DNA may be in the form of, e.g, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups.
  • RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid.
  • analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’- O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • PNAs peptide-nucleic acids
  • the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605- 2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).
  • “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • the term “gene” means the segment of DNA involved in producing a polypeptide chain.
  • the DNA segment may include regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons).
  • vector and “expression vector” refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression vector may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression vector includes a polynucleotide to be transcribed, operably linked to a promoter.
  • promoter is used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • Other elements that may be present in an expression vector include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators).
  • co-expression of multiple genes may be achieved by co-transfection of two or more vectors, the use of multiple or bidirectional promoters, or the creation of bicistronic or multi ci stronic vectors.
  • Gene co-expression may be driven by using a plasmid with multiple, individual expression cassettes.
  • each promoter creates unique mRNA transcripts for each gene that is expressed.
  • Bicistronic or multici stronic vectors simultaneously express two or more separate proteins from the same mRNA.
  • Bicistronic vectors may contain an Internal Ribosome Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA.
  • IRS Internal Ribosome Entry Site
  • Multicistronic vectors containing one or more self-cleaving 2A peptides are advantageous as they allow gene co-expression from the same cassette. In some instances, multicistronic vectors are preferred when only a portion of the plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is important.
  • “Recombinant” refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism.
  • a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated using well known methods.
  • a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook el al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)).
  • a recombinant expression cassette typically comprises polynucleotides in combinations that are not found in nature. For instance, human manipulated restriction sites or plasmid vector sequences can flank or separate the promoter from other sequences.
  • a recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).
  • a recombinant cell is one that has been modified (e.g., transfected or transformed), with a recombinant nucleotide, expression vector or cassette, or the like.
  • amino acid refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein.
  • Amino acids include naturally-occurring a-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers.
  • “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).
  • Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine.
  • Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and their combinations.
  • Stereoisomers of a naturally- occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D- His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D- methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D- serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D- Tyr), and their combinations.
  • D-alanine D-Ala
  • Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, TV-substituted glycines, and N- methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids.
  • amino acid analogs can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium.
  • Amino acids may be referred to by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • sequence identity refers to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated.
  • the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window” includes reference to a segment of any one of the number of contiguous positions (from 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150), in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known.
  • Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection.
  • Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site.
  • the algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989).
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993).
  • polypeptide “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • the terms encompass amino acid chains of any length, including full-length proteins (z.e., alleles), wherein the amino acid residues are linked by covalent peptide bonds.
  • the amino acid sequence of a polypeptide is presented from the N-terminus to the C-terminus. In other words, when describing an amino acid sequence of a polypeptide, the first amino acid at the N-terminus is referred to as the “first amino acid.”
  • Genome editing refers to a type of genetic engineering in which DNA is inserted, deleted, modified or replaced in the genome of a living organism. Genome editing can be site-specific.
  • Non-limiting examples of genome editing techniques include the use of nucleases such as clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs).
  • CRISPR/Cas9 clustered regularly interspaced short palindromic repeats/Cas9
  • TALENs transcription activator-like effector nucleases
  • ZFNs zinc-finger nucleases
  • Viral vectors such as integrasedefective lentiviral vectors (IDLVs), adenoviruses and adeno-associated viruses (AAVs) are typically used to deliver DNA for genome editing. Delivery technologies for genome editing are known in the art and any approach may be used for introducing exogenous MHC alleles in the engineered mammalian cells described herein. (See e.g., review by Yin et al., 2017. “Delivery technologies for genome editing.” Nature Review Drug Discovery. 16, 387-399).
  • cancer is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells.
  • the term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, recurrent, soft tissue, or solid, and cancers of all stages and grades including advanced, pre- and post- metastatic cancers.
  • cancers examples include, but are not limited to, gynecological cancers (e.g., ovarian, cervical, uterine, vaginal, and vulvar cancers); lung cancers (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, carcinoid tumors, lung adenocarcinoma); breast cancers (e.g., triple-negative breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, cribriform carcinoma, invasive lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, Paget’s disease, Phyllodes tumors); digestive and gastrointestinal cancers such as gastric cancer (e.g., stomach cancer), colorectal cancer, gastrointestinal stromal tumors (GIST), gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer
  • MHC Major Histocompatibility Complex
  • the MHC locus is present in all jawed vertebrates and contains about a hundred genes and pseudogenes. In humans, the MHC region occurs on chromosome 6, between the flanking genetic markers MOG and COL11A2 (from 6p22.1 to 6p21.3 about 29Mb to 33Mb on the hg38 assembly) and contains 224 genes spanning 3.6 megabase pairs (3,600,000 bases). In mice, the MHC region occurs on mouse chromosome 17.
  • the MHC genes encode MHC molecules/proteins/antigens.
  • MHC molecules MHC proteins
  • MHC antigens are used interchangeably herein to refer to the cell surface proteins that are encoded by the MHC genes.
  • the human MHC is also called the human leukocyte antigen (HLA) complex (often just the HLA).
  • HLA human leukocyte antigen
  • the swine MHC is called swine leukocyte antigens (SLA)
  • the bovine MHC is called bovine leukocyte antigens (BoLA)
  • the dog MHC is called dog leukocyte antigens (DLA)
  • the murine MHC is also called the Histocompatibility system 2 or just the H-2.
  • the rat MHC is called RT1, and the chicken MHC is called B-locus.
  • MHC gene family is divided into three groups: MHC class I, MHC class II, and MHC class III. Only MHC class I and class II genes encode the MHC molecules that are directly involved in the antigen presentation. MHC genes are highly polymorphic, for example, HLA class I genes contain 19,031 alleles and HLA class II genes contain 7,183 alleles as listed in the IMGT database.
  • HLA human leukocyte antigen
  • HLA alleles are named by the World Health Organization Naming Committee for Factors of the HLA system. Under this system, an HLA gene name is followed by a series of numerical fields. At a minimum, two numerical fields are included.
  • HLA- A*02: 101 denotes a specific allele of the HLA-A gene. The first field, separated from the gene name by an asterisk, denotes an allele group.
  • the second field denotes the specific HLA protein that is produced.
  • a longer name is used (e.g., HLA-A*02: 10L0L02N).
  • the third numerical field denotes whether a synonymous DNA substitution is present within the coding region
  • the fourth numerical field denotes differences between alleles that exist in the non-coding region.
  • an HLA allele name contains a letter at the end.
  • N denotes that the allele is a null allele (i.e., the allele produces a non-functional protein)
  • L denotes that the allele results in lower than normal cell surface expression of the particular HLA protein
  • S denotes that the allele produces a soluble protein not found on the cell surface
  • Q denotes a questionable allele (i.e., an allele that nay not affect normal expression)
  • C denotes that the allele produces a protein that is present in cell cytoplasm but is not present at the cell surface
  • A denotes an allele that results in aberrant expression (i.e., it is uncertain whether the particular HLA protein is expressed).
  • allele profile refers to a collection of alleles of one or more genes in a particular sample.
  • the sample may be obtained from a subject, a particular cell or cell type (e.g., a dendritic cell), or from an engineered cell (e.g., a dendritic cell that has been engineered to express one or more proteins).
  • an allele profile describes the alleles of a single gene that are present in a sample (e.g., in a cell obtained from a subject or a cell line), or may describe the alleles that are present for two or more genes in a sample.
  • an allele profile may list the alleles that are present for an HLA class II gene in a particular sample. For a diploid cell, only one allele may be present. Alternatively, two different alleles may be present. In other instances, the allele profile enumerates the alleles that are present for two or more genes.
  • human leukocyte antigen refers to a gene complex that encodes human major histocompatibility complex (MHC) proteins, which are a set of cell surface proteins that are essential for recognition of foreign molecules by the adaptive immune system.
  • MHC major histocompatibility complex
  • the HLA complex is found within a 3 Mbp stretch of chromosome 6p21.
  • the human MHC Class I proteins which present peptides from inside the cell, are encoded by the HLA-A, HLA- B, HLA-C, HLA-E, HLA-F, and HLA-G genes.
  • HLA-A, HLA-B, and HLA-C genes are more polymorphic, while HLA-E, HLA-F, and HLA-G genes are less polymorphic.
  • HLA-K and HLA- L are also known to exist as pseudogenes.
  • beta-2-microglobulin is an MHC class I protein, encoded by the (B2M) gene.
  • HLA-A nucleotide sequences are set forth under GenBank reference numbers NM_001242758 and NM_002116.
  • a nonlimiting example of an HLA-B nucleotide sequence is set forth under GenBank reference number NM_005514.
  • Non-limiting examples of HLA-C nucleotide sequences are set forth under GenBank reference numbers NM_001243042 andNM_002117.
  • Anon-limiting example of an HLA-E nucleotide sequence is set forth under GenBank reference number NM 005516.
  • a non-limiting example of an HLA-F nucleotide sequence is set forth under GenBank reference number NM_018950.
  • a non-limiting example of an HLA-G nucleotide sequence is set forth under GenBank reference number NM 002127.
  • a non-limiting example of a B2M nucleotide sequence is set forth under GenBank reference number NM 004048.
  • HLA-DP human MHC Class II proteins
  • HLA-DM human MHC Class II proteins
  • HLA-DO human MHC Class II proteins
  • HLA-DQ human MHC Class II proteins
  • HLA -DM genes include HLA-DMA and HLA-DMB.
  • HLA-DO genes include HLA- DOA and HLA-DOB.
  • HLA-DP genes include HLA-DP Al and HLA-DPBL
  • HLA-DQ genes include HLA-DQA 1, HLA-DQA2, HLA-DQB1, VX HLA-DQB2.
  • HLA-DR genes include HLA- DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5.
  • Non-limiting examples of HLA- DMA and HLA-DMB nucleotide sequences are set forth under GenBank reference numbers NM_006120 and NM_002118, respectively.
  • Non-limiting examples of HLA-DRA, HLA- DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 nucleotide sequences are set forth in GenBank reference numbers NM_01911, NM_002124, NM_022555, NM_021983, NM_002125, respectively.
  • the term “semi-allogeneic” refers to at least one MHC class I or class II molecule expressed by a subject’s dendritic cells or a dendritic cell line that is syngeneic to the recipient and at least one MHC class I or class II molecule that is allogeneic to the recipient.
  • “Syngeneic” refers to an MHC allele coding for an MHC molecule specificity that matches between the subject or the dendritic cell line and the recipient and is immunologically compatible with at least one of an MHC class I or class II allele of the recipient.
  • Allogeneic refers to at least one of an MHC class I or class II allele coding for an MHC molecule specificity that is unmatched and immunologically incompatible with at least one of an MHC class I or class II allele of the recipient.
  • vaccine refers to a biological composition that, when administered to a subject, has the ability to produce an acquired immunity to a particular pathogen or disease in the subject. Typically, one or more antigens, or fragments of antigens, that are associated with the pathogen or disease of interest are administered to the subject.
  • Vaccines can comprise, for example, inactivated or attenuated organisms (e.g., bacteria or viruses), cells, proteins that are expressed from or on cells (e.g., cell surface proteins), proteins that are produced by organisms (e.g., toxins), or portions of organisms (e.g., viral envelope proteins).
  • cells are engineered to express proteins such that, when administered as a vaccine, they enhance the ability of a subject to acquire immunity to that particular cell type (e.g, enhance the ability of a subject to acquire immunity to a cancer cell).
  • the term “vaccine” or “wholecell cancer vaccine” includes but is not limited to the engineered mammalian cell(s) of the present disclosure.
  • cytokine refers to small proteins released by cells that have a specific effect on the interactions and communications between cells.
  • Cytokines are generally known as lymphokines (e.g., cytokines made by lymphocytes), monokines (e.g., cytokines made by monocytes), or chemokines (e.g., cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (e.g., autocrine action), on nearby cells (e.g., paracrine action), or on distant cells (e.g., endocrine action).
  • cytokines may comprise a chemokine, an interferon, an interleukin, and/or a tumor necrosis factor (TNF).
  • cytokines may comprise an early T cell activation antigen- 1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin- 1 family member (IL-la, IL-P, IL-IRa, IL-18, IL-33, IL-36Ra, IL-36a, IL-36P, IL-36Y, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin- 10 (IL-10), an interleukin- 12 (IL-12), an interleukin
  • ETA-1 early
  • GM-CSF granulocyte macrophage colony-stimulating factor
  • CSF2 colony stimulating factor
  • GM-CSF functions as a cytokine that affects a number of cell types, in particular macrophages and eosinophils.
  • GM-CSF stimulates stem cells to produce granulocytes (i.e., neutrophils, eosinophils, and basophils) and monocytes.
  • the monocytes subsequently mature into macrophages and dendritic cells after tissue infiltration.
  • a non-limiting example of a CSF2 nucleotide sequence (the gene that encodes GM-CSF) in humans is set forth under GenBank reference number NM 000758.
  • Interferon refers to a cytokine that is produced in response to infection or other inflammatory stimuli.
  • Interferons are signaling proteins that are synthesized and released by host cells in response to a pathogen (e.g., viruses, bacteria, parasites, tumor cells).
  • pathogen e.g., viruses, bacteria, parasites, tumor cells.
  • Interferons are classified into three subgroups: type I interferons, type II interferon (IFNv), and type III interferons. Functionally, these cytokines modulate immune cell function.
  • type III interferons are structurally distinct from type I interferons, they have overlapping functions, and both signal through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway to induce transcription of interferon-stimulated genes (ISGs) and promote immune responses.
  • JK Janus kinase
  • STAT activator of transcription
  • Type I interferon proteins include IFN-a, IFN-P, IFN-s, IFN-K, IFN-T, IFN-5, IFN- , IFN-CO, and IFN- v.
  • Interferon alpha proteins are produced by leukocytes and are mainly involved in the innate immune response.
  • Genes that encode IFN-a proteins include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.
  • Nonlimiting examples ofIFNAl, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21 human nucleotide sequences are set forth in Gene Bank reference numbers NM_024013, NM_000605, NM_021068, NM_002169, NM_021002, NM_021057, NM_002170, NM_002171, NM_006900, NM_002172, NM_002173, NM_021268, and NM_002175, respectively.
  • the gene IFNA2 encodes IFN- a2a, IFN-a2b, and IFN-a2c variants.
  • IFN-a and “IFN-a2” are used interchangeably, and they refer to interferon proteins IFN-a2a or IFN-a2b.
  • Type III interferon proteins include interferon lambda 1 (IFN 1 (IL-29), interferon lambda 2 (IFN 2 (IL-28A)), interferon lambda 1 (IFNX3 (IL-28B)), and interferon lambda 4 (IFNA4).
