Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • 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
  • A61K40/33—Antibodies; T-cell engagers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/52—Constant or Fc region; Isotype
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/55—Fab or Fab'
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)

Definitions

• the claudin 4 protein is encoded by the CLDN4 gene.
• CLDN4 claudin 4
• the heavy chain comprises a CDR1 comprising GFTFNNYA (SEQ ID NO: 9), GFTFGGYA (SEQ ID NO: 12), GGTFSSYA (SEQ ID NO: 15), or Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 GGTFNNYA (SEQ ID NO: 18), CDR2 comprising IRDSGGST (SEQ ID NO: 10), LSNSGSNA (SEQ ID NO: 13), or IIPIVDIA (SEQ ID NO: 16), CDR3 comprising ARRGYSSSWYGDGYYYGMDV, (SEQ ID NO: 11), ARAVMSSSWYMRRYYYYYMDV (SEQ ID NO: 14), or ARGGSQGAYYMDV (SEQ ID NO: 17), or a combination of CDRs thereof; and wherein the light chain comprises a CDR1 comprising SGSIASSF (SEQ ID NO: 19), RSNIGSNT (SEQ ID NO:
• the antibody sequences have been determined according to IMGT numbering scheme.
• the claudin 4 protein is a human claudin 4 protein.
• the antibody is fully human or humanized.
• the antibody is monospecific, bispecific, or multispecific.
• the antibody is an IgG.
• the antibody is an IgG1, IgG2, IgG3 or IgG4 antibody.
• the antibody is a single chain antibody.
• the antibody has a binding affinity of at least 1.0 x10 -9 M.
• the antibody or fragment comprises a heavy chain constant region, a light chain constant region, an Fc region, and Fc variant, or a combination thereof.
• the antibody comprises Gly1-2-F4, Gly1-4-G3, Gly1-1-H9, or Gly1-1-B2.
• the antibody competes with binding of Gly1-2-F4, Gly1-4-G3, Gly1-1-H9, or Gly1-1-B2.
• the antibody or fragment is linked to a therapeutic agent.
• the antibody is a single chain fragment.
• the antibody comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:
• the antibody sequences have been determined according to IMGT numbering scheme.
• Aspects of the invention are also drawn towards an isolated scFv antibody that binds to a human claudin 4 protein comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1
• the antibody sequences have been determined according to IMGT numbering scheme.
• Aspects of the invention are drawn to an isolated antibody or fragment thereof that binds to a human claudin 4 protein comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, or a sequence at least 90% identical thereto.
• Aspects of the invention are drawn to an isolated antibody or fragment thereof that binds to a human claudin 4 protein comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.
• aspects of the invention are drawn to an isolated antibody or fragment thereof that binds to a human claudin 4 protein comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.
• aspects of the invention are drawn to an isolated scFv that binds to a human claudin 4 protein comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, or a sequence at least 90% identical thereto.
• Aspects of the invention are drawn to an isolated scFv that binds to a human claudin 4 protein comprising a light chain variable region comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.
• aspects of the invention are drawn to an isolated scFv that binds to a human claudin 4 protein comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.
• Embodiments of the invention comprise an isolated monoclonal antibody or antigen-binding fragment thereof that binds to a human claudin 4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 acid sequence about 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 2.
• Embodiments of the invention comprise an isolated scFv that binds to a human claudin 4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 2.
• Embodiments of the invention comprise an isolated monoclonal antibody or antigen-binding fragment thereof that binds to a human claudin 4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 4.
• Embodiments of the invention comprise an isolated scFv that binds to a human claudin 4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 5, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 6.
• Embodiments of the invention comprise an isolated scFv that binds to a human claudin 4 protein, comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 8. Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 [0037] Aspects of the invention are drawn towards an isolated bispecific antibody, comprising an antibody fragment as described herein and a second antigen-binding fragment having specificity to a molecule on an immune cell.
• the molecule is selected from the group consisting of CCR4, CXCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.
• the fragment and the second fragment each is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.
• the BiTE comprises: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 21; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 13, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 14, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:
• the antibody sequences have been determined according to IMGT numbering scheme.
• Aspects of the invention further comprise a bispecific T cell engager (BiTE) that binds to a human claudin 4 protein comprising a heavy chain variable region comprising an Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, or a sequence at least 90% identical thereto.
• BiTE bispecific T cell engager
• aspects of the invention further comprise a bispecific T cell engager (BiTE) that binds to a human claudin 4 protein comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.
• BiTE bispecific T cell engager
• aspects of the invention further comprise a bispecific T cell engager (BiTE) that binds to a human claudin 4 protein comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.
• BiTE bispecific T cell engager
• Embodiments as described herein can further comprise an Fc fragment.
• Aspects of the invention are further drawn to a nucleic acid encoding the antibody or fragment as described herein.
• the antibody or fragment thereof comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, and 7, or a sequence at least 90% identical thereto, and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, and 8, or a sequence at least 90% identical thereto.
• the cell comprises a T cell, an NK cell, an NKT cell, an iPS cell, an iPS derived cell, a cell line, or a B cell.
• the cell comprises a CD4+, CD8+, CD3+ pan T cells, or any combination thereof.
• contacting comprises immunohistochemistry.
• immunohistochemistry comprises precipitation, immunofluorescence, western blot, or ELISA.
• the sample is whole blood, a blood component, a body fluid, a biopsy, a tissue, serum, or one or more cells.
• the sample comprises a normal sample or a cancerous sample.
• the cancer expresses claudin 4.
• the cancer comprises biliary, breast, cervical, colorectal, esophageal, intestinal, lung, pancreatic, prostate, renal, rectal, stomach, thyroid, or uterine.
• the one or more cells comprise an in vitro culture.
• the antibody or fragment as described herein binds to claudin 3 with a binding affinity at least 5-, 10-, 25-, 50-, 100-, or 1000-fold less than the antibody, fragment or bispecific antibody binds to claudin 4.
• FIG. 1 shows non-limiting, exemplary data validating cytopathic effect flow cytometry.
• FIG.2 shows a non-limiting, exemplary schematic of panning against claudin-4.
• FIG.3 shows a chart of non-limiting, exemplary data of claudin-4 targeting scFv(s) by phage panning using solubilized claudin-4 expressed in insect cells.
• FIG. 4 shows a graph of non-limiting, exemplary data of EK01 (see, WO 2019/178359) phage binding specifically soluble claudin-4.
• FIG.5 shows a graph of non-limiting, exemplary data of F4 biding claudin-4.
• FIG.6 shows histograms of non-limiting, exemplary flow cytometry data.
• FIG.7 shows a non-limiting, exemplary Western blot from protein purification.
• FIG. 8 shows non-limiting, exemplary data of EK01 (see, WO 2019/178356) minibody binding data.
• FIG.9 shows a non-limiting, exemplary schematic of paramagnetic proteoliposomes (PMPL) panning.