  • Interferon lambda family members signal through the common IL-10 receptor subunit 2 (IL-10R2).
  • Human interferon lambda proteins are encoded by four IFNL genes, IFNL1 (IL29 IFNL2 I2 A ⁇ .
  • IFNL3 (IL28B), and IFNL4.
  • co-stimulatory molecule refers to a cell surface molecule that amplifies or counteracts the initial activating signals provided to T cells from the T cell receptor (TCR) following its interaction with an antigen/major histocompatibility complex (MHC).
  • Costimulatory molecules generally may influence T cell differentiation and fate.
  • Co-stimulatory molecules belong to three major families, namely the immunoglobulin (Ig) superfamily, the tumor necrosis factor (TNF) - TNF receptor (TNFR) superfamily, and the T cell Ig and mucin (TIM) domain family.
  • Ig immunoglobulin
  • TNF tumor necrosis factor
  • TNFR tumor necrosis factor receptor
  • TIM T cell Ig and mucin
  • Exemplary costimulatory molecules and ligands include, but are not limited to, CD28 and ligands B7-1 (CD80), CTLA-4, PDL-1, orB7-2 (CD86), CTLA-4 and ligands B 7-1 (CD80) orB7-2 (CD86), ICOS and ligand ICOS-L, CD27 and ligand CD70, CD30 and ligand CD30L, CD40 and ligand CD40L (a.k.a.
  • CD 154 0X40 and ligand OX40L, GITR and ligand GITRL, TIM-1 and ligands TIM-1, TIM-4, IgA, or phosphatidylserine (PtdSer), TIM-2 and ligands H-ferritin or semaphorin 4A (Sem4A), and TIM-4 and ligand phosphatidylserine (PtdSer).
  • co-stimulatory molecules may comprise a CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL a.k.a CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), 0X40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), CD30 molecule (CD30), and combinations thereof See e.g., FIG. 5).
  • CD86 CD86
  • CD80 CD80
  • 4-1BB ligand molecule 4-1BB ligand molecule
  • 4-1BBL a.k.a CD137L 4-1BB ligand molecule
  • ICOS-L ICOS-L
  • CD70 molecule CD70 a.k.a
  • Tregs refer to a highly immunosuppressive subset of CD4 + CD25 + T cells that are essential to establish and maintain homeostasis and self-tolerance.
  • Tregs can inhibit T cell proliferation and cytokine production and prevent autoimmunity.
  • Treg cells express the biomarkers CD4, CD25, and Forkhead box protein P3 (FoxP3).
  • CD4 is a marker for some thymic-derived populations of Tregs in addition to helper T cells.
  • CD25 is a component of the IL-2 receptor and can serve as a marker for activated T cells.
  • FoxP3 is a transcription factor that represses the expression of several cytokines such as IL-2, IL-4 and IFNy with concomitant activation of the IL-2 receptor (CD25), cytotoxic T lymphocyte-associated protein-4 (CTLA4) and glucocorticoid-induced TNF receptor (GITR).
  • CD25 IL-2 receptor
  • CTL4 cytotoxic T lymphocyte-associated protein-4
  • GITR glucocorticoid-induced TNF receptor
  • FoxP3 is the master protein involved in the differentiation and functional expression of regulatory T cells and can be used as a Treg molecular marker (see, e.g, Science (2003) 299: 1057-61). Further description and information on human CD4+CD25+ regulatory T cells can be found in the following references, which are all hereby incorporated by reference: Jonuleit et al. (2001) J Exp Med.
  • regulatory T cell inhibitory agent refers to an agent that: (1) inhibits or decreases the activity or function of a regulatory T cell; (2) decreases the population of regulatory T cells in a subject (in one embodiment, the decrease can be temporary, for example, for a few hours, a day, a few days, a week, or a few weeks); or (3) substantially ablates or eliminates the population of regulatory T cells in a subject (in one embodiment, the ablation or elimination can be temporary, for example, for a few hours, a day, a few days, a week, or a few weeks).
  • a Treg agent can decrease the suppression of immune system activation and can decrease prevention of self-reactivity.
  • Exemplary Treg agents include, but are not limited to, a compound, antibody, fragment of an antibody, or chemical that targets a Treg cell surface marker (such as CD25, CD4, CD28, CD38, CD62L (selectin), OX-40 ligand (OX-40L), CTLA4, CCR4, CCR8, FOXP3, LAG3, CD 103, NRP-1, glucocorticoid-induced TNF receptor (GITR), galectin-1, TNFR2, or TGF- PR1).
  • a Treg agent targets a Treg cell surface marker that is involved in Treg activation such that the Treg inhibitor prevents Treg activation.
  • Treg agents include, but are not limited to, antibodies, fusion proteins, ONTAK, HuMax-Tac, Zenapax, or MDX-010, aptamers, siRNA, ribozymes, antisense oligonucleotides, and the like.
  • the administration of a Treg agent or derivatives thereof can block the action of its target, such as a Treg cell surface marker.
  • a Treg agent can have an attached toxic moiety such that upon internalization of the inhibitor, the attached toxic moiety can kill the regulatory T cell.
  • Tumor antigen refers to an antigenic substance produced in tumor cells that may trigger an immune response in the host.
  • Tumor antigens generally refer to tumor- associated antigen (TAAs) or tumor-specific antigens (TSAs).
  • TSAs are found in cancer cells only and are not in healthy (e.g., non-cancerous) cells. TSAs may arise from oncogenic driver mutations that generate novel peptide sequences (e.g., neoantigens).
  • a nonlimiting example of a TSA is alphafetoprotein (AFP) expressed in germ cell tumors and hepatocellular carcinoma.
  • TAAs have elevated levels in tumor cells and may express at lower levels in healthy cells.
  • a non-limiting example of a TAA is melanoma-associated antigen (MAGE) expressed in the testis along with malignant melanoma.
  • MAGE melanoma-associated antigen
  • the term “survival” refers to a length of time following the diagnosis of a disease and/or beginning or completing a particular course of therapy for a disease (e.g., cancer).
  • the term “overall survival” includes the clinical endpoint describing patients who are alive for a defined period of time after being diagnosed with or treated for a disease, such as cancer.
  • the term “disease-free survival” includes the length of time after treatment for a specific disease (e.g., cancer) during which a patient survives with no sign of the disease (e.g., without known recurrence).
  • disease-free survival is a clinical parameter used to evaluate the efficacy of a particular therapy, which is usually measured in units of 1 or 5 years.
  • progression-free survival includes the length of time during and after treatment for a specific disease (e.g, cancer) in which a patient is living with the disease without additional symptoms of the disease. In some embodiments, survival is expressed as a median or mean value.
  • the present disclosure provides an engineered mammalian dendritic cell comprising one or more exogenous alleles of an MHC class II gene.
  • the engineered mammalian dendritic cell comprises one exogenous allele of an MHC class II gene.
  • the engineered mammalian dendritic cell comprises more than one exogenous MHC class II allele.
  • the engineered mammalian dendritic cell comprises a first exogenous allele of a first MHC class II gene and a second exogenous allele of a second MHC class II gene.
  • the engineered mammalian dendritic cell comprises a first exogenous allele of an MHC class II gene and a second exogenous allele of the same MHC class II gene. In some embodiments, the engineered mammalian dendritic cell further comprises one or more exogenous alleles of an MHC class I gene.
  • the one or more exogenous alleles are introduced into the mammalian dendritic cell through homologous recombination.
  • one or more exogenous MHC class II alleles are introduced into the mammalian dendritic cell through homologous recombination.
  • the homologous recombination replaces an endogenous allele of the MHC class II gene with an exogenous allele.
  • the homologous recombination inserts an exogenous allele into the MHC class II gene without deleting the related endogenous allele.
  • one or more exogenous MHC class I alleles are also introduced through homologous recombination into the mammalian dendritic cell.
  • the homologous recombination is triggered by a nuclease creating a double-stranded break (DSB) at a specific site in the genome.
  • the nuclease can be an endonuclease, a Zinc finger nuclease (ZEN), a transcription activator-like effector nuclease (TALEN), site-specific recombinase, transposase, topoisomerase, and modified derivatives and variants thereof. Descriptions of nucleases that can be used in the present disclosure are provided further herein.
  • the nuclease can be an RNA-guided nuclease, such as a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the one or more exogenous alleles are introduced through transfection or transduction of one or more expression vectors into the cell.
  • one or more exogenous MHC class II alleles are introduced through transfection or transduction of one or more expression vectors into the cell.
  • one or more exogenous MHC class II alleles are introduced through transfection of one or more expression vectors into the cell.
  • transfection of one or more expression vectors comprising MHC class II alleles into the cell includes transfection of plasmids or expression cassettes.
  • one or more exogenous MHC class II alleles are introduced through transduction of one or more expression vectors into the cell.
  • transduction of one or more expression vectors comprising MHC class II alleles into the cell includes transduction of viral vectors such as, but not limited to, adenoviral vectors, adeno- associated viral vectors, retroviral vectors, lentiviral vectors.
  • viral vectors such as, but not limited to, adenoviral vectors, adeno- associated viral vectors, retroviral vectors, lentiviral vectors.
  • only one exogenous MHC class II allele is introduced through transfection or transduction of one vector into the cell.
  • more than one exogenous MHC class II alleles are introduced into the cell.
  • all the exogenous MHC class II alleles can be present on the same vector.
  • each exogenous MHC class II allele can be present on a separate vector.
  • two, three, four, five, six, or more exogenous MHC class II alleles can be present on the same vector. Any number of combinations of exogenous alleles on a single vector and any number of vectors in a cell is permitted. Descriptions of expression vectors and the methods of transfection or transduction that can be used in the present disclosure are provided further herein.
  • two exogenous MHC class II alleles selected from HLA-DR, HLA-DP, and/or HLA-DQ alleles are introduced into the cell on the same vector or separate vectors.
  • one or more exogenous MHC class I alleles are also introduced through transfection or transduction of one or more expression vectors into the mammalian dendritic cell. In certain instances, all the exogenous MHC class II alleles can be present on one vector, and all the exogenous MHC class I alleles can be present on another vector.
  • the engineered mammalian dendritic cell described herein is an engineered human dendritic cell.
  • the engineered mammalian dendritic cell is derived from a non-human dendritic cell.
  • the non-human dendritic cell can be from a mouse, a rat, a simian, a farm animal, a sport animal, or a pet animal.
  • farm animals include cattle, goats, pigs, sheep, dogs, horses, and rabbits.
  • sport animals include horses, bovines (calves, bulls, and steers), and dogs.
  • Non-limiting examples of pet animals include dogs, cats, rabbits, rats, pigs, horses, and guinea pigs.
  • the present disclosure provides an engineered human dendritic cell comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous HLA class II alleles of an MHC class II gene or a plurality thereof.
  • the MHC class II gene is an HLA class II alpha subunit gene.
  • the MHC class II gene is an HLA class II beta subunit gene.
  • the MHC class II gene is a combination of HLA class II alpha subunit and HLA class II beta subunit genes.
  • the MHC class II gene is an HLA-DR gene, an HLA-DP gene, an HLA-DQ gene, an HLA-DM gene, and/or an HLA-DO gene.
  • the HLA-DR gene is an HLA-DRA gene, an HLA-DRB1 gene, an HLA-DRB3 gene, an HLA-DRB4 gene, and/or an HLA-DRB5 gene.
  • the engineered human dendritic cell comprises one or more exogenous alleles of one or more (e.g., 1, 2, 3, 4, 5, or more) HLA-DR gene(s).
  • the HLA-DP gene is an HLA-DPA1 gene. In other instances, the HLA-DP gene is an HLA-DPB1 gene.
  • the engineered human dendritic cell comprises one or more exogenous alleles of both HLA-DPA1 and HLA-DPB1 gene alleles.
  • the HLA-DQ gene is an HLA-DQA1 gene. In other instances, the HLA-DQ gene is an HLA-DQB1 gene. In certain instances, the engineered human dendritic cell comprises one or more exogenous alleles of both HLA-DQA1 and HLA-DQB1 gene alleles.
  • the HLA-DM gene is an HLA- DMA gene. In other instances, the HLA-DM gene is an HLA-DMB gene.
  • the engineered human dendritic cell comprises one or more exogenous alleles of both HLA- DMA and HLA-DMB gene alleles.
  • the HLA-DO gene is an HLA-D0A1 gene.
  • the HLA-DO gene is an HLA-D0B1 gene.
  • the engineered human dendritic cell comprises one or more exogenous alleles of both HLA-D0A1 and HLA-D0B1 gene alleles.
  • suitable HLA-DRB3 alleles include but are not limited to HLA- DRB3*02:02, HLA-DRB3*01:01, and HLA-DRB3*03:01.
  • suitable HLA-DRB4 alleles include but are not limited to HLA-DRB4*01 :01 and HLA-DRB4*01 :03.
  • suitable HLA-DRB5 alleles include but are not limited to HLA-DRB5*01 :02, HLA- DRB5*01:01, and HLA-DRB5*02:02.
  • the engineered human dendritic cells of the present disclosure can comprise one or more e.g., 1, 2, 3, 4, 5, or more) exogenous alleles of HLA- DRB3/4/5 alleles.
  • suitable HLA-DPA1 alleles include but are not limited to HLA- DPAl*01:05, HLA-DPA1 *02:08, and HL A-DP Al *04:05.
  • suitable HLA-DPB1 alleles include but are not limited to HLA-DPB1 *32:01 and HLA-DPBl *1454:01.
  • suitable HLA-DQA1 alleles include but are not limited to HLA-DQAl*01 :06, HLA- DQA1 *02:29, and HLA-DQAl*06:04.
  • suitable HLA-DQB1 alleles include but are not limited to HLA-DQB 1*05:04, HLA-DQB 1*06 TOO, and HLA-DQB 1*04:95.
  • suitable HLA-DMA alleles include but are not limited to HLA- DMA*01 :01 :01 :01, HLA-DMA*01 :01 :02, and HLA-DMA*01 :02:01 :09.
  • suitable HLA-DQB alleles include but are not limited to HLA-DMB*01 :03:01 :05, HLA- DMB*01 :01 :01 :20, and HLA-DMB*01 :01 :01 :01.
  • the present disclosure provides an engineered human dendritic cell further comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous HLA class I alleles of an MHC class I gene or a plurality thereof.
  • the MHC class I gene is an HL A- A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, or a B2M gene.
  • the MHC class I gene is a combination of an HLA-A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, and/or a B2M gene.
  • Engineered human dendritic cells of the present disclosure can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more) exogenous HLA-A alleles.
  • HLA-B alleles include but are not limited to HLA-B* 13:02, HLA-B*41:01, HLA-B*18:03, HLA-B*44:02, HLA-B*07:02, HLA-B*35:01, HLA-B*40:01, HLA-B*35:08, HLA-B*55:01, HLA-B*51:01, HLA-B*44:03, HLA-B*58:01, HLA-B*08:01, HLA-B*18:01, HLA-B*15:01, and HLA-B*52:01.
  • Engineered human dendritic cells of the present disclosure can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more) exogenous HLA-B alleles.
  • HLA-C alleles include but are not limited to HLA-C*04:01, HLA-C*07:02, HLA-C*07:01, HLA-C*06:02, HLA-C*03:04, HLA-C*01:02, HLA-C*02:02, HLA-C*08:02, HLA-C*15:02, HLA-C*03:03, HLA-C*05:01, HLA-C*08:01, HLA-C*16:01, HLA-C* 12:03, and HLA-C* 14:02.
  • Engineered human dendritic cells of the present disclosure can comprise one or more (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) exogenous HLA-C alleles.
  • Murine MHC is composed of 11 subclasses.
  • the “classical MHC class I” also called MHC-Ia comprises H-2D, H-2K and H-2L subclasses located in the K region and D region of the murine H-2 loci (FIG. 2D).
  • the “non-classical MHC class I” (MHC- lb) comprises H-2Q, H-2M and H-2T subclasses in the Q/T/M region.
  • the “classical MHC class II” (MHC-IIa) includes H-2A(I-A), and H-2E(I-E) subclasses, and the “non-classical MHC class II” (MHC- Ilb) comprises H-2P (P), H-2M (DM) and H-2O (DO). All murine MHC class II subclasses are located in the I region. The murine MHC class III is located in the S region.
  • Murine MHC class I molecules consist of a 45 kD highly glycosylated heavy chain non-covalently associated with a 12 kD 2-microglobulin, a polypeptide that is also found free in serum.
  • Murine MHC class II antigen is composed of a 33 kD chain and a 28 kD chain.
  • MHC class I molecules are expressed on almost all nucleated cells. They play an important role in presentation of altered self-cell antigens (virally infected or tumor cells) to CD8+ cytotoxicity T cells.
  • the MHC class II molecules are expressed on antigen presenting cells (B cells, monocytes/ macrophages, dendritic cells, and Langerhans cells, etc.). They are involved in presentation of processed peptide antigens to CD4+ cells.
  • the present disclosure provides an engineered murine dendritic cell comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous MHC class II alleles of an MHC class II gene or a plurality thereof.
  • the engineered murine dendritic cell further comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) exogenous MHC class I alleles of an MHC class I gene or a plurality thereof.
  • MHC haplotype antigens of BALB/c mice are H-2K d , H-2D d , H-2L d , H2- IA d , and H2-//? / and MHC haplotype antigens of C57BL/6J mice are H2-D b , H2-K b , and H2- IA b .
  • Some variant murine strains are also generated in the laboratory.
  • B6.C-7/2- K bml I y] (bml) is generated through the introgression of a spontaneous variant (BALB/cBy x C57BL/6By)F 1 -derived H2 bml allele on chromosome 17 onto the C57BL/6By background for 10 generations.
  • the variant allele, H2-K bml differs from H2-K b by 7 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide binding groove in positions 152, 155, and 156 of the a2 domain.
  • B6(C)-J/2MZ>7 fem72 /KhEgJ (bml2) is also generated through the introgression of a spontaneous variant (C57BL/6Kh x BALB/cKh) F 1 -derived H2- Abl bmI2 allele onto the C57BL/6Kh background for 10 generations.
  • This variant MHC class II allele differs from H2-Abl b by 3 nucleotides resulting in 3 amino acid substitutions (Ile67Phe, Arg70Gln, Thr71Lys) occurring along the edge of the peptide binding groove of the pi domain.
  • the engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • the engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprises one or more exogenous MHC class I genes selected from any class I gene, a codon optimized version thereof, a variant thereof, or a fragment thereof.
  • the engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • the engineered mammalian dendritic cell comprises one exogenous MHC class II gene selected from any MHC class II gene, a codon optimized version thereof, a variant thereof, or a fragment thereof.
  • the engineered mammalian dendritic cell comprises at least two exogenous MHC class II gene selected from any class II gene, a codon optimized version thereof, a variant thereof, or a fragment thereof.
  • the engineered mammalian dendritic cell may comprise a first recombinant polynucleotide encoding a first MHC class II gene and a second recombinant polynucleotide encoding a second MHC class II gene.
  • the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprises one exogenous MHC class I gene selected from any MHC class I gene, a codon optimized version, a variant thereof, or a fragment thereof.
  • the engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprises at least two recombinant polynucleotides each encoding an MHC class I gene selected from any MHC class I gene, a codon optimized version, a variant thereof, or a fragment thereof.
  • the engineered mammalian dendritic cell may comprise a recombinant polynucleotide encoding a first MHC class I gene and a second recombinant polynucleotide encoding a second MHC class I gene.
  • the present disclosure provides an engineered mammalian dendritic cell (e.g., an engineered human dendritic cell) further comprising one or more recombinant polynucleotides encoding a cytokine, a co-stimulatory molecule, a variant thereof, a fragment thereof, or a combination thereof.
  • an engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • polynucleotides encoding a cytokine, a co-stimulatory molecule, a variant thereof, a fragment thereof, or a combination thereof.
  • the cytokine can be a chemokine, an interferon, an interleukin, or a tumor necrosis factor.
  • the cytokine can be selected from an early T cell activation antigen-1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin-1 family member (IL-la, IL-0, IL-IRa, IL- 18, IL-33, IL-36Ra, IL-36a, IL-36 , IL-36 Y, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin- 10 (IL- 10), an interleukin- 12 (IL-12), an interleukin- 13 (IL-13),
  • the co-stimulatory molecule can be selected from at least one of a CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL, also known as TNFSF9 or CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), 0X40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), a CD30 molecule (CD30), and a combination thereof.
  • CD86 CD86
  • CD80 CD80
  • 4-1BB ligand molecule 4-1BB ligand molecule
  • 4-1BBL also known as TNFSF9 or CD137L
  • ICOS-L ICOS-L
  • CD70 molecule CD70 a.k.a.
  • one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be introduced into the cell through homologous recombination. In other embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be introduced into the cell through transfection or transduction of one or more expression vectors.
  • one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules are present on one or more vectors in the cell and one or more exogenous MHC alleles are in the genome of the same cell. In some embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules are in the genome of the cell and one or more exogenous MHC alleles are present on one or more vectors in the same cell.
  • one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules and one or more exogenous MHC alleles are in the genome of the cell. In some embodiments, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules and one or more exogenous MHC alleles are present on one or more vectors in the cell.
  • one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be present on the same vector with one or more exogenous MHC alleles. In other instances, one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules can be present on a separate vector with one or more exogenous MHC alleles.
  • the cell comprises: (a) a vector comprising one or more exogenous HLA class II alleles selected from HLA-DR, HLA-DP, and/or HLA-DQ alleles; and (b) a vector comprising one or more recombinant polynucleotides encoding cytokines and/or co-stimulatory molecules.
  • the cell further comprises: (c) a vector comprising one or more exogenous HLA class I alleles.
  • the present disclosure provides the engineered mammalian dendritic cell e.g., an engineered human dendritic cell) further comprising one or more recombinant polynucleotides encoding a heterologous antigen (e.g., an antigen of a pathogen, a tumor- associated antigen, a neo-antigen, an allergen, an antigen that is the target of an immune response), a variant thereof, or a fragment thereof.
  • a heterologous antigen e.g., an antigen of a pathogen, a tumor- associated antigen, a neo-antigen, an allergen, an antigen that is the target of an immune response
  • one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be introduced into the cell through homologous recombination. In other embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be introduced into the cell through transfection or transduction of one or more expression vectors.
  • one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide are present on one or more vectors in the cell and one or more exogenous MHC alleles are in the genome of the same cell. In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide are in the genome of the cell and one or more exogenous MHC alleles are present on one or more vectors in the same cell.
  • one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide and one or more exogenous MHC alleles are in the genome of the cell. In some embodiments, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide and one or more exogenous MHC alleles are present on one or more vectors in the cell.
  • one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be present on the same vector with the exogenous MHC alleles. In other instances, one or more recombinant polynucleotides encoding a heterologous antigen and/or an antigen peptide can be present on a separate vector with the exogenous MHC alleles.
  • the cell comprises: (a) a vector comprising one or more HLA class II alleles selected from HLA-DR, HLA-DP, and/or HLA-DQ alleles; and (b) a vector comprising recombinant polynucleotides encoding a unique antigen peptide of a pathogenic antigen, a tumor-associated antigen, a neo-antigen, an allergen, or an antigen that is the target of an immune response.
  • the cell further comprises: (c) a vector comprising one or more exogenous HLA class I alleles (e.g., HLA-A alleles).
  • an engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • one or more heterologous antigens e.g., an antigen of a pathogen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is the target of an immune response
  • one or more antigens and/or an antigen peptide can be introduced into the cell through incubation of the antigen and/or the antigen peptide with the cell in the same culture.
  • an engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • a primary patient cell is pulsed by a heterologous antigen and/or an antigen peptide of a pathogen that the patient is suffering from.
  • an engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • an engineered mammalian dendritic cell e.g., an engineered human dendritic cell
  • LPS Lipopolysaccharide
  • the engineered mammalian dendritic cell is derived from a dendritic cell line (e.g., a human dendritic cell line).
  • a dendritic cell line e.g., a human dendritic cell line
  • human dendritic cell lines include the following cell lines and subclones thereof: HL-60, THP-1, K562, MUTZ3, or an immortalized dendritic cell.
  • the immortalized dendritic cell expresses HTLV-1 transactivator (Tax) protein, SV40 proteins, and/or hTERT.
  • the engineered mammalian dendritic cell is engineered from a primary cell of a patient, for example, a blood or biopsy sample from a patient.
  • the patient has a cancer.
  • the engineered mammalian dendritic cell described herein contains one or more exogenous alleles in the genomic DNA.
  • the one or more exogenous MHC class II alleles are introduced into the cell through homologous recombination.
  • the homologous recombination replaces an endogenous allele of the MHC class II gene with an exogenous allele.
  • the homologous recombination inserts an exogenous allele into the MHC class II gene.
  • a nuclease can be used to create a double-stranded break (DSB) at a specific site in the genome and trigger homologous recombination.
  • nucleases include, but are not limited to, an endonuclease, a Zinc finger nuclease (ZEN), a transcription activator-like effector nuclease (TALEN), site-specific recombinase, transposase, topoisomerase, and modified derivatives and variants thereof.
  • the nuclease can be an RNA-guided nuclease, such as a clustered regularly interspaced short palindromic repeats (CRISPR) nuclease.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the nuclease used in methods and compositions of the disclosure is a CRISPR-associated (Cas) protein.
  • a Cas protein refers to an RNA-guided double-stranded DNA-binding nuclease protein or nickase protein. Wild-type Cas nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands.
  • a Cas protein can induce double strand breaks in genomic DNA (target nucleic acid) when both functional domains are active.
  • the Cas protein can comprise one or more catalytic domains of a Cas protein derived from bacteria belonging to the group consisting of Corynebacter , Sullerella.
  • the Cas protein can be a fusion protein, e.g., the two catalytic domains are derived from different bacteria species.
  • Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, homologs thereof, variants thereof, mutants thereof, and derivatives thereof.
  • Type II Cas proteins include Casl, Cas2, Csn2, Cas9, and Cfpl. These Cas proteins are known to those skilled in the art.
  • the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. NP 269215, and the amino acid sequence of Streptococcus thermophilus wildtype Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP_011681470.
  • Cas proteins can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma syn
  • Torquens Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp.
  • Jejuni Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.
  • the Cas protein can be a high-fidelity or enhanced specificity Cas9 polypeptide variant with reduced off-target effects and robust on-target cleavage.
  • Cas9 polypeptide variants with improved on-target specificity include the SpCas9 (K855A), SpCas9 (K810A/K1003A/R1060A) (also referred to as eSpCas9(1.0)), and SpCas9 (K848A/K1003A/R1060A) (also referred to as eSpCas9(l.
  • a Cas protein can be guided to its target nucleic acid by a guide RNA (gRNA).
  • gRNA is a version of the naturally occurring two-piece guide RNA (crRNA and tracrRNA) engineered into a two-piece gRNA or a single, continuous sequence.
  • a gRNA can contain a guide sequence (e.g., the crRNA equivalent portion of the gRNA) that targets the Cas protein to the target nucleic acid and a scaffold sequence that interacts with the Cas protein (e.g., the tracrRNAs equivalent portion of the gRNA).
  • a gRNA can be selected using a software.
  • considerations for selecting a gRNA can include, e.g., the PAM sequence for the Cas protein to be used, and strategies for minimizing off-target modifications.
  • Tools such as NUPACK® and the CRISPR Design Tool, can provide sequences for preparing the gRNA, for assessing target modification efficiency, and/or assessing cleavage at off-target sites.
  • the guide sequence in the gRNA may be complementary to a specific sequence within a target nucleic acid (e.g., one allele of an MHC Class II gene).
  • the 3’ end of the target nucleic acid sequence can be followed by a PAM sequence.
  • Approximately 20 nucleotides upstream of the PAM sequence is the target nucleic acid.
  • a Cas9 protein or a variant thereof cleaves about three nucleotides upstream of the PAM sequence.
  • the guide sequence in the gRNA can be complementary to either strand of the target nucleic acid.
  • the guide sequence of a gRNA comprises about 100 nucleic acids at the 5’ end of the gRNA that can direct the Cas protein to the target nucleic acid site using RNA-DNA complementarity base pairing. In some embodiments, the guide sequence comprises about 20 nucleic acids at the 5’ end of the gRNA that can direct the Cas protein to the target nucleic acid site using RNA-DNA complementarity base pairing. In other embodiments, the guide sequence comprises less than 20, e.g., 19, 18, 17, 16, 15 or less, nucleic acids that are complementary to the target nucleic acid site. In some instances, the guide sequence in the gRNA contains at least one nucleic acid mismatch in the complementarity region of the target nucleic acid site. In some instances, the guide sequence contains about 1 to about 10 nucleic acid mismatches in the complementarity region of the target nucleic acid site.
  • the scaffold sequence in the gRNA can serve as a protein-binding sequence that interacts with the Cas protein or a variant thereof.
  • the scaffold sequence in the gRNA can comprise two complementary stretches of nucleotides that hybridize to one another to form a double-stranded RNA duplex (dsRNA duplex).
  • the scaffold sequence may have structures such as lower stem, bulge, upper stem, nexus, and/or hairpin.