• PMPL paramagnetic proteoliposomes
• three rounds panning were performed on soluble claudin-4 and screened with claudin-4. After confirmation ELISAs and dilution series on soluble claudin- 4 one hit was initially identified. Panning initially failed on PMPLs. New PMPLs were then made.
• FIG.10 shows non-limiting, exemplary data of PMPL production.
• FIG.11 shows non-limiting, exemplary schematic of panning.
• FIG. 12 shows non-limiting, exemplary results 6 plates from re-rescue of the panning in Fig.11.
• FIG. 13 shows non-limiting, exemplary results of clones and frequencies from rounds 1 and 2 of panning in Fig.11.
• FIG. 14 shows non-limiting, exemplary data of next-generation sequencing (NGS) of round 1 and round 2 from Fig.11.
• FIG.15 shows non-limiting, exemplary data of phage binding solubilized claudin-4 ELISA.
• FIG.16 shows non-limiting, exemplary data of phage binding in Claudin-4 positive cells and cells that are not transfected.
• FIG. 17 shows non-limiting, exemplary data on phage binding on cells that are not transduced.
• FIG.18 shows a non-limiting, exemplary ELISA data.
• FIG.19 shows non-limiting, exemplary minibody binding data.
• FIG.20 shows non-limiting, exemplary minibody binding data.
• FIG. 21 shows non-limiting, exemplary schematic of claudin-4 whole cell panning path.
• FIG.22 shows non-limiting, exemplary purified phage binding curves (CLDN-4). Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 [0086] FIG.
• FIG. 23 shows non-limiting, exemplary CLDN-4 minibody binding of MB231 CLDN-4.
• KM is an anti-CLDN4 antibody synthesized from US 8,076,458 and cloned into our scFv-Fc vector as a control. Miltenyi and R&D were purchased and used as is.
• FIG. 24 shows a non-limiting, exemplary schematic of CLDN protein structure. In embodiments, structure and expression of CLDN-3 and CLDN-4 can be similar. In embodiments, expression levels can also be similar in cancer tissues.
• FIG.25 shows a non-limiting, exemplary schematic of Human CLDN-3 and CLDN- 4 extracellular domains.
• homology in extracellular domains can be used to discover CLDN-4 specific antibodies. Homology: ECL198%, ECL279%, Both 93%.
• FIG.26 shows examples of commercial antibody stabilities.
• FIG.27 shows non-limiting, exemplary quantitation data of cell line staining.
• FIG. 28 shows non-limiting, exemplary data of CLDN-3 binding of aCLDN-4 minibodies.
• FIG. 29 shows non-limiting, exemplary data of specificity of claudin antibodies. Similar results were seen with 0.1 mg/ml.
• FIG.30 shows non-limiting, exemplary data of staining of MB231 with aCXCR4 to KO. [0094] FIG.
• FIG. 31 shows a non-limiting, exemplary graph of CD8+ anti-CLDN-4 CART killing for CLDN-4 expressing MB231 cells.
• Positive controls KM3900 and CPE killed tumors non-specifically aC4-G3 and aC4-F4 did recognize and kill CLDN+ tumors specifically (9- fold higher). Negative control background killing is low ( ⁇ 10%).
• Our anti-CLDN-4 CARTs kill tumor cells expressing high levels of CLDN-4 specifically.
• FIG. 32 shows graphs of non-limiting, exemplary data of kinetics of CD8+ anti- CLDN-4 CART killing at E:T ratio of 2:1.
• Recombinant as it pertains to polypeptides (such as antibodies) or polynucleotides can refer to a form of the polypeptide or polynucleotide that does not exist naturally, a non- limiting example of which can be created by combining polynucleotides or polypeptides that cannot normally occur together.
• the amino acid sequence of the monoclonal CLDN4 antibodies are provided herein, in addition to an exemplary wildtype IgG constant region useful in combination with the VH and VL sequences provided herein (see Tables 1-4); the amino acid sequences of the heavy and light chain complementary determining regions CDRs of the CLDN4 antibodies are underlined (CDR1), underlined and bolded (CDR2), or underlined, italicized, and bolded (CDR3) below: Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 Table 1.
• Gly1-2-F4_PelB-F_2019-12-08_C03 Ab Variable Region amino acid sequences
• V H chain of Gly1-2-F4 QVQLVQSGGGLVQPGGSLRLSCAASGFTFNNYAMSWVRQAPGKGLEWVSTIRDSGGSTYYT DSVKGRFTISRDSSKNTLYLQMNSLRADDTAVYYCARRGYSSSWYGDGYYYGMDVWGQGTT PD
• V H chain of Gly1-4-G3 EVQLVESGGGVVQPGRSLRLSCAASGFTFGGYAMHWVRQAPGKGLEWVAELSNSGSNAFYA GT PD Table 3.
• Gly1-1-H9_PelB-F_2019-12-08_B03 Ab Variable Region amino acid sequences V H chain of Gly1-1-H9 YA PD Table 4.
• Gly1-1-B2_PelB-F_2019-12-08_A03 Ab Variable Region amino acid sequences YA DR Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 [00105]
• the amino acid sequences of the heavy and light chain complementary determining regions of the CLDN4 antibodies are shown in Table 5A-B below: Table 5A.
• Heavy chain (VH) complementary determining regions (CDRs) of the CLDN4 antibodies Sequence ID V H CDR1 V H CDR2 V H CDR3 GFTFNNYA IRDSGGST V DV Ta ble 5B.
• Light chain (VL) complementary determining regions (CDRs) of the CLDN4 antibodies Sequence ID V L CDR1 V L CDR2 V L CDR3 [00106]
• the amino acid sequences of the heavy and light chain framework regions of the CLDN4 antibodies are shown in Table 6A-B below: Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 Table 6A.
• VH framework regions of the CLDN4 antibodies Seq ID VH FR1 VH FR2 VH FR3 VH FR4 Gly1-2- QVQLVQSGGGL MSWVRQAPGK YYTDSVKGRFTI WGQGTTVTVS F4 VQPGGSLRLSC GLEWVST SRDSSKNTLYLQ (SEQ ID NO: AAS (SEQ ID MNSLRADDTAVY 34) S O: S O: S O: Ta ble 6B.
• VL framework regions (FRs) of the CLDN4 antibodies Seq ID VL FR1 VL FR2 VL FR3 VL FR4 L O: L O: L O: Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 Seq ID VL FR1 VL FR2 VL FR3 VL FR4 ETTLTQSPATL TRATGIPDRFSG FGQGTKVEIK SVSPGERATLS LAWYRQKPGQ SGSGTDFTLTIS (SEQ ID NO: CRAS APRLLIY RLEPEDFAVYFC 61) [0 rovided herein; the nucleic acid sequences of the heavy and light chain complementary determining regions CDRs of the CLDN4 antibodies are underlined (CDR1), underlined and bolded (CDR2), or underlined, italicized, and bolded (CDR3) below: [00108] Table 7A.
• CDR1 underlined
• VH Heavy chain (VH) complementary determining regions (CDRs) of the CLDN4 antibodies Seq ID VH CDR1 VH CDR2 VH CDR3 GGATTCACCTTTAA ATTCGTGATAGTGGT GCGAGGCGCGGGTATAG TG GT AG GA AC CA GG CA GG [00109] Table 7B.
• VH Heavy chain (VH) complementary determining regions (CDRs) of the CLDN4 antibodies
• CLDN4 antibodies Seq ID V ⁇ J ⁇ REGION Gly1-2-F4 AATTTTATGCTGACGCAGCCCCACTCTGTGTCGGAGTCTCCGGGGGA (SEQ ID NO: 90) GACGGTAACCATCTCCTGCGCCCGCAGCAGTGGCAGCATTGCCAGCA GCTTTGTGCAGTGGTACCAGCAGCGCCCGGGCGCTTCCCCCACCACT TT CT AT CT CA ( TA TC TT GC AC GT AA ( CA CT TT CT AT GT GG ( CT TC AG GG TA [00112]
• the CLDN4 antibodies described herein bind to CLDN4.
• the CLDN4 antibodies have high affinity and high specificity for CLDN4.
• Some embodiments also feature antibodies that have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the anti-CLDN4 antibodies described herein.
• “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which can be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
• the Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity when compared to a specified region or the full length of any one of the anti-CLDN4 antibodies described herein.
• the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleic acid identity when compared to a specified region or the full length of any one of the anti-CLDN4 antibodies described herein.
• Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and/or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the invention.
• sequence comparison algorithms i.e., BLAST or BLAST 2.0
• manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the invention.
• Polypeptide as used herein can encompass a singular “polypeptide” as well as plural “polypeptides,” and can refer to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
• polypeptide can refer to any chain or chains of two or more amino acids and does not refer to a specific length of the product.
• peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term can be used to refer to a chain or chains of two or more amino acids, can refer to “polypeptide” herein, and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
• Polypeptide can also refer to the products of post- expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.
• a polypeptide can be derived from a natural biological source or produced by recombinant technology but is not necessarily translated from a nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
• amino acid sequences one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant".
• the alteration results in the substitution of an amino acid with a chemically similar amino acid.
• Conservative substitution tables providing functionally similar amino acids are well known in the art.
• Such conservatively modified variants of the anti-CLDN4 antibodies disclosed herein can exhibit increased cross-reactivity to CLDN4 in comparison to an unmodified CLDN4 antibody.
• a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
• Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
• an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen.
• An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof.
• antibody can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen.
• Non-limiting examples a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein.
• CDR complementarity determining region
• the term “antibody” can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen.
• immunoglobulin immunoglobulin
• immunologically active portions of an immunoglobulin (Ig) molecule i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen.
• Ig immunoglobulin
• antibody fragment or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F (ab′)2 , F (ab)2 , F ab ′, F ab , Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody.
• antibody fragment can include aptamers (such as spiegelmers), minibodies, and diabodies.
• antibody fragment can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.
• Antibody molecules obtained from humans fall into five classes of immunoglobulins: IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
• immunoglobulins Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon ( ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ) with some subclasses among them (e.g., ⁇ 1- ⁇ 4).
• Certain classes have subclasses as well, such as IgG1, IgG2, IgG3 and IgG 4 and others.
• immunoglobulin subclasses e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgG 5 , etc. are well characterized and are known to confer functional specialization.
• IgG a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 of molecular weight 53,000-70,000.
• the four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.
• Immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of an immunoglobulin molecule.
• Light chains are classified as kappa or lambda ( ⁇ , ⁇ ). Each heavy chain class can be bound with a kappa or lambda light chain.
• FR can refer to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins.
• the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface.
• the antigen-binding surface is complementary to the three- dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity-determining regions,” or "CDRs.”
• CDRs complementarity-determining regions
• the six CDRs present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 as the antibody assumes its three-dimensional configuration in an aqueous environment.
• the remainder of the amino acids in the antigen-binding domains, the FR regions, show less inter- molecular variability.
• the framework regions largely adopt a ⁇ -sheet conformation and the CDRs form loops which connect, and in some cases form part of, the ⁇ -sheet structure.
• the framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions.
• the antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope.
• the amino acids comprising the CDRs and the framework regions, respectively can be readily identified for a heavy or light chain variable region by one of ordinary skill in the art, since they have been previously defined (See, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
• the CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein.
• the appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a CDR given the variable region amino acid sequence of the antibody.
• the CDRs of an antibody can be determined according to the IMGT numbering system.
• the IMGT unique numbering has been defined to compare the variable domains whatever the antigen receptor, the chain type, or the species [Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., The Immunologist, 7, 132-136 (1999)/Lefranc, M.-P., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)].
• epitopes can include any protein determinant that can specifically bind to an immunoglobulin, a scFv, or a T-cell receptor.
• the variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens.
• the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three-dimensional antigen- binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y.
• Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen, which is the target of the Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 immunoglobulin sought, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Engineer, published by The Engineer, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28).
• the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen.
• the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
• RIA radioimmunoassay
• ELISA enzyme-linked immunoabsorbent assay
• the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).
• the monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
• Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No.4,816,567 (incorporated herein by reference in its entirety).
• DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can bind specifically to genes encoding the heavy and light chains of murine antibodies).
• the hybridoma cells of the invention serve as a source of such DNA.
• the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
• Human monoclonal antibodies such as fully human and humanized antibodies, can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies can be utilized and can be produced by using human hybridomas (see Cote, et al, 1983.
• humanized antibodies can be produced in transgenic plants, as an inexpensive production alternative to existing mammalian systems.
• the transgenic plant can be a tobacco plant, i.e., Nicotiania benthamiana, and Nicotiana tabaccum.
• the antibodies are purified from the plant leaves.
• Stable transformation of the plants can be achieved through the use of Agrobacterium tumefaciens or particle bombardment.
• nucleic acid expression vectors containing at least the heavy and light chain sequences are expressed in bacterial cultures, i.e., A. tumefaciens strain BLA4404, via transformation.
• Infiltration of the plants can be accomplished via injection.
• Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of the antibodies can be performed by many of the methods known to the skilled artisan in the art. Other methods for antibody production in plants are described in, for example, Fischer et al., Vaccine, 2003, 21:820-5; and Ko et al, Current Topics in Microbiology Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 and Immunology, Vol. 332, 2009, pp. 55-78.
• the invention further provides any cell or plant comprising a vector that encodes the antibody of the invention or produces the antibody of the invention.
• “Humanization” also called Reshaping or CDR-grafting is a well-established technique understood by the skilled artisan for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (such as rodent) and for improving their activation of the human immune system (See, for example, Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling”. J Biochem.144 (1): 115–20).
• antibodies can also be produced using other techniques, including phage display libraries.
• phage display libraries See Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)).