  • the scaffold sequence in the gRNA can be between about 90 nucleic acids to about 120 nucleic acids.
  • the nuclease is a Zinc-finger nuclease (ZFN).
  • ZFNs typically comprise a zinc finger DNA binding domain and a nuclease domain.
  • ZFNs include two zinc finger arrays (ZFAs), each of which is fused to a single subunit of a non-specific endonuclease, such as the nuclease domain from the FokI enzyme, which becomes active upon dimerization.
  • ZFAs zinc finger arrays
  • a single ZFA consists of 3 or 4 zinc finger domains, each of which is designed to recognize a specific nucleotide triplet (GGC, GAT, etc. .
  • a ZFN composed of two "3-finger" ZFAs is therefore capable of recognizing an 18 base pair target site (i.e., recognition sequence); an 18 base pair recognition sequence is generally unique, even within large genomes such as those of humans and plants.
  • ZFNs By directing the co-localization and dimerization of the two FokI nuclease monomers, ZFNs generate a functional site-specific endonuclease that can target a particular locus (e.g., gene, promotor or enhancer).
  • Zinc-finger nucleases useful in the methods disclosed herein include those that are known and ZFN that are engineered to have specificity for one or more target sites described herein (e.g., promotor or enhancer nucleotide sequence).
  • Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence within a target site of the host cell genome.
  • ZFN can comprise an engineered DNA- binding zinc finger domain linked to a non-specific endonuclease domain, for example, a nuclease domain from a Type Ils endonuclease such as HO or FokI.
  • a zinc finger DNA binding domain can be fused to a site-specific recombinase, transposase, or a derivative thereof that retains DNA nicking and/or cleaving activity.
  • additional functionalities can be fused to the zinc-finger binding domain, including but not limited to, transcriptional activator domains (such as VP 16, VP48, VP64, VP160 and the like) or transcription repressor domains (such as KRAB).
  • the zinc finger nuclease is engineered such that the zinc finger nuclease comprises a transcriptional activator domain selected from VP16, VP48, VP64 or VP160.
  • the zinc finger nuclease is engineered such that the zinc finger nuclease comprises a transcriptional activator domain selected from HSF1, VP 16, VP64, p65, RTA, MyoDl, SET7, VPR, histone acetyltransferase p300, TET1 hydroxylase catalytic domain, LSD1, CIB1, AD2, CR3, GATA4, p53, SP1, MEF2C, TAX, PPAR-gamma, and SET9.
  • a transcriptional activator domain selected from HSF1, VP 16, VP64, p65, RTA, MyoDl, SET7, VPR, histone acetyltransferase p300, TET1 hydroxylase catalytic domain, LSD1, CIB1, AD2, CR3, GATA4, p53, SP1, MEF2C, TAX, PPAR-gamma, and SET9.
  • engineered zinc finger transcriptional activator that interact with a promoter region of the gamma-globulin gene was shown to enhance fetal hemoglobin production in primer adult erythroblasts (Wilber et al., Blood, 115(15):3033-3041).
  • Other polydactyl zinc-finger transcription factors are also known in the art, including those disclosed in Beerli and Barbas (see, Nature Technology, (2002) 20: 135-141).
  • Each zinc finger domain recognizes three consecutive base pairs in the target DNA. For example, a three finger domain recognizes a sequence of nine contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind a 18 nucleotide recognition sequence.
  • Useful zinc finger modules include those that recognize various GNN and ANN triplets (Dreier et al., (2001) J Biol Chem 276:29466-78; Dreier et al., (2000) J Mol Biol 303:489-502; Liu et al., (2002) J Biol Chem 277:3850-6), as well as those that recognize various CNN or TNN triplets (Dreier et al., (2005 J Biol Chem 280:35588-97; Jamieson et al., (2003) Nature Rev Drug Discovery 2:361-8).
  • a ZFN comprises a fusion protein having a cleavage domain of a Type IIS restriction endonuclease fused to an engineered zinc finger binding domain, wherein the binding domain further comprises one or more transcriptional activators.
  • the type IIS restriction endonuclease is selected from a HO endonuclease or a FokI endonuclease.
  • the zinc finger binding domain comprises 3, 4, 5 or 6 zinc fingers.
  • the zinc finger binding domain specifically binds to a recognition sequence corresponding to a promoter or enhancer disclosed herein (e.g., SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A and PAX6 promotor or enhancer).
  • the one or more transcriptional activators is selected from VP 16, VP48, VP64, or VP 160.
  • the DNA-binding domain of a ZFN contains between 3 and 6 individual zinc finger repeats and can recognize between 9 and 18 contiguous nucleotides.
  • Each ZFN can be designed to target a specific target site in the host cell genome, e.g., a promotor sequence, an enhancer sequence, or exon/intron within a gene.
  • the nuclease is a TALEN.
  • TAL effectors are proteins secreted by Xanthomonas bacteria and play an important role in disease or triggering defense mechanisms, by binding host DNA and activating effector-specific host genes, see, e.g., Gu et al. (2005) Nature 435: 1122-5; Yang et al., (2006) roc. Natl. Acad. Set. USA 103: 10503-8; Kay et al., (2007) Science 318:648-51; Sugio et al., (2007) Proc. Natl. Acad. Sci.
  • a TALEN comprises a TAL effector DNA-binding domain fused to a DNA cleavage domain.
  • the DNA binding domain interacts with DNA in a sequence-specific manner through one or more tandem repeat domains.
  • the repeated sequence typically comprises 33-34 highly conserved amino acids with divergent 12 th and 13 th amino acids.
  • RVD Repeat Variable Diresidue
  • the TAL-effector DNA binding domain can be engineered to bind to a target DNA sequence and fused to a nuclease domain, e.g., a Type IIS restriction endonuclease, such as FokI (see e.g., Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160).
  • a nuclease domain e.g., a Type IIS restriction endonuclease, such as FokI (see e.g., Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160).
  • the nuclease domain can comprise one or more mutations (e.g., FokI variants) that improve cleavage specificity (see, Doyon et al., (2011) Nature Methods, 8 (1): 74-9) and cleavage activity (Guo et al., (2010) Journal of Molecular Biology, 400 (1): 96-107).
  • Other useful endonucleases that can be used as the nuclease domain include, but are not limited to, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and AlwI.
  • the TALEN can comprise a TAL effector DNA binding domain comprising a plurality of TAL effector repeat sequences that bind to a specific nucleotide sequence (i.e., recognition sequence) in the target DNA.
  • TALENs useful for the methods provided herein include those described in W010/079430 and U.S. Patent Application Publication No. 2011/0145940.
  • the TAL effector DNA binding domain can comprise 10 or more DNA binding repeats, and preferably 15 or more DNA binding repeats.
  • each DNA binding repeat comprises a RVD that determines recognition of a base pair in the target DNA, and wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA.
  • the RVD comprises one or more of HD for recognizing C; NG for recognizing T; NI for recognizing A; NN for recognizing G or A; NS for recognizing A or C or G or T; N* for recognizing C or T, where * represents a gap in the second position of the RVD; HG for recognizing T; H* for recognizing T, where * represents a gap in the second position of the RVD; IG for recognizing T; NK for recognizing G; HA for recognizing C; ND for recognizing C; HI for recognizing C; HN for recognizing G; NA for recognizing G; SN for recognizing G or A; and YG for recognizing T.
  • the TALEN is engineered such that the TAL effector comprises one or more transcriptional activator domains (e.g., VP 16, VP48, VP64 or VP 160).
  • transcriptional activator domains e.g., VP 16, VP48, VP64 or VP 160.
  • engineered TAL effectors having a transcriptional activator domain at the c- terminus of the TAL effector were shown to modulate transcription of Sox2 and Klf4 genes in human 293FT cells (Zhang et al., Nature Biotechnology, 29(2):149-153 (2011).
  • TALE-TFs Other TAL effector transcription factors
  • TALE-TFs are also known in the art, including those disclosed in Perez-Pinera et al., (Nature Methods, (2013) 10(3):239-242) that demonstrated modulation of IL1RN, KLK3, CEACAM5 and ERBB2 genes in human 293T cells using TALE-TFs.
  • the one or more transcriptional activator domains are located adjacent to the nuclear localization signal (NLS) present in the C-terminus of the TAL effector.
  • the TALE-TFs can bind nearby sites upstream or downstream of the transcriptional start site (TSS) for a target gene.
  • TSS transcriptional start site
  • the TAL effector comprises a transcriptional activator domain selected from VP 16, VP48, VP64 or VP 160. In another embodiment, the TAL effector comprises a transcriptional activator domain selected from HSF1, VP16, VP64, p65, RTA, MyoDl, SET7, VPR, histone acetyltransferase p300, TET1 hydroxylase catalytic domain, LSD1, CIB1, AD2, CR3, GATA4, p53, SP1, MEF2C, TAX, PPAR-gamma, and SET9.
  • the TALEN comprises a TAL effector DNA-binding domain fused to a DNA cleavage domain, wherein the TAL effector comprises a transcriptional activator.
  • the DNA cleavage domain is of a Type IIS restriction endonuclease selected from a HO endonuclease or a FokI endonuclease.
  • the TAL effector DNA-binding domain specifically binds to a recognition sequence corresponding to a promoter region or enhancer region disclosed herein (e.g., SIM1, MC4R, PKD1, SETD5, THUMPD3, SCN2A and PAX6 promotor or enhancer).
  • the engineered mammalian dendritic cell described herein contains one or more expression vectors for expressing the exogenous MHC class II alleles.
  • expression vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons.
  • Viral vectors that may be used, for example, include vectors based on HIV, SV40, EBV, HSV or BPV.
  • the expression vectors may be replication-defective by design such that the viral vector is defective for one or more functions that are essential for viral genome replication or synthesis and assembly of viral particles. Many of the currently existing replication-defective viruses can carry large therapeutic genes, effectively transduce various types of cells, and provide longterm and stable expression of genes of interest.
  • Lentiviruses are a subset of retroviruses commonly used in research. Lentiviruses can transduce both dividing and non-dividing cells without a significant immune response. These viruses also integrate stably into the host genome, enabling long term transgene expression.
  • a common lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.
  • HIV human immunodeficiency virus
  • an infectious viral particle may comprise plasmids that components of the viral capsid and envelope (typically called the packaging and envelope plasmids), and plasmid that encodes the viral genome (typically called the transfer plasmid).
  • Common lentiviral packaging and envelope plasmids that can be used herein include, but are not limited to, pRSV-Rev, pMDLg/pRRE, psPAX2, pCMV delta R8.2, pMD2.G, pCMV-VSV-G, pCMV-dR8.2 dvpr, pCI-VSVG, pCPRDEnv, pLTR-RD114A, pLTR-G, pCD/NL-BH*DDD, psPAX2-D64V, pCEP4-tat, pHEF-VSVG, pNHP, pCAG-Eco, and pCAG-VSVG.
  • Common lentiviral transfer plasmids that can be used herein include, but are not limited to, pLKO.l puro, pLKO.l - TRC cloning plasmid, pLKO.3G, Tet-pLKO-puro, pSico, pLJMl-EGFP, FUGW, pLVTHM, pLVUT-tTR-KRAB, pLL3.7, pLB, pWPXL, pWPI, EF.CMV.RFP, pLenti CMV Puro DEST, pLenti-puro, pLOVE, pULTRA, pLX301, plnducer20, pHIV-EGFP, Tet-pLKO-neo, pLV- mCherry, pCW57.1, pLionll, pSLIK-Hygro, and plnducerlO-mir-RUP-PheS.
  • lentiviral vectors may be purchased from commercial providers.
  • lentiviral vectors In general, production of lentiviral vectors involves multiple steps including plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, fill and finish. See e.g., www. addgene . org/viral-vectors/lenti virus/ ; www. therm ofi sher. com/us/ en/home/ clinical/ cell- gene-therapy/gene-therapy/lv-production-workflow.html).
  • the lentiviral vector may be designed to express one or more genes of interest simultaneously.
  • Various molecular strategies are available, including the use of multiple promoters, signals of splicing, fusion of genes, cleavage factors and multi ci stronic vectors. (See e.g., review by Shaimardanova et al., “Production and application of multi ci stronic constructs for various human disease therapies.” Pharmaceutics 2019, 11, 580.)
  • the engineered mammalian dendritic cells described herein are expressed using non-viral approaches.
  • Exemplary methods include, but are not limited to, cationic lipids such as liposomes and lipoplexes, polymers or polyplexes and dendrimers, naked plasmids for direct delivery, electroporation, ultrasound and micro bubbles, magnetofections, inorganic molecules.
  • the present disclosure provides a composition comprising an engineered mammalian dendritic cell.
  • the engineered mammalian dendritic cell comprises one or more exogenous alleles of an MHC class II gene.
  • the composition comprises at least 10,000 cells, at least 100,000 cells, at least 1,000,000 cells, at least 1,250,000 cells, at least 1,500,000 cells, at least 2,000,000 cells, at least 2,500,000 cells, at least 3,000,000 cells, at least 3,500,000 cells, at least 4,000,000 cells, at least 4,500,000 cells, at least 5,000,000 cells, at least 10,000,000 cells, at least 12,500,000 cells, at least 15,000,000 cells, at least 20,000,000 cells, at least 25,000,000 cells, at least 30,000,000 cells, at least 35,000,000 cells, at least 40,000,000 cells, at least 45,000,000 cells, or at least 50,000,000 cells.
  • the composition comprises at least 1,000,000 cells. In some embodiments, the composition comprises at least 20,000,000 cells.
  • the composition comprises at most 10,000 cells, at most 100,000 cells, at most 1,000,000 cells, at most 1,250,000 cells, at most 1,500,000 cells, at most 2,000,000 cells, at most 2,500,000 cells, at most 3,000,000 cells, at most 3,500,000 cells, at most 4,000,000 cells, at most 4,500,000 cells, at most 5,000,000 cells, at most 10,000,000 cells, at most 12,500,000 cells, at most 15,000,000 cells, at most 20,000,000 cells, at most 25,000,000 cells, at most 30,000,000 cells, at most 35,000,000 cells, at most 40,000,000 cells, at most 45,000,000 cells, or at most 50,000,000 cells. In some embodiments, the composition comprises at most 20,000,000 cells. In some embodiments, the composition comprises at most 40,000,000 cells.
  • the composition comprises about 1,000,000 to about 50,000,000 cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the composition comprises about 1,000,000 cells. In some embodiments, the composition comprises about 20,000,000 cells. In some embodiments, the composition comprises about 40,000,000 cells.
  • the present disclosure provides a pharmaceutical composition.