• human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
• Human antibodies can additionally be produced using transgenic nonhuman animals which are modified to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
• an antibody of interest such as a human antibody, is disclosed in U.S. Patent No.5,916,771.
• This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell.
• the hybrid cell expresses an antibody containing the heavy chain and the light chain.
• the antibody of interest can also be expressed by a vector containing a DNA segment encoding the single chain antibody described herein.
• Vectors include, but are not limited to, chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vectors (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618), which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g.
• the vectors can be chromosomal, non-chromosomal or synthetic. Retroviral vectors can also be used and include moloney murine leukemia viruses. DNA viral vectors can also be used, and include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (See Geller, A. I. et al, J. Neurochem, 64:487 (1995); Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A.
• HSV herpes simplex I virus
• Examples of modes of gene transfer include e.g., naked DNA, CaP0 4 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors. Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 [00155]
• the vector can be employed to target essentially any target cell.
• stereotaxic injection can be used to direct the vectors (e.g., adenovirus, HSV) to a preferred location.
• the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System.
• a method based on bulk flow termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell.
• convection A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell.
• Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.
• These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of CLDN4 in a sample.
• Antibody fragments that contain the idiotypes to a protein antigen can be produced by techniques known in the art including, but not limited to: (i) an F (ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F (ab')2 fragment; (iii) an F ab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments.
• Heteroconjugate antibodies are also within the scope of the invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies can, for example, target immune system cells to unwanted cells (see U.S. Patent No.
• the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
• immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No.4,676,980.
• the antibody of the invention can be modified with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer.
• cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region.
• the homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC).
• ADCC complement-mediated cell killing and antibody- dependent cellular cytotoxicity
• an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.
• the antibody of the invention has modifications of the Fc region, such that the Fc region does not bind to the Fc receptors.
• the Fc receptor is Fc ⁇ receptor.
• Antibodies with modification of the Fc region such that the Fc region does not bind to Fc ⁇ , but still binds to neonatal Fc receptor are useful as described herein.
• an antibody of the invention can comprise an Fc variant. See, for example, WO2018/145075 and WO2019/183362, which provide Fc variant compositions for augmenting antibody mediated receptor signaling.
• the Fc variant can comprise an amino acid substitution which alters the antigen-independent effector functions of the antibody, such as the circulating half-life of the antibody.
• Such antibodies exhibit increased or decreased binding to FcRn when compared to antibodies lacking these substitutions, therefore, have an increased or decreased half-life in serum, respectively.
• Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered antibody is preferred, e.g., to treat a chronic disease or disorder.
• Fc variants with decreased FcRn binding affinity have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time can be advantageous, e.g., for in vivo diagnostic imaging or in situations where the starting antibody has toxic side effects when present in the circulation for prolonged periods.
• Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women.
• other applications in which reduced FcRn binding affinity can be preferred include those applications in which localization to the brain, kidney, and/or liver is preferred.
• the Fc variant-containing antibodies can exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the Fc variant- containing antibodies can exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space.
• BBB blood brain barrier
• an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of an Fc domain.
• the FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn binding activity are disclosed in PCT Publication No.
• the antibodies, or fragments thereof, of the invention comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering).
• mutations are introduced to the constant regions of the mAb such that the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the mAb is altered.
• the mutation is a LALA mutation in the CH2 domain.
• the antibody e.g., a human mAb, or a bispecific Ab
• the mAb contains mutations on one scFv unit of the heterodimeric mAb, which reduces the ADCC activity.
• the mAb contains mutations on both chains of the heterodimeric mAb, which completely ablates the ADCC activity.
• the mutations introduced into one or both scFv units of the mAb are LALA mutations in the CH2 domain.
• antibodies of the invention for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, which can be altered to reduce or eliminate glycosylation.
• an antibody of the invention can also comprise an Fc variant comprising an amino acid substitution which alters the glycosylation of the antibody.
• the Fc variant can have reduced glycosylation (e.g., N- or O-linked glycosylation).
• the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering).
• the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS.
• the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 numbering).
• the antibody comprises an IgGl or IgG4 constant region comprising an S228P and a T299A mutation (EU numbering).
• Exemplary amino acid substitutions which confer reduced or altered glycosylation are described in PCT Publication No, WO05/018572, which is incorporated by reference herein in its entirety.
• the antibodies of the invention, or fragments thereof are modified to eliminate glycosylation.
• Such antibodies, or fragments thereof can be referred to as "agly” antibodies, or fragments thereof, (e.g. "agly” antibodies).
• agly antibodies, or fragments thereof can have an improved safety and stability profile in vivo.
• Exemplary agly antibodies, or fragments thereof comprise an aglycosylated Fc region of an IgG 4 antibody which is devoid of Fc-effector function thereby eliminating the potential for Fc mediated toxicity to the normal vital tissues and cells that express CLDN4.
• antibodies of the invention, or fragments thereof comprise an altered glycan.
• the antibody can have a reduced number of fucose residues on an N-glycan at Asn297 of the Fc region, i.e., is afucosylated.
• the antibody can have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.
• a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
• Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
• Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates Docket No.: 5031461-133-
• a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987).
• Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody.
• MX-DTPA l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid
• See PCT Publication No. WO94/11026, and U.S. Patent No.5,736,137 See PCT Publication No. WO94/11026, and U.S. Patent No.5,736,137.
• Coupling can be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In one embodiment, binding is, covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by the incorporation of external bridging molecules.
• bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the invention, to other molecules.
• representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines.
• This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133: 1335- 2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al, Science 238: 1098 (1987)).
• Non-limiting examples of linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker.
• MBS M-maleimidobenzoyl-N-hydroxysuccinimide ester
• Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (l-ethyl-3- (3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido]hexanoate (Pierce Chem.
• sulfo- NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates.
• NHS-ester containing linkers are less soluble than sulfo-NHS esters.
• the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available.
• Sulfo-NHS can enhance the stability of carbodimide couplings.
• Carbodimide couplings when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
• the antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556.
• Non-limiting examples of useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the preferred diameter.
• Fab' fragments of the antibody of the invention can be conjugated to the liposomes as described in Martin et al, J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.
• multispecific antibodies such as bi-specific and trispecific antibodies, that Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 recognize CLDN4 and a second antigen and/or a third antigen.
• multispecific antibodies e.g., bi-specific antibodies and trispecific antibodies
• Exemplary second and or third antigens include tumor associated antigens (e.g., LINGO1), cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Reference No. NP_000873.2; IL- 12B (p40 subunit) protein sequence having NCBI Reference No.
• NP_002178.2 protein sequence having NCBI Reference no. NP_001553.1
• IL-15 protein sequence having NCBI Reference No. NP_000576.1
• IL-7 protein sequence having NCBI Reference No. NP_000871.1
• IL-2 protein sequence having NCBI Reference No. NP_000577.2
• IL-21 protein sequence having NCBI Reference No. NP_068575.1
• cytokine cognate receptors eg., IL-12R
• Non-limiting examples of second and/or third antigens include CTLA-4, CXCR4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47 and CD73.
• the bispecific and trispecific antibodies comprise CLDN4 fusion proteins.
• the fusion protein comprises an antibody comprising a variable domain or scFv unit and a ligand or antigen and/or a third ligand or antigen as described herein such that the resulting antibody recognizes an antigen and binds to the ligand-specific receptor.
• Exemplary antibody compositions that are useful for the design of CLDN4 fusion proteins as described herein include, but are not limited to, anti-CAIX antibodies described in PCT/US2006/046350 and PCT/US2015/067178; anti-CXCR4 antibodies described in PCT/US20006/005691 and PCT/US2019/022272; anti-CCR4 antibodies described in PCT/US2008/088435, PCT/US2013/039744, and PCT/US2015/054202; anti-PD-L1 antibodies described in PCT/US2008/088435 and PCT/US2020/062815; anti-PD-1 antibodies described in PCT/US2020/037791 and PCT/US2020/037781; anti-GITR antibodies described in PCT/US2017/043504; anti-claudin-4 antibodies described in PCT/US2019/022272; and anti-MUC1 antibodies described in PCT/US2020/037783
• the fusion protein further comprises a constant region, and/or a linker as described herein.
• multispecific antibodies e.g., bispecific antibodies and trispecific antibodies such as a fusion protein comprises an antibody that recognizes CLDN4 and a ligand
• Ligands can be tumor associated antigens (e.g., LINGO1, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-l, Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 MN-CA IX, RET1, RET2 (AS), prostate specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate-carcinoma tumor antigen-l (PCTA-1), GAGE, MAGE, mesothelin, ⁇ -catenin, TGF- ⁇ RII, BRCA1/2, SAP-1, HPV-E6, HPV-E7 (see also, PCT/US2015/067225 and PCT/US2019/022272 for additional
• each of the anti- CLDN4 fragment and the second antigen-specific fragment and/or the third antigen-specific fragment is each independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.
• the bispecific or trispecific antibody further includes a Fc fragment (e.g., as described in PCT/US2015/021529 and PCT/US2019/023382, each of which are incorporated by reference in their entireties).
• a bispecific or trispecific antibody of the invention can comprise a heavy chain and a light chain combination or scFv of the CLDN4 antibodies described herein.
• Multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies) of the invention (for example, an anti-CLDN4-scFv fusion protein) can be constructed using methods known art.
• the bi-specific antibody is a single polypeptide wherein the two scFv fragments are joined by a long linker polypeptide, of sufficient length to allow intramolecular association between the two scFv units to form an antibody.
• the bi-specific antibody is more than one polypeptide linked by covalent or non- covalent bonds.
• the amino acid linker depicted herein (GGGGSGGGGS; “(G4S)2”) can be generated with a longer G4S linker to improve flexibility.
• the linker can also be: “(G 4 S) 3 ” (e.g., GGGGSGGGGSGGGGS); “(G4S)4” (e.g., GGGGSGGGGSGGGGSGGGGS); “(G 4 S) 5 ” (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS); “(G4S)6” (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS); Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 “(G4S)7” (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGGGSGGGGS); and the like.
• the multispecific antibodies e.g., bispecific antibodies and trispecific antibodies such as anti-CLDN4-scFv fusions
• the multispecific antibodies can be constructed through exchange of heavy-light chain dimers from two or more different antibodies to generate a hybrid antibody where the first heavy-light chain dimer recognizes CLDN4 and the second heavy-light chain dimer recognizes a second antigen and/or third antigen.
• IgG 4 molecules can also be altered such that the heavy and light chains recognize CLDN4 or a second and/or third antigen, as disclosed herein.
• Use of this method for constructing the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CLDN4-scFv fusions) of the invention can be beneficial due to the intrinsic characteristic of IgG 4 molecules wherein the Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain white blood cells.
• the multispecific antibodies e.g., bispecific antibodies and trispecific antibodies such as anti-CLDN4-scFv fusions
• the multispecific antibodies can be engineered with a non-depleting heavy chain isotype, such as IgG1-LALA or stabilized IgG4 or one of the other non-depleting variants.
• the multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered as modular tetrameric bispecific antibodies (tBsAb).
• tBsAb modular tetrameric bispecific antibodies
• the tetravalent antibody can be a dimer of a bispecific scFv fragment including a first binding site for a first antigen, and a second binding site for a second antigen.
• the anti- CLDN4 antibody can be the first binding site for a first antigen.
• the anti- CLDN4 antibody can be the second binding site for a second antigen. The two binding sites can be joined together via a linker domain.
• the scFv fragment is a tandem scFv
• the linker domain includes an immunoglobulin hinge region (e.g., an IgGl, an IgG2, an IgG3, or an IgG4 hinge region) amino acid sequence.
• the immunoglobulin hinge region amino acid sequence can be flanked by a flexible linker amino acid sequence, e.g., having the linker amino acid sequence (GGGS) x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF.
• the linker domain includes at least a portion of an immunoglobulin Fc domain, e.g., an IgGl, an IgG2, an IgG3, or an IgG4 Fc domain.
• the at least a portion of the immunoglobulin Fc domain does not include a CH2 domain.
• the at least a portion of the immunoglobulin Fc domain can be a CH2 domain.
• An exemplary CH2 domain amino acid sequence includes APELLGGPDVFLF (SEQ ID NO: 95).
• the Fc domain can be linked to the C-terminus of an immunoglobulin hinge region (e.g., an IgGl, an IgG2, an IgG3, or an IgG4 hinge region) amino acid sequence.
• the linker domain can include a flexible linker amino acid sequence (e.g., (GGGS)x1-6, (GGGGS)x1-6, or GSAGSAAGSGEF) at one terminus or at both termini.
• the tBsAb can be specific for CLDN4, and also a target selected from the group consisting of B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CCR4, CXCR4, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-L1, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, and KIR.
• a target selected from the group consisting of B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CCR4, CXCR4, CTLA-4, GITR, GITRL, ICOS, ICOSL, L
• the multispecific antibody can be a bi-specific T-cell engager (BiTE).
• BiTEs (a bispecific T-cell engager) refers to a single polypeptide chain molecule with two antigen binding domains, one of which binds to a T-cell antigen.
• the BiTE can comprise a CLDN4 antibody disclosed herein, or a functional fragment thereof, and an antibody or fragment thereof that binds to a T-cell antigen.
• the antibody or fragment thereof that binds to a T-cell antigen can be specific for CD3.
• the multispecific antibody can be a tri-specific T-cell engager (TriTE).
• TriTEs can refer to a single polypeptide chain molecule with three antigen binding domains, one or more of which binds to a T-cell antigen.
• the TriTE can comprise a CLDN4 antibody disclosed herein, or a functional fragment thereof, and an antibody or fragment thereof that binds to a T-cell antigen.