  • the pharmaceutical composition comprises any of the compositions described herein and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may comprise an engineered mammalian dendritic cell or cell line comprising at least 1, 2, 3, 4, 5, or more exogenous alleles encoding at least one MHC class II gene.
  • the engineered mammalian dendritic cell or cell line may also comprise at least 1, 2, 3, 4, 5, or more exogenous alleles encoding at least one MHC class I gene.
  • the engineered mammalian dendritic cell or cell line may further comprise one or more co-stimulatory molecules, heterologous antigens, and/or cytokine described herein.
  • the at least 1, 2, 3, 4, 5, or more recombinant polynucleotides may comprise a heterologous sequence encoding, e.g., a co-stimulatory molecule, a heterologous antigen, a cytokine, or a 2A splicing peptide.
  • the recombinant polynucleotide encoding the one or more MHC alleles, co-stimulatory molecule, heterologous antigen, and/or cytokine may be separated by a sequencing encoding a 2A splicing peptide (e.g., T2A, P2A, E2A).
  • a sequencing encoding a 2A splicing peptide (e.g., T2A, P2A, E2A).
  • an expression vector e.g., a replication defective lentiviral vector
  • the engineered mammalian dendritic cell or cell line provided in the pharmaceutical composition may have at least 1, 2, 3, 4, 5, or more expression vectors, each comprising at least 1, 2, 3, 4, 5 or more exogenous alleles encoding the MHC alleles, co- stimulatory molecule, heterologous antigen, and/or cytokine.
  • the pharmaceutical composition further comprises a cryoprotectant, an interferon alpha (e.g., IFN-a2a or IFN-a2b), and/or an interferon lambda family member (e.g., an interferon lambda 1 (IFNLI (IL-29)), an interferon lambda 2 (IFNZ2 (IL-28A)), an interferon lambda 3 (IFNLI (IL-28B)), an interferon lambda 4 (IFNX4)).
  • an interferon lambda 1 IFNLI (IL-29)
  • IFNZ2 interferon lambda 2
  • IFNLI interferon lambda 3
  • IFNX4 interferon lambda 4
  • the interferon alpha e.g., IFN-a2a or IFN-a2b
  • the interferon alpha is expressed by a vector comprising a polynucleotide sequence of the IFNA2 gene in the engineered mammalian dendritic cell as described herein.
  • the interferon alpha is a pegylated IFN-a2a provided exogenously.
  • the pharmaceutical composition further comprises one or more excipients.
  • the pharmaceutical composition further comprises CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation media.
  • the pharmaceutical composition comprises cells cryopreserved in CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation media.
  • the pharmaceutical composition is formulated in a dosage form comprising a total number of engineered mammalian dendritic cell per dose for administration to a subject in need therefor.
  • the pharmaceutical composition is formulated as an “off-the-shelf’ product for self-administration to a subject in need thereof.
  • the pharmaceutical composition may have at least at least 10,000 cells, at least 100,000 cells, at least 1,000,000 cells, at least 1,250,000 cells, at least 1,500,000 cells, at least 2,000,000 cells, at least 2,500,000 cells, at least 3,000,000 cells, at least 3,500,000 cells, at least 4,000,000 cells, at least 4,500,000 cells, at least 5,000,000 cells, at least 10,000,000 cells, at least 12,500,000 cells, at least 15,000,000 cells, at least 20,000,000 cells, at least 25,000,000 cells, at least 30,000,000 cells, at least 35,000,000 cells, at least 40,000,000 cells, at least 45,000,000 cells, or at least 50,000,000 cells.
  • the pharmaceutical composition comprises at least 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises at least 20,000,000 cells.
  • the pharmaceutical composition comprises at most 10,000 cells, at most 100,000 cells, at most 1,000,000 cells, at most 1,250,000 cells, at most 1,500,000 cells, at most 2,000,000 cells, at most 2,500,000 cells, at most 3,000,000 cells, at most 3,500,000 cells, at most 4,000,000 cells, at most 4,500,000 cells, at most 5,000,000 cells, at most 10,000,000 cells, at most 12,500,000 cells, at most 15,000,000 cells, at most 20,000,000 cells, at most 25,000,000 cells, at most 30,000,000 cells, at most 35,000,000 cells, at most 40,000,000 cells, at most 45,000,000 cells, or at most 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises at most 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises at most 40,000,000 cells.
  • the pharmaceutical composition comprises about 1,000,000 to about 50,000,000 cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 40,000,000 cells.
  • the pharmaceutical composition is formulated in the form of a suspension.
  • the formulation of pharmaceutical compositions is generally known in the art (see, e.g., REMINGTON’SPHARMACEUTICALSCIENCES, 18TH ED., Mack Publishing Co., Easton, PA (1990)). Prevention against microorganism contamination can be achieved through the addition of one or more of various antibacterial and antifungal agents.
  • the pharmaceutical composition is a liquid formulation comprising cells resuspended in Lactated Ringer’s solution.
  • compositions suitable for administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Typical carriers include a solvent or dispersion medium containing, for example, water-buffered aqueous solutions (z.e., biocompatible buffers, non-limiting examples of which include Lactated Ringer’s solution and CryoStor cry opreservation media (e.g., CS2, CS5, and CS10, containing 2%, 5%, and 10%, respectively of DMSO; available from BioLife Solutions, Bothell, WA)), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils.
  • biocompatible buffers non-limiting examples of which include Lactated Ringer’s solution and CryoStor cry opreservation media (e.g., CS2, CS5, and CS10, containing 2%,
  • Sterilization can be accomplished by an art-recognized technique, including but not limited to addition of antibacterial or antifungal agents, for example, paraben, chlorobutanol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.
  • antibacterial or antifungal agents for example, paraben, chlorobutanol, sorbic acid or thimerosal.
  • isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.
  • sterile injectable solutions containing engineered mammalian dendritic cell(s), and/or other composition(s) of the present disclosure can be accomplished by incorporating the compound(s) in the required amount(s) in the appropriate solvent with various ingredients enumerated above, as required, followed by sterilization.
  • the above sterile solutions can be vacuum-dried or freeze-dried as necessary.
  • the engineered mammalian dendritic cell(s), and/or other composition(s) provided herein are formulated for administration, e.g., intradermal injection, intralymphatic injection, oral, nasal, topical, or parental administration in unit dosage form for ease of administration and uniformity of dosage.
  • Unit dosage forms refers to physically discrete units suited as unitary dosages for the subjects, e.g., humans or other mammals to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some instances, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced.
  • the more concentrated dosage forms thus will contain substantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount of the engineered mammalian dendritic cell(s), and/or other composition(s).
  • the engineered mammalian dendritic cell(s), and/or other composition(s) provided herein are formulated for administration e.g., one or more doses over a period of time.
  • the engineered mammalian dendritic cell(s), and/or other composition(s) are formulated for administration every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks.
  • the engineered mammalian dendritic cell(s), and/or other composition(s) are formulated for administration every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 12 months, every 18 months, or every 24 months.
  • a dose may include, for example, about 50,000 to 50,000,000 (e.g., about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, or more) engineered mammalian dendritic cells.
  • 50,000 to 50,000,000 e.g.
  • a dose may contain about 1,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may contain about 5,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may contain about 10,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may contain about 20,000,000 engineered mammalian dendritic cells.
  • a dose may also include, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000, or more) engineered mammalian dendritic cells.
  • a dose may include at least about 1,000,000 engineered mammalian dendritic cells.
  • a dose may include at least about 5,000,000 engineered mammalian dendritic cells.
  • a dose may include at least about 10,000,000 engineered mammalian dendritic cells. In some embodiments, a dose may include at least about 20,000,000 engineered mammalian dendritic cells. [0147] A dose may alternatively include, for example, at least about 100,000,000 to 1,000,000,000 (e.g, about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000,000, 550,000,000, 600,000,000,
  • the dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like.
  • Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES, supra).
  • excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.
  • Carbopols e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc.
  • the dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents.
  • lubricating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens)
  • pH adjusting agents such as inorganic and organic acids and bases
  • sweetening agents and flavoring agents.
  • the dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.
  • the therapeutically effective dose may further comprise other components, for example, anti-allergy drugs, such as antihistamines, steroids, bronchodilators, leukotriene stabilizers and mast cell stabilizers. Suitable anti-allergy drugs are well known in the art.
  • the present disclosure provides a method for semi-allogeneic dendritic cell-based immunotherapy in a subject.
  • the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising engineered mammalian dendritic cells of the present disclosure) described herein.
  • a pharmaceutical composition of the present disclosure e.g., a pharmaceutical composition comprising engineered mammalian dendritic cells of the present disclosure
  • the method comprises, prior to the administering, (i) obtaining an MHC class II allele profile by genotyping a plurality of MHC class II genes in a biological sample from the subject; and (ii) selecting an engineered mammalian dendritic cell for administering to the subject, wherein the engineered mammalian dendritic cell comprises one or more mismatches to the MHC class II allele profile of the subject.
  • the subject is a human.
  • the method for semi-allogeneic dendritic cellbased immunotherapy is for treating a cancer in a human.
  • the engineered human dendritic cell or a plurality thereof for the cancer treatment comprises a tumor-specific antigen or a fragment thereof.
  • the present disclosure also provides a method for autologous dendritic cell-based immunotherapy in a subject.
  • the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising engineered mammalian dendritic cells of the present disclosure), wherein the engineered mammalian dendritic cell is derived from a primary cell of the subject.
  • the primary cell is a primary immune cell, including but not limit to, a dendritic cell, a monocyte, a macrophage, B cell, a T cell, a nature killer (NK) cell, and a neutrophil.
  • NK nature killer
  • a particular example of the primary immune cell is dendritic cell.
  • the method for autologous dendritic cell-based immunotherapy further comprises, prior to the administering, (i) obtaining the primary immune cell or a plurality from the subject; (ii) genotyping a plurality of MHC class II genes of the primary immune cell to determine an endogenous MHC class II allele profile; and (iii) engineering the primary immune cell into an engineered mammalian dendritic cell by (a) introducing into the primary immune cells one or more exogenous MHC class II alleles comprising at least one mismatch to the endogenous MHC class II allele profile of the subject; and (b) introducing into the primary immune cell an antigen of the cancer or the pathogen the subject is suffering or a fragment thereof.
  • the engineering of the mammalian dendritic cells further comprises a step of: (c) incubating the primary immune cell with Lipopolysaccharide (LPS), interferon-gamma (IFN-y), or a combination of LPS and IFN-
  • LPS Lipopolysaccharide
  • IFN-y interferon-gamma
  • the disclosure provides a method for enhancing or inducing anti-cancer response in a subject via using regulatory T cell inhibitory agents (Treg inhibitory agents, or Treg agents) that inhibit or decrease the activity or function of Tregs in order to promote the efficacy of the cancer vaccines described herein.
  • the method comprises administering to a subject an effective amount of a Treg agent to a subject, wherein the Treg agent decreases the activity or function of Tregs, and administering an effective amount of the pharmaceutical composition comprising engineered mammalian dendritic cells disclosed herein.
  • the Treg agent and the pharmaceutical composition are administered to the subject at the same time.
  • the Treg agent is administered to the subject before administering the pharmaceutical composition.
  • the Treg agent is administered to the subject after administering the pharmaceutical composition.
  • the Treg agent is administered to the subject an hour, a few hours, a day, a few days, a week, a few weeks, a month, or a few months, after administering the pharmaceutical composition.
  • the regulatory T cell inhibitory agent can be any Treg-targeting agent, including but not limited to, any compound, small molecule, toxin, polynucleotide (such as aptamer, RNAi, siRNA, or antisense oligonucleotide), polypeptide, protein (such as antibodies), fusion protein, drug conjugate, chemotherapeutic agents and the like, that (i) inhibits or decreases Treg cell functions, and/or (ii) depletes or diminishes regulatory T cell populations.
  • a Treg-targeting agent including but not limited to, any compound, small molecule, toxin, polynucleotide (such as aptamer, RNAi, siRNA, or antisense oligonucleotide), polypeptide, protein (such as antibodies), fusion protein, drug conjugate, chemotherapeutic agents and the like, that (i) inhibits or decreases Treg cell functions, and/or (ii) depletes or diminishes regulatory T cell populations.
  • the Treg agent is selected from the group consisting of an antibody, a small molecule, an antibody-drug conjugate, an immunotoxin, a peptide-drug conjugate, a peptide, a small interfering RNA, a siRNA conjugate, a chemotherapeutic agent, and any derivative, fragment or fusion thereof.
  • Treg agents include, but are not limited to, as those described in Yang, J., Bae, H. Drug conjugates for targeting regulatory T cells in the tumor microenvironment: guided missiles for cancer treatment. Exp Mol Med 55, 1996-2004, Kumar P, Kumar A, Parveen S, Murphy JR, Bishai W.
  • a Treg agent can comprise an antibody, or a fragment thereof, which specifically binds to a regulatory T cell surface protein.
  • Antibodies that comprise a Treg agent can target a surface protein of the Treg cell, which include, for example, CD25, CD4, CD28, CD38, CD62L (selectin), OX-40 ligand (OX-40L), cytotoxic T lymphocyte-associated antigen 4 (CTLA4), CCR4, CCR8, FOXP3, LAG3, CD 103, NRP-1, glucocorticoid-induced TNF receptor (GITR), galectin-1, TNFR2, or TGF-PR1.
  • the Treg agent can be, for example, ONTAK, HuMax-Tac, Zenapax, or MDX-010 or a combination thereof.
  • ONTAK is a monoclonal antibody that binds to the CD25 subunit of the IL-2 receptor.
  • HuMax- TAC is a fully human monoclonal antibody that targets the TAC antigen.
  • TAC is also known as CD25 or the alpha subunit of the interleukin-2 receptor (IL-2Ra) and is overexpressed by activated T-cells.
  • Zenapax is an immunosuppressive, humanized IgGl monoclonal antibody that binds to the CD25 subunit of the human high-affinity IL-2 receptor expressed on the surface of activated lymphocytes.
  • MDX-010 is a monoclonal antibody directed against CTLA4.
  • the Treg agent can comprise an antibody-drug conjugate.
  • the antibody, or fragment thereof can further comprise a radionuclide or toxic moiety such that the antibody can kill the regulatory T cell.
  • radionuclides suitable for use in the present disclosure can include those having suitable emission properties to provide ablation of targeted Tregs in situ, while not unduly exposing the surrounding cells and tissues to damaging levels of irradiation.
  • An ideal radionuclide for use in such therapeutic compositions is a relatively short-lived a-emitter, a y-emitter, or a P-emitter that emits enough gamma irradiation to cause local destruction.