• the antibody or fragment thereof that binds to a T-cell antigen can be specific for CD3, CD28, or both.
• the multispecific antibodies e.g., bispecific antibodies and trispecific antibodies such as anti-CLDN4-scFv fusions
• the invention provides a fusion protein containing a CLDN4 antibody disclosed herein, or a functional fragment thereof, operably linked to a second protein.
• the second protein can be, for example, a cytokine or a growth factor.
• the cytokine is IL-2 or TGF-beta and variants thereof.
• the second protein can be a therapeutic agent, such as a toxin, a detectable moiety, such as a fluorescent protein for detection, or a biological agent, such as an agent that stimulates T cells (i.e., CD3).
• the CLDN4 antibodies of the invention can be operably linked to more than one additional protein or peptide, for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional proteins or peptide sequences.
• the CLDN4 antibody disclosed herein, or functional fragment thereof is joined directly to the second protein.
• the CLDN4 antibody, or functional fragment thereof is joined to the second protein via a linker, such as a flexible polypeptide chain.
• the linker can be any suitable linker of any length, but can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids in length.
• the linker is an amino acid sequence that is naturally present in immunoglobulin molecules of the host, such that the presence of the linker cannot result in an immune response against the linker sequence by the mammal.
• Fusion proteins of the invention that include more than one additional protein to the CLDN4 antibody can have multiple linker sequences that join each additional protein or peptide sequence.
• the fusion proteins of the invention can be constructed by recombinant methods known to the skilled artisan.
• an expression vector containing the nucleic acid sequence encoding a CLDN4 antibody of the invention can be operably linked to the nucleic acid sequence encoding the second protein and can be introduced to an expression Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 system to translate and produce the fusion protein.
• an expression Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 system to translate and produce the fusion protein.
• one skilled in the art can readily utilize de nova protein synthesis techniques to produce the fusion proteins described herein.
• Antibodies of the invention specifically binding a CLDN4 protein, or a fragment thereof can be administered for the treatment of a CLDN4 associated disease or disorder.
• A"CLDN4-associated disease or disorder” includes disease states and/or symptoms associated with a disease state, where increased levels of CLDN4 and/or activation of cellular signaling pathways involving CLDN4 are found.
• Exemplary CLDN4-associated diseases or disorders include, but are not limited to, diseases where T cells are suppressed, such as in cancer and infectious diseases.
• the cancer can be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer.
• the cancer can be a CLDN4-associated cancer.
• the CLDN-4 associated cancer can be triple negative breast cancer.
• Antibodies of the invention can be used as therapeutic agents. Such agents can be employed to treat cancer in a subject, increase vaccine efficiency or augment a natural immune response.
• An antibody preparation for example, one having high specificity and high affinity for its target antigen, is administered to the subject and will have an effect due to its binding with the target. Administration of the antibody can abrogate or inhibit or interfere with an activity of the CLDN4 protein.
• Pharmaceutical Compositions [00195] Antibodies of the invention specifically binding a CLDN4 protein or fragment thereof can be administered for the treatment of a cancer in the form of pharmaceutical compositions.
• a specific dosage and treatment regimen for any patient will depend upon a variety of factors, including the specific antibodies, variant or derivative thereof used, the patient's age, Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the preferred effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
• a therapeutically effective amount of an antibody of the invention can be the amount needed to achieve a therapeutic objective. As noted herein, this can be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target.
• the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
• the dosage administered to a subject (e.g., a patient) of the antigen-binding polypeptides described herein is about 0.1 mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of the patient's body weight.
• Human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the disclosure can be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
• Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention can be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week.
• antibody fragments the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred.
• peptide molecules can be designed that retain the ability to bind the target protein sequence.
• Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci.
• the formulation can also contain more than one active compound as necessary for the indication being treated, for example, those with complementary activities that do not adversely affect each other.
• the composition can comprise an agent that enhances its function, such as, for example, a Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 cytotoxic agent, cytokine (e.g., IL-15), chemotherapeutic agent, or growth-inhibitory agent.
• cytotoxic agent e.g., IL-15
• chemotherapeutic agent e.g., chemotherapeutic agent
• growth-inhibitory agent e.g., chemotherapeutic agent
• the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules
• macroemulsions for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules
• the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
• Sustained-release preparations can be prepared.
• sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
• sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
• copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
• compositions suitable for administration can comprise the antibody or agent and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
• Non-limiting examples of such carriers or diluents include water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used.
• the use of such media and agents for pharmaceutically active substances is well known Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is intended. Supplementary active compounds can also be incorporated into the compositions.
• a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
• routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• a sterile diluent such as water for injection, saline solution, fixed oils, poly
• compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
• the composition is sterile and is fluid to the extent that easy syringeability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
• isotonic agents can be included, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional ingredient from a previously sterile-filtered solution thereof.
• Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
• the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
• a lubricant such as magnesium stearate or Sterotes
• a glidant such as colloidal silicon dioxide
• the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
• a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
• Systemic administration can also be by transmucosal or transdermal means.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
• Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
• the active compounds are formulated into ointments, salves, gels, or creams as known in the art.
• the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 [00210]
• the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations are apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. [00211] Oral or parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage.
• the sample can be a cancer sample or a sample from a subject at risk of having cancer.
• the cancer can be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer.
• the antibody can contain a detectable label.
• Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) can be used.
• the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
• in vitro techniques for detection of an analyte mRNA includes Northern hybridizations and in situ hybridizations.
• in vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
• In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations.
• the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
• Antibodies directed against a CLDN4 protein can be used in methods known within the art relating to the localization and/or quantitation of a CLDN4 protein (e.g., for use in measuring levels of the CLDN4 protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
• antibodies specific to a CLDN4 protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain are utilized as pharmacologically active compounds (referred to herein as "therapeutics").
• An antibody of the invention specific for a CLDN4 protein can be used to isolate a CLDN4 polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation.
• Antibodies directed against a CLDN4 protein can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
• Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
• detectable substances include, but are not limited to, various Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
• Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 I, 131 I, 35 S, 32 P or 3 H.
• the invention provides methods (also referred to herein as “screening assays") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that modulate or otherwise interfere with a CLDN4 activity. Also provided are methods of identifying compounds useful to treat cancer. The invention also encompasses compounds identified using the screening assays described herein. [00221] For example, the invention provides assays for screening candidate or test compounds which modulate CLDN4 expression and/or activity.
• a candidate compound is introduced to an antibody-antigen complex and determining whether the candidate compound disrupts the antibody-antigen complex, wherein a disruption of this complex indicates that the candidate compound modulates an CLDN4 activity.
• at least one CLDN4 protein is provided, which is exposed to at least one monoclonal antibody.
• the antibody can be a CLDN4 antibody.
• the antigen can be a CLDN4 protein or a portion thereof.
• the screening methods disclosed herein can be performed as a cell-based assay or as a cell-free assay. In the case of cell-free assays comprising the membrane-bound forms of the CLDN4 proteins, it can be desirable to utilize a solubilizing agent such that the membrane- bound form of the proteins is maintained in solution.
• a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
• GST-antibody fusion proteins or GST- antigen fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
• the antibody or the antigen can be immobilized utilizing conjugation of biotin and streptavidin.
• Biotinylated antibody or antigen molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well-known within the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
• the invention further pertains to new agents identified by any of the screening assays described herein and uses thereof for treatments as described herein.
• CAR cell therapies such as chimeric antigen receptor (CAR) cell therapies, are also provided herein.
• the cell can be a CAR T-cell or a CAR NK-cell.
• CAR cell therapies redirect a patient’s T-cells and/or NK-cells to kill tumor cells by the exogenous expression of a CAR on a T-cell or NK-cell, for example.
• a CAR can be a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor or NK-cell receptor.
• monospecific CAR cells are provided.
• the anti- CLDN4 antibodies described herein can be used as the targeting moiety for the CAR cell.
• the CLDN4 antibody can have low affinity but high avidity for its antigen.
• the CLDN4 antibody can have high affinity but low avidity for its antigen.
• Antibodies with fewer binding sites can have high affinity and low avidity, while those with greater binding sites can have low affinity and high avidity.
• bispecific (or dual-targeted) CAR cells are provided.
• the CAR cell is an engineered cell comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular ligand binding domain that is specific for a first antigen and a second antigen on the surface of a cancer cell, Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 wherein the first antigen comprises an antigen that is not CLDN4 and the second antigen comprises CLDN4.
• the anti-CLDN4 antibodies or the CLDN4 fusion proteins described herein can be used as a payload for armored CAR-cell therapies.
• a suitable cell can be used, for example, that can secrete an anti-CLDN4 antibody of the invention (or alternatively engineered to express an anti-CLDN4 antibody as described herein to be secreted).
• the anti- CLDN4 “payloads” to be secreted can be, for example, minibodies, scFvs, IgG molecules, bispecific fusion molecules, and other antibody fragments as described herein.
• the cell described herein can then be introduced to a patient in need of a treatment by infusion therapies known to one of skill in the art.
• the patient can have a CLDN4-associated disease or disorder as described herein, such as cancer.
• the cell e.g., a T cell
• the cell can be, for instance, T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation.
• Exemplary CARs and CAR factories useful in aspects of the invention include those disclosed in, for example, PCT/US2015/067225 and PCT/US2019/022272, each of which are hereby incorporated by reference in their entireties.
• the CLDN4 antibodies discussed herein can be used in the construction of multi-specific antibodies or as the payload for a CAR-T cell or CAR NK-cell.
• the anti-CLDN4 antibodies discussed herein can be used for the targeting of the CARs (i.e., as the targeting moiety).
• the anti-CLDN4 antibodies discussed herein can be used as the targeting moiety, and a different CLDN4 antibody that targets a different epitope can be used as the payload.
• the payload can be an immunomodulatory antibody payload.
• TME tumor microenvironment
• the cell can then be introduced to a cancer patient in need of a treatment by infusion therapies known to one of skill in the art.
• the cancer patient can have a cancer of any of the types as disclosed herein.
• the cell e.g., a T cell
• CAR cells i.e., CAR T cells or CAR NK cells
• CAR T cells can be generated according to methods known in the art using lentivirus systems (via transduction), retrovirus systems (via transfection (electroporation)), and transposon systems (via PiggyBac).
• promoters for payloads that can be used in the generating of CAR-Ts include, for example, constitutive promoters (where the promoter is the same as for CAR-T, such as EF1a then IRES or 2A); inducible promoters (where the promoter is different from the promoter for CAR-T, such as NFAT, IL-2 prom); and genetically engineered promoters (such as a CLDN4 locus “knock in” of cytokine and/or a promoter that is under the control of an endogenous promoter).
• constitutive promoters where the promoter is the same as for CAR-T, such as EF1a then IRES or 2A
• inducible promoters where the promoter is different from the promoter for CAR-T, such as NFAT, IL-2 prom
• genetically engineered promoters such as a CLDN4 locus “knock in” of cytokine and/or a promoter that is under the control of an endogenous promote
• the anti-CLDN4 antibodies or the CLDN4 fusion proteins discussed herein can be used for the targeting of the CARS (i.e., as the targeting moiety).
• the anti-CLDN4 antibodies or the CLDN4 fusion proteins discussed herein can be used as a payload to be secreted by a CAR cell.
• the anti-CLDN4 antibodies or the CLDN4 fusion proteins discussed herein can be used as the targeting moiety, and a different CLDN4 antibody that targets a different epitope can be used as the payload.
• the payload can be an immunomodulatory antibody payload.
• the CLDN4 antibodies or the CLDN4 fusion proteins as described herein for use in CAR-T compositions are not high-affinity CLDN4 antibodies (for example, so that the antibody does not bind strongly to its CLDN4 target).
• the CLDN4 antibodies or the CLDN4 fusion proteins described herein can be used as a payload secreted by the CAR cell, with the two targeting moieties (for example, tumor-associated surface antigens) selected for a specific cancer.
• Non-limiting examples of a tumor-associated surface antigen include ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-l, MN-CAIX, RET1, RET2 (AS), prostate specific antigen (PSA), TAG- 72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate-carcinoma tumor antigen-l (PCTA-1), GAGE, MAGE, mesothelin, ⁇ -catenin, TGF- ⁇ RII, BRCA1/2, SAP-1, HPV-E6, HPV-E7 (see also, PCT/US2015/067225 and PCT/US2019/022272 for additional tumor- associated surface antigens, which are incorporated by reference in their entireties).
• Exemplary armored CAR-T cells are listed in the table below.
• CART Payload Format Promoter Publication Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 PSMA DN-TGFb Molecular Therapy 26: 1855 (2016) GD2 cJun cDNA Nature 576: 293(2019 ) Fibronectin CD47 VHH Cancer Immunol Res. 8:518-529 (2020) PD-L1 PD-L1 CTLA-4 GPC3 IL-12 J Immunol 2019; 203:198 CD20 PD-1 Cancer Science.
• Chimeric B-cell Receptor A modified B cell receptor called chimeric B cell receptor, such as a B cell receptor containing an antibody or antibody fragment previously selected by high affinity against a specific disease associated antigen, is a powerful new approach against diseases.
• B cells serve as professional antigen presenting cells, they can process and present antigens on MHC class II molecules, enhancing immune cell recognition of the tumor and assisting in neoantigen spreading.
• the B cell can include a receptor that is chimeric, non-natural and engineered at least in part by the hand of man.
• the engineered chimeric B cell receptor has one, two, three, four, or more components, and in some embodiments the one or more components facilitate targeting or binding of the B cell to one or more antigen-comprising cells.
• aspects of the invention include genetically engineered B cells that are modified to express and bear on its surface a chimeric B cell receptor.