  • Radionuclides include lutetium-177, iodine-131, iodine-125, and phosphorus-32 (y-emitters); actinium-225, astatine-211, and bismuth-212 and bismuth-213 (a-emitters); iodine-123, copper-64, iridium-192, osmium-194, rhodium-105, rhodium-186, samarium-153, and yttrium-88, yttrium-90, and yttrium-91.
  • the Treg agent can comprise a fusion protein.
  • the fusion protein can comprise a targeting moiety and a toxic moiety.
  • the targeting moiety can comprise a ligand or portion thereof of a regulatory T cell surface protein.
  • the ligand can be, for example, IL2, T cell receptor (TCR), MHCII, CD80, CD86, TARC, CCL17, CKLF1, CCL1, TCA-3, eotaxin, TER-1, E-cadherin, VEGF, semaphorin3a, CD134, CD31, CD62, CD38L, or glucocorticoid-induced TNF receptor ligand (GITRL).
  • the toxic moiety can comprise, for example, lectin, ricin, abrin, viscumin, modecin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, botulinum toxin, tetanus toxin, calicheamicin, or pokeweed antiviral protein.
  • the Treg agent can comprise an immunotoxin.
  • An immunotoxin can be an immunoconjugate that induces death of a target cell by combining an antibody with target-specific high-affinity binding activity with other molecules, such as radioisotopes, chemicals, siRNA, and cytotoxic proteins.
  • the immunotoxin comprises Denileukin diftitox (Ontak), Tagraxofusp (Elzonris), Moxetumomab pasudotox (Lumoxiti), or any derivative, combination thereof.
  • the Treg agent can comprise a peptide-drug conjugate (PDC).
  • PDC peptide-drug conjugate
  • a PDC comprises a peptide linked to a payload.
  • the peptide in the PDC can be target cell specific and induce receptor-mediated endocytosis of the conjugate.
  • the payload can be highly toxic drug such as maytansine, camptothecin derivatives, auristatin, or doxorubicin.
  • the Treg agent can comprise a small interfering RNA (siRNA).
  • siRNA is a double-stranded RNA (dsRNA) molecule 21-23 nucleotides in length that specifically causes RNA interference (RNAi), a posttranscriptional method of silencing gene expression.
  • RNAi RNA interference
  • Aptamers are single-stranded oligonucleotides that recognize their targets through their unique three- dimensional complementarity.
  • the Treg agent can comprise an siRNA conjugate, such as CTLA4apt-STAT3 siRNA, NPsiCTLA-4, or a hybrid SNP.
  • CTLA4apt- STAT3 siRNA is a siRNA conjugate in which CTLA4 binding RNA aptamer and mouse STAT3 siRNA are linked.
  • NPsiCTLA-4 is a nanostructure material-siRNA conjugate in which siRNA-targeting CTLA-4 mRNA is surrounded by nanoparticles composed of PEG5k- PLA1 Ik and BHEM-Chol.
  • Hybrid SNPs are spherical nucleotide nanoparticles (SNPs) loaded with a CTLA-4-siRNA aptamer (cSNP) and PD-1 siRNA (pSNP) in a nanoparticle comprising an amphiphilic polymer of PLGA-S-S-PEG as the core and the cationic lipid DOTAP.
  • SNPs spherical nucleotide nanoparticles
  • cSNP CTLA-4-siRNA aptamer
  • pSNP PD-1 siRNA
  • a Treg agent modulates a regulatory T cell via either decreasing the activity or function of a Treg after the Treg agent is administered to a subject; or a Treg agent that is attached to a toxic moiety can kill or ablate regulatory T cells.
  • the administration of a Treg agent or derivatives thereof can block the action of its target (for example a Treg cell surface marker).
  • a Treg agent can decrease the suppression of immune system activation and can decrease the prevention of self-reactivity. Such a decrease can be measured via techniques established in the art.
  • Non-limiting examples of assays used for the detection of T cell responses include delayed- type hypersensitivity responses; in vitro T cell proliferation responses (e.g., measured by incorporation of radioactive nucleotides); library screens; expression arrays; T cell cytokine responses (e.g., measured by ELISA or other related immuno-assays or RT-PCR for specific cytokine mRNA); as well as any other assay established in the art for measuring a B cell and/or T cell immune response in a subject.
  • Methods for detecting an immune response can include, but are not limited to, antibody detection assays such as, for example, EIA (enzyme immunoassay); ELISA (enzyme linked immunosorbent assay); agglutination reactions; precipitation/flocculation reactions, immunoblots (Western blot; dot/slot blot); radioimmunoassays; immunodiffusion assays (RIA); histochemical assays; immunofluorescence assays (FACS); chemiluminescence assays, library screens, expression arrays, etc.
  • EIA enzyme immunoassay
  • ELISA enzyme linked immunosorbent assay
  • agglutination reactions precipitation/flocculation reactions
  • immunoblots Western blot; dot/slot blot
  • radioimmunoassays immunodiffusion assays (RIA); histochemical assays; immunofluorescence assays (FACS); chemiluminescence assay
  • the method further comprising administering to the subject one or more doses of cyclophosphamide intravenously at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or longer, prior to administering to the subject the pharmaceutical composition described herein.
  • the cyclophosphamide is administered at least about 2-3 days prior to administering to the subject the pharmaceutical composition described herein.
  • a low-dose of cyclophosphamide at about 100, 150, 200, 250, 300, or 450 mg/m 2 is administered to the subject.
  • the method further comprising administering to the subject one or more doses of an interferon-alpha-2b (IFN-a2b), IFN-a2a, or a pegylated IFN-a2a intradermally at the inoculation site of the pharmaceutical composition described herein.
  • the method further comprising administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, or 84 hours following administering to the subject the pharmaceutical composition described herein.
  • the method further comprising administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally at about 1-4 hours, about 2-6 hours, about 8-12 hours, about 10-24 hours, about 20-48 hours, or about 60-72 hours following administering to the subject the pharmaceutical composition described herein. In some embodiments, the method further comprising administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally no later than 5, 10, 15, 20, 25, 30, 45, 50, 60, 72, or 84 hours after administering to the subject the pharmaceutical composition.
  • the method further comprising administering to the subject one or more doses of IFN-a2b, IFN- a2a or pegylated IFN-a2a intradermally no later than 1, 2, 3, 4, 5, or 6 days following administering to the subject the pharmaceutical composition. In some embodiments, the method further comprising administering to the subject one or more doses of IFN-a2b, IFN- a2a or pegylated IFN-a2a intradermally no later than about 1-6 days, 2-3 days, or 3-5 days following administering to the subject the pharmaceutical composition.
  • the method further comprising administering to the subject a first doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally between 1 to 4 hours and a second dose of IFN-a2b, IFN- a2a or pegylated IFN-a2a intradermally between 1-3 days following administering to the subject the pharmaceutical composition.
  • the IFN-a2b, administered is at a low-dose between about 1-20,000 IU, 100-15,000 IU, 5000-12,000 IU, or 9,000-11,000 IU.
  • the IFN-a2b administered is at dose of about 10,000 IU.
  • the IFN-a2a or pegylated IFN-a2a, administered is at a low-dose between about 0.01-0.1 micrograms (mcg), 0.05 - 0.15 mcg, 0.06 - 0.12 mcg, or 0.09 -0.11 mcg.
  • the IFN-a2b administered is at dose of about 0.1 mcg.
  • the method further comprises administering to the subject one or more additional therapies.
  • additional types include, but are not limited to, chemotherapy, immunotherapy, radiotherapy, hormone therapy, a differentiating agent, and a small-molecule drug.
  • suitable additional types include, but are not limited to, chemotherapy, immunotherapy, radiotherapy, hormone therapy, a differentiating agent, and a small-molecule drug.
  • Chemotherapeutic agents that can be used in the present disclosure include but are not limited to alkylating agents (e.g., nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (e.g., streptozocin, carmustine (BCNU), lomustine), alkyl sulfonates (e.g., busulfan), triazines (e.g., dacarbazine (DTIC), temozlomide), ethylenimines (e.g., thiotepa, altretamine (hexamethylmelamine)), platinum drugs (e.g., cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., 5 -fluorouracil (5-FU), 6- mercaptopurine (6-MP), capecitabine, cytar
  • Topoisomerase inhibitors are compounds that inhibit the activity of topoisomerases, which are enzymes that facilitate changes in DNA structure by catalyzing the breaking and rejoining of phosphodiester bonds in the backbones of DNA strands. Such changes in DNA structure are necessary for DNA replication during the normal cell cycle. Topoisomerase inhibitors inhibit DNA ligation during the cell cycle, leading to an increased number of single- and double-stranded breaks and thus a degradation of genomic stability. Such a degradation of genomic stability leads to apoptosis and cell death.
  • Topoisomerases are often divided into type I and type II topoisomerases.
  • Type I topoisomerases are essential for the relaxation of DNA supercoiling during DNA replication and transcription.
  • Type I topoisomerases generate DNA single-strand breaks and also religate said breaks to re-establish an intact duplex DNA molecule.
  • Examples of inhibitors of topoisomerase type I include irinotecan, topotecan, camptothecin, and lamellarin D, which all target type IB topoisomerases.
  • Type II topoisomerase inhibitors are broadly classified as topoisomerase poisons and topoisomerase inhibitors. Topoisomerase poisons target topoisomerase-DNA complexes, while topoisomerase inhibitors disrupt enzyme catalytic turnover. Examples of type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, and fluoroquinolones.
  • the chemotherapeutic agent is a topoisomerase inhibitor.
  • the topoisomerase inhibitor is a topoisomerase I inhibitor, a topoisomerase II inhibitor, or a combination thereof.
  • the topoisomerase inhibitor is selected from the group consisting of doxorubicin, etoposide, teniposide, daunorubicin, mitoxantrone, amsacrine, an ellipticine, aurintricarboxylic acid, HU-331, irinotecan, topotecan, camptothecin, lamellarin D, resveratrol, genistein, quercetin, epigallocatechin gallate (EGCG), and a combination thereof.
  • EGCG is one example of a plant-derived natural phenol that serves as a suitable topoisomerase inhibitor.
  • the topoisomerase inhibitor is doxorubicin.
  • Immunotherapy refers to any treatment that uses the subj ect’ s immune system to fight a disease (e.g., cancer). Immunotherapy methods can be directed to either enhancing or suppressing immune function. In the context of cancer therapies, immunotherapy methods are typically directed to enhancing or activating immune function.
  • an immunotherapeutic agent comprises a monoclonal antibody that targets a particular type or part of a cancer cell. In some cases, the antibody is conjugated to a moiety such as a drug molecule or a radioactive substance.
  • Antibodies can be derived from mouse, chimeric, or humanized, as non-limiting examples.
  • Non-limiting examples of therapeutic monoclonal antibodies include alemtuzumab, bevacizumab, cetuximab, daratumumab, ipilimumab (MDX-101), nivolumab, ofatumumab, panitumumab, pembrolizumab, retifanlimab, rituximab, tositumomab, and trastuzumab.
  • Immunotherapeutic agents can also comprise an immune checkpoint inhibitor, which modulates the ability of the immune system to distinguish between normal and “foreign” cells.
  • Programmed cell death protein 1 (PD-1) and protein death ligand 1 (PD-L1) are common targets of immune checkpoint inhibitors, as disruption of the interaction between PD1 and PD- L1 enhance the activity of immune cells against foreign cells such as cancer cells.
  • PD-1 inhibitors include pembrolizumab, retifanlimab and nivolumab.
  • An example of a PD- L1 inhibitor is atezolizumab.
  • CTLA-4 cytotoxic T lymphocyte-associated protein 4
  • ipilimumab is a monoclonal antibody that binds to and inhibits CTLA-4.
  • Radiotherapy refers to the delivery of high-energy radiation to a subject for the treatment of a disease (e.g., cancer).
  • Radiotherapy can comprise the delivery of X-rays, gamma rays, and/or charged particles.
  • Radiotherapy can be delivered locally (e.g. to the site or region of a tumor), or systemically (e.g., a radioactive substance such as radioactive iodine is administered systemically and travels to the site of the tumor).
  • hormone therapy can refer to an inhibitor of hormone synthesis, a hormone receptor antagonist, or a hormone supplement agent.
  • Inhibitors of hormone synthesis include but are not limited to aromatase inhibitors and gonadotropin releasing hormone (GnRH) analogs.
  • Hormone receptor antagonists include but are not limited to selective receptor antagonists and antiandrogen drugs.
  • Hormone supplement agents include but are not limited to progestogens, androgens, estrogens, and somatostatin analogs.
  • Aromatase inhibitors are used, for example, to treat breast cancer. Non-limiting examples include letrozole, anastrozole, and aminoglutethimide.
  • GnRH analogs can be used, for example, to induce chemical castration.
  • Selective estrogen receptor antagonists which are commonly used for the treatment of breast cancer, include tamoxifen, raloxifene, toremifene, and fulvestrant.
  • Antiandrogen drugs which bind to and inhibit the androgen receptor, are commonly used to inhibit the growth and survival effects of testosterone on prostate cancer.
  • Non-limiting examples include flutamide, apalutamide, and bicalutamide.
  • the term “differentiating agent” refers to any substance that promotes cell differentiation, which in the context of cancer can promote malignant cells to assume a less stem cell-like state.
  • a non-limiting example of an anti-cancer differentiating agent is retinoic acid.
  • Small molecule drugs generally are pharmacological agents that have a low molecular weight (i.e., less than about 900 daltons).
  • Non-limiting examples of small molecule drugs used to treat cancer include bortezomib (a proteasome inhibitor), imatinib (a tyrosine kinase inhibitor), and seliciclib (a cyclin-dependent kinase inhibitor), and epacadostat (an indoleamine 2,3 -dioxygenase (IDO1) inhibitor).
  • the method comprises administering to the subject an effective amount of the pharmaceutical composition intradermally in the upper back or thighs.
  • the upper back and thighs are chosen for patient acceptability as these areas have less nerves in the skin and are thus less sensitive.
  • the draining lymph nodes in the proximity may convey antigens from breast tumors in the upper and lower torso, which are common sites for breast cancer metastases.
  • the method may further comprise administering to the subject the pharmaceutical composition in an interval of every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks.
  • the method comprises administering to be subject the pharmaceutical composition in an interval of every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 12 months, every 18 months, or every 24 months. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for at least 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for not more than 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 weeks, or 52 weeks. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for not more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 12 months.
  • the method comprises administering to the subject an effective amount of the pharmaceutical composition through oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration.
  • administration of the effective amount of the pharmaceutical composition is performed by parenteral administration (e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial) or transmucosal administration (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal).
  • the method comprises the use of liposomal formulations, intravenous infusion, or transdermal patches.
  • Therapy such as engineered mammalian dendritic cell(s), composition(s), and pharmaceutical composition(s) of the present disclosure can be administered using routes, dosages, and protocols that will readily be known to one of skill in the art. Administration can be conducted once per day, once every two days, once every three days, once every four days, once every five days, once every six days, or once per week. Therapy can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more times per week.
  • engineered mammalian dendritic cell(s), composition(s), and/or pharmaceutical composition(s) of the present disclosure are administered as a single dose, co-administered (e.g., administered in separate doses or by different routes, but close together in time), or administered separately (e.g., administered in different doses, including the same or different route, but separated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours).
  • administration can occur, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more times in a day.
  • therapeutic administration can occur about once per week, about every two weeks, about every three weeks, or about once per month. In other cases, therapeutic administration can occur about 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, or more times per month. Treatment can continue for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; or longer. At any time during treatment, the therapeutic plan can be adjusted as necessary.
  • a different vaccine may be selected, one or more additional therapeutic agents or drugs may be chosen, or any aspect of the therapeutic plan can be discontinued.
  • One of skill in the art will readily be able to make such decisions, which can be informed by, for example, the results of allele profile comparison, changes in the activity and/or number of an immune cell, and/or changes in the the presence or level of one or more biomarkers.
  • the engineered mammalian dendritic cell(s), composition(s), and pharmaceutical composition(s) of the present disclosure can be administered by any suitable route, including those described herein.
  • the administration is by intradermal or intralymphatic injection.
  • the whole-cell cancer vaccine e.g., comprising engineered mammalian dendritic cells of the present disclosure
  • IFNa interferon alpha
  • the IFNa is injected locally. IFNa can be given before and/or after the vaccine is administered. Timing of the separate injections can be any suitable interval, including those described herein.
  • a dose may include, for example, about 50,000 to 50,000,000 (e.g., about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 25,000,000, 30,000,000,
  • a dose may contain about 1,000,000 engineered mammalian dendritic cells. In some embodiments a dose may contain about 5,000,000 engineered mammalian dendritic cells. In some embodiments a dose may contain about 10,000,000 engineered mammalian dendritic cells. In some embodiments a dose may contain about 20,000,000 engineered mammalian dendritic cells.
  • a dose may also include, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000, or more) engineered mammalian dendritic cells.
  • 5,000,000 to 100,000,000 e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000, or more engineered mammalian dendritic cells.
  • a dose may alternatively include, for example, at least about 100,000,000 to 1,000,000,000 (e.g., about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000,000, 550,000,000, 600,000,000,
  • the engineered mammalian dendritic cells are irradiated.
  • the irradiation dose may be, for example, between about 2 and 2,000 Gy (e.g., about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 Gy).
  • the engineered mammalian dendritic cells are irradiated with a dose of about 100 Gy.
  • the method of treating cancer of the present disclosure further comprises selecting a whole-cell cancer vaccine for the subject according to a method of the present disclosure described herein.
  • the subject has stage I, stage II, stage III, and/or stage IV cancer.
  • the cancer is transitioning between stages.
  • the subject has a pre-cancerous lesion.
  • the subject does not have cancer.
  • treating the subject comprises inhibiting cancer cell growth, inhibiting cancer cell proliferation, inhibiting cancer cell migration, inhibiting cancer cell invasion, ameliorating or eliminating the symptoms of cancer, reducing the size (e.g., volume) of a cancer tumor, reducing the number of cancer tumors, reducing the number of cancer cells, inducing cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death, or enhancing the therapeutic effects of a composition or pharmaceutical composition.
  • treating the subject results in an increased survival time. In some instances, overall survival is increased. In other instances, disease-free survival is increased. In some instances, progression-free survival is increased. In certain embodiments, treating the subject results in a reduction in tumor volume and/or increased survival time.
  • treating the subject enhances the therapeutic effects of an anti-cancer therapy such as a chemotherapeutic agent, an immunotherapeutic agent, radiotherapy, hormone therapy, a differentiating agent, and/or a small-molecule drug.
  • an anti-cancer therapy such as a chemotherapeutic agent, an immunotherapeutic agent, radiotherapy, hormone therapy, a differentiating agent, and/or a small-molecule drug.
  • treating the subject results in a decrease in the presence or level of one or more heterologous antigens measured or detected in a sample obtained from the subject. In some embodiments, treating the subject results in an increase in the presence or level of one or more biomarkers measured or detected in a sample obtained from the subject. In certain embodiments, treating the subject results in no change the presence or level of the one or more biomarkers.
  • treating the subject results in an increase in the activity and/or number of one or more immune cells.
  • the increase is produced in one cell type.
  • the increase is produced in multiple cell types.
  • the cell in which the level of activity and/or number is increased is selected from the group consisting of a peripheral blood mononuclear cell (PBMC), a lymphocyte (e.g. T lymphocyte, B lymphocyte, NK cell), a monocyte, a dendritic cell, a macrophage, a myeloid-derived suppressor cell (MDSC), and a combination thereof.
  • PBMC peripheral blood mononuclear cell
  • lymphocyte e.g. T lymphocyte, B lymphocyte, NK cell
  • monocyte e.g. T lymphocyte, B lymphocyte, NK cell
  • monocyte e.g. T lymphocyte, B lymphocyte, NK cell
  • MDSC myeloid-derived suppressor cell
  • the level of activity and/or number of immune cell(s) is measured using methods of the present disclosure described herein.
  • an increase in immune cell activity and/or number indicates that the subject should be administered one or more additional doses of the pharmaceutical composition (e.g., comprising engineered mammalian dendritic cells of the present disclosure).
  • a different vaccine is administered.
  • an increase in immune cell activity and/or number will occur, in some instances, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more doses of the vaccine have been administered.
  • a sample is obtained from the subject. In other embodiments, a sample is obtained from a different subject or a population of subjects. Samples can be used for the purposes of selecting an appropriate cancer vaccine of the present disclosure, monitoring the response to vaccine therapy, and/or predicting how the subject will respond to vaccine therapy. Samples obtained from a different subject and/or a population of subjects can be used, for example, to establish reference ranges to facilitate comparisons that are part of the methods of the present disclosure. Samples can be obtained at any time, including before and/or after administration of the engineered mammalian dendritic cell(s), pharmaceutical composition(s), and/or other composition(s) of the present disclosure.
  • the sample comprises whole blood, plasma, serum, cerebrospinal fluid, tissue, saliva, buccal cells, tumor tissue, urine, fluid obtained from a pleural effusion, hair, skin, or a combination thereof.
  • the sample can comprise any biofluid.
  • any cell, tissue, or biofluid type is suitable, as long as it contains a sufficient amount of DNA or RNA to allow typing.
  • the sample comprises circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • the sample can also be made up of a combination of normal and cancer cells.
  • the sample comprises circulating tumor cells (CTCs).
  • the sample can be obtained, for example, from a biopsy, from a surgical resection, and/or as a fine needle aspirate (FNA). Samples can be used to determine, measure, or detect MHC allele(s), immune cell activity and/or number, and/or biomarker(s), as described herein.
  • FNA fine needle aspirate
  • the results of the MHC typing e.g., the alleles present in an allele profile, the results of a comparison of allele profiles
  • immune cell activity and/or number measurement, and/or biomarker presence or level determinations are recorded in a tangible medium.
  • the results of assays e.g., the alleles present in an allele profile, the results of a comparison of allele profiles, the activity level and/or number of immune cells, the presence or level (e.g., expression) of one or more biomarkers and/or a prognosis or diagnosis (e.g., of whether or not there is the presence of cancer, the prediction of whether the subject will respond to a vaccine, or whether the subject is responding to a vaccine) can be recorded, e.g., on paper or on electronic media (e.g., audio tape, a computer disk, a CD, a flash drive, etc.).
  • the methods further comprise the step of providing the results of assays, prognosis, and/or diagnosis to the patient (i.e., the subject) and/or the results of treatment.
  • the present disclosure provides a kit for treating a subject with a cancer.
  • the kit comprises an engineered mammalian dendritic cell line, a composition, and/or a pharmaceutical composition of the present disclosure described herein.
  • kits are useful for treating any cancer, some non-limiting examples of which include breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, liver cancer, brain cancer, eye cancer, soft tissue cancer, renal cancer, bladder cancer, head and neck cancer, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, and any other cancer described herein, including a combination thereof.
  • cancer some non-limiting examples of which include breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, liver cancer, brain cancer, eye cancer, soft tissue cancer, renal cancer, bladder cancer, head and neck cancer, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, and any other cancer described herein, including a
  • kits Materials and reagents to carry out the various methods of the present disclosure can be provided in kits to facilitate execution of the methods.
  • kit includes a combination of articles that facilitates a process, assay, analysis, or manipulation.
  • the kits of the present disclosure find utility in a wide range of applications including, for example, diagnostics, prognostics, therapy, and the like.
  • Kits can contain chemical reagents as well as other components.
  • the kits of the present disclosure can include, without limitation, instructions to the kit user, apparatus and reagents for sample collection and/or purification, apparatus and reagents for product collection and/or purification, apparatus and reagents for administering engineered mammalian dendritic cell(s) or other composition(s) of the present disclosure, apparatus and reagents for determining the level(s) of biomarker(s) and/or the activity and/or number of immune cells, apparatus and reagents for detecting MHC alleles, sample tubes, holders, trays, racks, dishes, plates, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples.
  • Kits of the present disclosure can also be packaged for convenient storage and safe shipping, for example, in a box having a lid.
  • the kits may be stored and shipped at room temperature, on wet ice or with cold packs, or frozen in the vapor phase of liquid nitrogen or in dry ice.
  • the kits also contain negative and positive control samples for detection of MHC alleles, immune cell activity and/or number, and/or the presence or level of biomarkers.
  • the negative control samples are non-cancer cells, tissue, or biofluid obtained from the subject who is to be treated or is already undergoing treatment.
  • the negative control samples are obtained from individuals or groups of individuals who do not have cancer.
  • kits contain samples for the preparation of a titrated curve of one or more biomarkers in a sample, to assist in the evaluation of quantified levels of the activity and/or number of one or more immune cells and/or biomarkers in a biological sample.
  • This example illustrates how to generate syngeneic or semi-allogeneic bone marrow dendritic cells (BMDCs) from three mouse strains.
  • BMDCs bone marrow dendritic cells
  • BMDCs bone marrow dendritic cells
  • Both B6.C-7/2-A" / ’"' / /ByJ (bml) and ⁇ 6(C)-H2-Ab l bm12 /KhEgJ (bml2) mouse strains are derived originally from C57BL/6J (WT) mouse strain.
  • the B6.C-7/2-A" / ’"' / /ByJ (bml) mouse strain has point mutations in the MHC class I H2-K b allele.
  • the B6(C)-J/2- H&7 fem72 /KhEgJ (bml2) mouse strain has point mutations in the MHC class II H2-IA b allele.
  • the mouse strains and their MHC haplotypes and alloantigens are listed in Table 1.
  • C57BL/6J is a wild-type (WT) mouse strain with haplotype H2b (FIG. 1A).
  • MHC haplotype antigens of C57BL/6J mice are H-2D b , H-2K b , and I-A b .
  • B6.C-H2-K bml IBy] (bml) is an MHC class I mutant with a variant allele, H2-K bml (FIG. IB).
  • the H2-K bml allele differs from H2-K b by 7 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide-binding groove in positions 152, 155 and 156 of the a2 domain.
  • B6.C-H2- K bml /ByJ (bml) bears the haplotype H-2D b , H-2K bml , and I-A b .
  • B6(C)-772MZ>7 fem72 /KhEgJ (bml2) is an MHC class II mutant with a variant allele, H2-Abl bm12 (FIG. 1C).
  • the H2- Abl bm i 2 allele differs from the H2-IA b allele by 3 nucleotides resulting in 3 amino acid substitutions occurring along the edge of the peptide-binding groove in positions 67, 70 and 71 of the pi domain.
  • B6(C)-772MZ>7 fem72 /KhEgJ (bml2) bears the haplotype H-2D b , H-2K b , and
  • Antigen presenting cells from a C57BL/6J WT mouse comprise wild type MHC alloantigens H2-D b , H2-K b , and H2-IA b .
  • APCs are pulsed by a E7 peptide from human papilloma virus (HPV) protein E7, only MHC class I alloantigen, H2-D b presents the E7 peptide (FIG. 2A).
  • APCs from a B6 When such APCs are pulsed by a E7 peptide from human papilloma virus (HPV) protein E7, only MHC class I alloantigen, H2-D b presents the E7 peptide (FIG. 2A).
  • HPV human papilloma virus
  • C-H2-K bml /By (bml) mouse comprise wild type H2-D b which presents the E7 peptide, and wild type H2-IA b , and mutant H2-K b which stimulates MHC class I allogeneic help (FIG. 2B).
  • APCs from a B6(C)-772- HZ>7 fem72 /KhEgJ (bml2) mouse contains wild type H2-D b which presents the E7 peptide, and wild type H2-K b , and mutant H2-IA b which stimulates MHC class II allogeneic help (FIG. 2C).
  • FIG. 2D indicates the genetic loci of the murine H-2 complex, also known as the murine major histocompatibility complex (MHC).
  • MHC murine major histocompatibility complex
  • Syngeneic BMDCs are the BMDCs collected from one mouse strain (e.g., C57BL/6J WT), pulsed with an antigen peptide (e.g., H-2D b -restricted E743-77 peptide), and injected back to the same mouse strain (e.g., C57BL/6J WT).
  • Semi-allogeneic BMDCs can be the BMDCs collected from one mouse strain (e.g., C57BL/6J WT), pulsed with an antigenic peptide, and injected to a different mouse strain which has at least one different MHC alloantigen (e.g., B6.
  • Semi- allogeneic BMDCs can be the BMDCs collected from B6.
  • an antigenic peptide e.g., 7/-2/U-restricted E743-77 peptide
  • BMDCs Bone-Marrow Derived Dendritic Cells
  • This example illustrates a method to generate dendritic cells (DCs) from murine bone marrow (BM) progenitors.
  • DCs dendritic cells
  • BM murine bone marrow
  • DCs Dendritic cells
  • BM bone marrow
  • B6.C-H2-K bml /By] bml
  • B6(C)-H2-Abl bml2 /KhEg] bml2
  • FBS fetal bovine serum
  • the BM cells were centrifuged at 1500 rpm for 5 min and then lysed red blood cells in Ammonium-Chloride- Potassium lysing buffer (Lonza, USA). The cells were strained through a 40 pm filter and washed twice with cell culture medium by centrifuging. At Day 0, 2 x 10 6 cells were seeded in 100 mm-bacteriological petri dishes in a total volume of 10 ml culture medium containing 20 ng/ml recombinant murine granulocyte- macrophage colony-stimulating factor (GM-CSF; PeproTeck/Tebu, Frankfurt, Germany). Additional 10 ml of culture medium containing 20 ng/ml GM-CSF were added on Day 3.
  • GM-CSF murine granulocyte- macrophage colony-stimulating factor
  • This example illustrates a method of stimulating BMDCs using an antigen peptide and maturing BMDCs.
  • BMDCs from C57BL/6J (WT), 6.C-H2-K bml /ByJ (bml), or B6(C)- 2-/lZ>7 6m72 /KhEgJ (bml2) were collected, resuspended at 1-2 x 10 6 cells/ml in fresh culture medium and were incubated for 1- 2 hours with a peptide from the E7 protein, E743-77 (10 pg/ml, United biosystems #241093) for antigen pulsing.
  • E743-77 10 pg/ml, United biosystems #241093
  • GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO: 1).
  • the E7 peptide binds to the H-2D b MHC molecules on the BMDCs (FIG. 2).
  • These antigen-pulsed BMDCs can function as antigen presenting cells to present antigen through the 77-27) fe -E7 peptide complex to CD8+ cells, thus stimulating CD8+ cells to attack E7-expressing TC-1 cancer cells, eventually slowing or stopping the growth of the cancer.
  • BMDCs from C57BL/6J (WT), 6.C-H2-K bml /ByJ (bml), or B6(C)- H2-Abl bml2 /KhEgJ (bml2) were matured with 100 ng/ml LPS overnight. After overnight incubation, the cells were detached from petri dishes by 2 mM EDTA and washed with PBS to remove the peptide and LPS and resuspended in PBS for further use. The expression of CD11c in the BMDCs were examined by flow cytometry.
  • This example illustrates that a peptide-pulsed MHC class II mutant dendritic cell vaccine has superior efficacy in a murine tumor model.
  • BMDCs Syngeneic bone marrow dendritic cells
  • BMDCs Syngeneic bone marrow dendritic cells
  • BMDCs were generated from C57BL/6 and semi-allogeneic BMDCs were generated from two mouse strains, ⁇ 6.C-H2-K bml /By J and B6(C)-772-24Z>7 fem72 /KhEgJ which had limited point mutations in the MHC class I H2-K b allele or MHC class II H2-IA b allele, respectively.
  • Each BMDC was pulsed with 77-27) fe -restricted E743-77 peptide and matured before injection.
  • mice received intradermal injections of syngeneic or one of the semi-allogeneic E7-pulsed BMDC vaccines starting 8-9 days after the TC-1 implantation.
  • the MHC class I mutant BMDC vaccine had efficacy similar to the syngeneic BMDC vaccine in suppressing TC-1 tumor growth.
  • the MHC class II mutant BMDC vaccine had efficacy significantly superior to that of the other BMDC vaccines.
  • MHC class II semi- allogeneic BMDCs may be more effective than syngeneic DC-based cancer vaccines, presumably because the class II alloantigens induce additional T cell help for anti-tumor immunity.
  • TC-1 cells are a mouse lung cancer cell line expressing human papilloma virus (HPV) proteins E6 and E7. Eight days after the TC-1 cell inoculation, all tumor growth on mice reached around 5 mm in diameter.
  • HPV human papilloma virus
  • mice were vaccinated intradermally 2 x 10 6 (1.6 x 10 6 on Day 19) syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi-allogeneic E7-pulsed BMDCs from B6.C-772-7f fem7 /ByJ (E7-mBMDC bml), or semi- allogeneic E7-pulsed BMDCs from B6(C)-7/2-/lZ>7 6m72 /KhEgJ (E7-mBMDC bml2).
  • the control group was injected with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • FIG. 5A compared to the PBS control group, vaccination with the E7-pulsed BMDC WT slowed tumor growth.
  • the E7-pulsed BMDCs of bml (MHC class I mutant) showed a similar effect as the E7-pulsed BMDCs (WT), but the E7-pulsed BMDCs of bml2 (MHC class II mutant) showed a greater effect than either the E7-pulsed BMDCs (WT) or the E7-pulsed BMDCs of bml (MHC class I mutant).
  • FIG. 6 More specific tumor measurements (tumor volume growth and tumor weight) of the mice after TC-1 cell inoculation and E7-pulsed mBMDCs vaccinations are shown in FIG. 6. Tumor volumes on Day 19, 21, 34 post-TC-1 cell inoculation indicate that all vaccinated mice exhibited slower tumor growth compared with the PBS control mice, and after the third vaccination on Day 19, the mice vaccinated with the E7-pulsed mBMDCs of bml2 (MHC class II mutant) exhibited the best response compared with all other groups (FIGS.
  • mice 6A-C All the mice were euthanized, and all the tumor weights were measured on Day 35.
  • mice Female C57BL/6J mice (9 weeks old) were inoculated subcutaneously with TC-1 cells and later vaccinated intradermally with either syngeneic or semi-allogeneic E7-pulsed BMDC vaccines for 4 times.
  • mice Nine days after the inoculation, all tumor growth on mice reached around 5 mm in diameter.
  • mice were vaccinated intradermally 2 x 10 6 (1.6 x 10 6 on Day 14) syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT), or semi- allogeneic E7-pulsed BMDCs from B6.C-J/2-7/ fem7 /ByJ (E7-mBMDC bml), or semi-allogeneic E7-pulsed BMDCs from B6(C)-.H2-n&7 6m72 /KhEgJ (E7-mBMDC bml2).
  • the control group was injected with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • mice vaccinated with the E7-BMDC WT slowed tumor growth compared with the PBS control group.
  • the mice vaccinated with the E7-BMDC bml showed a similar effect as the mice vaccinated with the E7-BMDC WT.
  • the mice vaccinated with the E7-BMDC bml2 (MHC class II mutant) showed a greater effect than either the E7- BMDCs WT or the E7-BMDC bml (MHC class I mutant), as indicated by the tumor growth (FIG. 7A) of the mice.
  • This example illustrates that CD4 T cell depletion assists semi-allogeneic cancer vaccines to limit a late-stage tumor growth in a murine tumor model.
  • mice Female C57BL/6J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 5 TC-1 cells. Eight days after the inoculation, all tumors on the mice reached around 5 mm in diameter. Intraperitoneal injection of anti-CD4 antibody at an early stage of tumor growth (Early aCD4), or anti-CD8 antibody (aCD8), was started on day 6 (200 pg/mouse) after the inoculation and continued every 2-4 days (100 pg/mouse) to the end of the experiment.
  • Early aCD4 antibody at an early stage of tumor growth (Early aCD4)
  • aCD8 antibody anti-CD8 antibody
  • Intraperitoneal injection of anti-CD4 antibody at a late stage of tumor growth was started on day 17 (200 pg/mouse) after the inoculation and continued every 2-4 days (100 pg/mouse) to the end of the experiment.
  • mice were vaccinated intradermally with 2 x 10 6 syngeneic E7-pulsed BMDCs from C57BL/6J (E7-mBMDC WT) or semi-allogeneic E7-pulsed BMDCs from B6(C)-7/2- HZ>7 6 '” 72 /KhEgJ (E7-mBMDC bml2).
  • the control group was injected with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • mice vaccinated with E7- BMDC WT with isotype control antibody (Isotype + the E7-BMDC WT) and the mice vaccinated with E7-BMDC WT and treated with anti-CD4 antibody at an early stage of tumor growth (Early aCD4 + E7-BMDC WT) showed similar tumor growth curves (FIG. 8B).
  • the mice vaccinated with E7-BMDC bml2 with isotype control antibody (Isotype + E7-mBMDC bml2) showed a great effect on limiting tumor growth (FIG. 8C).
  • FIG. 9 shows tumor volume growth of the mice on day 14 (FIG. 9A), 20 (FIG.9B), and 29 (FIG. 9C) after TC-1 cell inoculation in the CD4 or CD8 T cell depletion study.
  • FIG. 9A shows tumor volume growth of the mice on day 14 (FIG. 9A), 20 (FIG.9B), and 29 (FIG. 9C) after TC-1 cell inoculation in the CD4 or CD8 T cell depletion study.
  • FIG. 9A shows tumor volume growth of the mice on day 14 (FIG. 9A), 20 (FIG.9B), and 29 (FIG. 9C) after TC-1 cell inoculation in the CD4 or CD8 T cell depletion study.
  • FIG. 9A shows tumor volume growth of the mice on day 14 (FIG. 9A), 20 (FIG.9B), and 29 (FIG. 9C) after TC-1 cell inoculation in the CD4 or CD8 T cell depletion study.
  • This example illustrates that Treg depletion assists semi-allogeneic cancer vaccines to limit tumor growth in a murine tumor model.
  • mice To further confirm whether the depletion of CD4 + CD25 + suppressor T cells or Regulatory T cells (Treg cells) could contribute E7-mBMDC bml2 to suppress tumor growth in mice, 6 groups of female B6.129(Cg)-Foxp3tm3(Hbegf/GFP)Ayr/J mice (10 weeks old) were inoculated subcutaneously with 1 x 10 5 TC-1 cells. Seven days after the inoculation, all tumors on the mice reached around 5 mm long diameter. The first group of mice was injected with phosphate-buffered saline (PBS) only as a control group.
  • PBS phosphate-buffered saline
  • mice Two groups of mice (2 doses of E7-mBMDC bml2) were vaccinated intradermally 2 x 10 6 semi-allogeneic E7-pulsed BMDCs from B6(C)-Z72-HZ>7 6m72 /KhEgJ (E7-mBMDC bml2) on day 3 and 13 after the TC-1 cell inoculation.
  • Three groups of mice (5 doses of E7-mBMDC bml2) received the same vaccine on day 8, 13, 18, 23, and 28.
  • These Foxp3 DTR knock-in mice express the human diphtheria toxin (DT) receptor, and they are depleted for Treg cells when they are injected with DT.
  • DT diphtheria toxin
  • mice received late DT injection in which intraperitoneal injection of 10 pg/kg DT was started on day 15 after the inoculation, and the other group did not receive DT treatment (2 doses of E7-mBMDC bml2).
  • mice (Early DT + 5 doses of E7-mBMDC bm!2) received early DT injection in which intraperitoneal injection of 10 pg/kg DT was started on day 6 after the inoculation; one group of mice (late DT + 5 doses of E7-mBMDC bml2) received late DT inj ection started on day 15 after the inoculation; and the last group did not receive DT treatment (5 doses of E7-mBMDC bml2). All the mice treated with DT continued receiving DT treatment every 2-4 days to the end of the experiment.
  • mice vaccinated with E7-BMDC bml2 only displayed a reduced tumor growth: the treatment of “early DT + 5 doses of E7-BMDC bml2” showed a slower tumor growth at an early stage, the treatment of “late DT + 2 doses E7-BMDC bml2” showed a slower tumor grow at a late stage of tumor growth, and the treatment of “late DT + 5 doses E7-BMDC bml2” showed the best effect on controlling last-stage tumor grow and also exhibited tumor suppression at the end of the experiment.
  • Treg cell depletion assists semi-allogeneic cancer vaccine to limit tumor growth in mice.
  • Example 7 IFNy Production in TC-1 Bearing T cells Co-cultured With BMDC Vaccines
  • This example illustrates IFNy production in TC-1 bearing mouse T cells co-cultured with mBMDC WT, E7-mBMDCs WT, mBMDC bml2, E7-mBMDC bml2.
  • CD4 and CD8 T cells were isolated from spleens of TC-1 bearing mice 8 days after TC-1 inoculation and incubated at a concentration of 2 x 10 5 cells per well, alone or co-cultured with BMDCs (1 x 10 5 cells per well, with or without E7-pulsing), in a 96-well round plate for 24 hrs or 72 hrs.
  • the isolated CD8 T cells either alone (CD8) or co-cultured with mBMDC WT (WT CD8), E7-pulsed mBMDCs WT (E7-WT CD8), mBMDC bml2 (bml2 CD8), or E7- pulsed mBMDC bml2 (E7-bml2 CD8), were tested for IFNy production via intracellular staining and flow cytometry (FIG. 11 A) and for IFNy production in supernatant by ELISA (FIG. 11C).
  • the isolated CD4 T cells either alone (CD4) or co-cultured with mBMDC WT (WT CD4), E7-pulsed mBMDCs WT (E7-WT CD4), mBMDC bml2 (bml2 CD4), or E7- pulsed mBMDC bml2 (E7-bml2 CD4), were tested for IFNy production via intracellular staining and flow cytometry (FIG. 11B) and for IFNy production in supernatant by ELISA (FIG. 11C).
  • CD8 T cells co-cultured with E7-pulsed mBMDC WT (E7- WT CD8) generated significantly higher level of IFNy in comparison to a mix of CD8 and CD4 T cells co-cultured with E7-pulsed mBMDC WT (E7-WT CD8+CD4).
  • CD8 T cells co- cultured with E7-mBMDC bml2 (E7-bml2 CD8) and a mix of CD8 and CD4 T cells co- cultured with E7-mBMDC bml2 (E7-bml2 CD8+CD4) showed similar high IFNy production to CD8 T cells co-cultured with E7-mBMDC WT (E7-WT CD8).
  • E7-WT CD8+CD4 T cells co-cultured with E7-mBMDC WT E7-WT CD8.
  • CD4 T cells co-cultured with E7-mBMDC bml2 (E7-BM12 CD4) generated significantly higher level of IFNy in comparison to CD4 T cells co- cultured with E7-mBMDC WT (E7-WT CD4).
  • FIG. 11C showed IFNy production in supernatant from CD4 and/or CD8 T cell culture by ELISA 72hrs after the co-culture. While no IFNy was detected in most samples, CD4 T cells co-cultured with E7-mBMDC bml2 (E7- BM12 CD4) showed a good amount of IFNy release, significantly higher than CD8 T cells co- cultured with E7-mBMDC bml2 (E7-BM12 CD8).
  • E743-77 GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR (SEQ ID NO: 1).

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Abstract

La présente divulgation concerne des cellules dendritiques de mammifère modifiées qui expriment des allèles de classe II du complexe majeur d'histocompatibilité (CMH) exogène. La divulgation concerne également des méthodes d'utilisation des cellules dendritiques de mammifère modifiées de la présente divulgation pour une immunothérapie à base de cellules dendritiques semi-allogéniques et autologues chez un sujet.
PCT/US2024/022230 2023-03-31 2024-03-29 Méthodes d'amélioration de l'immunogénicité de vaccins cellulaires Ceased WO2024206821A1 (fr)

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