• the genetically modified B cell can comprise a single chimeric B cell receptor targeting one antigen, such as CLDN4, or a single chimeric B cell receptor targeting two or more antigens (e.g., a bi-specific chimeric B cell receptor, or a multispecific chimeric B cell receptor).
• the cells comprise a split chimeric B cell receptor, such as two different scFvs expressed on the B cell surface with different co-stimulation domains. Further, some embodiments comprise a fine-tuned chimeric B cell receptor.
• the chimeric B cell receptor comprises an extracellular domain, a transmembrane domain, and an intracellular signaling domain; such that the polypeptides assemble together to form a chimeric B cell receptor.
• the extracellular ligand-binding domain can be chosen to recognize a ligand, such as CLDN4, that acts as a cell surface marker on target cells associated with a disease state.
• the disease state can be cancer
• the target ligand can be a cancer associated antigen, such as CLDN4.
• the extracellular ligand-binding domain can comprise an antigen binding domain or antigen recognition domain derived from an antibody against an antigen of the target, such as an anti-CLDN4 antibody described herein.
• the extracellular ligand-binding domain can comprise an antibody or fragment thereof described herein.
• the transmembrane domain comprises a stalk region.
• the stalk region can be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region (such as CH1, CH2, CH3, or both CH2 and CH3 for an IgG antibody, or CH1, CH2, CH3, CH4, or any combination thereof for an IgM antibody).
• the stalk region can be a synthetic sequence that corresponds to a naturally occurring stalk sequence or can be an entirely synthetic stalk sequence. In an embodiment said stalk region is a part of human CD8 alpha chain. Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 [00252]
• the signal transducing domain or intracellular signaling domain of the chimeric B cell receptor of the invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response. In other words, the signal transducing domain is responsible for the activation of at least one of the normal functions of the B cell in which the chimeric B cell receptor is expressed.
• the term "signal transducing domain” can refer to the portion of a protein which directs the cell to perform a specialized function, such as early activation of Lyn and Syk and late activation of NFAT and NF ⁇ B as examples.
• the chimeric B cell receptor can comprise native transmembrane and intracellular domains. In native B cells, engagement of the B cell receptor leads to rapid tyrosine phosphorylation of the intracellular domains and calcium ion polarization, resulting in downstream activation of NFAT and NF-kB.
• NFAT/NF-kB response elements to drive expression of our secreted proteins, we have designed an inducible expression system that will be activated by antigens associated with disease states, such as cancer.
• transmembrane polypeptides comprise the ability to be expressed at the surface of an immune cell, such as B cells, and to interact together for directing cellular response of immune cell against a predefined target cell.
• the different transmembrane polypeptides of the chimeric B cell receptor comprising an extracellular ligand- binding domain and/or a signal transducing domain interact together to take part in signal transduction following the binding with a target ligand and induce an immune response.
• the transmembrane domain can be derived from a natural or from a synthetic source.
• the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
• the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an unsought physiological change or disorder, such as the progression of cancer.
• Beneficial or preferred clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
• Treatment can refer to prolonging survival as compared to expected survival if not receiving treatment.
• the invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a cancer (for example, if an early detection cancer biomarker is identified in such a subject), or other cell proliferation-related diseases or disorders.
• diseases or disorders include but are not limited to, e.g., those diseases or disorders associated with aberrant expression of CLDN4 and/or aberrant activation of cellular signaling pathways involving CLDN4.
• diseases or disorders are included in CLDN- associated disease or disorders.
• the methods are used to treat, prevent, or alleviate a symptom of cancer.
• the methods are used to treat, prevent, or alleviate a symptom of a solid tumor.
• tumors that can be treated by compositions described herein comprise lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or stomach cancer.
• the methods of the invention can be used to treat hematologic cancers such as leukemia and lymphoma.
• the methods can be used to treat, prevent, or alleviate a symptom of a cancer that has metastasized.
• cancers that can be treated or prevented or for which symptoms can be alleviated include B- cell chronic lymphocytic leukemia (CLL), non-small-cell lung cancer, melanoma, ovarian cancer, lymphoma, or renal-cell cancer.
• CLL B- cell chronic lymphocytic leukemia
• cancers that can also be treated or prevented or for which symptoms can be alleviated include those solid tumors with a high mutation burden and WBC in filtrate.
• the invention provides methods for preventing, treating, or alleviating a symptom cancer or a cell proliferative disease or disorder in a subject by administering to the subject a monoclonal antibody, scFv antibody or bi- specific antibody of the invention.
• an anti-CLDN4 antibody can be administered in therapeutically effective amounts.
• Subjects at risk for cancer or cell proliferation-related diseases or disorders can include patients who have a family history of cancer or a subject exposed to a known or suspected cancer-causing agent. Administration of a prophylactic agent can occur prior to the manifestation of cancer such that the disease is prevented or, alternatively, delayed in its progression.
• tumor cell growth is inhibited by contacting a cell with an anti- CLDN4 antibody of the invention.
• the cell can be any cell that expresses CLDN4.
• the invention further provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a chronic or acute viral, bacterial, or parasitic infection.
• the invention also provides for therapeutic methods for both prophylactic and Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 therapeutic methods of treating a subject at risk of a disease or disorder or condition associated with T-cell exhaustion or a risk of developing T-cell exhaustion.
• the invention also provides for therapeutic methods for both prophylactic and therapeutic methods of treating a subject at risk of a disease or disorder or condition associated with T-cell exhaustion or a risk of developing T-cell exhaustion.
• diseases or disorder include, but are not limited to HIV, AIDS, and chronic or acute bacterial, viral or parasitic infections.
• HBV hepatitis B virus
• HCV hepatitis C virus
• H. pylori herpes simplex virus 1
• H. pylori herpes simplex virus 1
• Toxoplasma gondii Other acute infections included are those caused by, for example, microorganisms, such as a Gram-positive bacterium, a Gram-negative bacterium, a protozoan, or a fungus, as described herein.
• methods of increasing or enhancing an immune response to an antigen An immune response is increased or enhanced by administering to the subject a monoclonal antibody, scFv antibody, or bi-specific antibody of the invention.
• the immune response is augmented for example by augmenting antigen specific T effector function.
• the antigen is a viral (e.g., HIV), bacterial, parasitic or tumor antigen.
• the immune response is a natural immune response.
• natural immune response is meant an immune response that is a result of an infection.
• the infection is a chronic infection.
• Increasing or enhancing an immune response to an antigen can be measured by a number of methods known in the art.
• an immune response can be measured by measuring any one of the following: T cell activity, T cell proliferation, T cell activation, production of effector cytokines, and T cell transcriptional profile.
• the immune response is a response induced due to a vaccination.
• compositions of the invention as described herein can be administered in combination with cytokines.
• Cytokines that can be administered with the compositions include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL- 12, IL-13, IL-15, anti-CD40, CD40L, and TNF- ⁇ .
• the compositions described herein can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
• the compositions described herein can be administered in combination with other immunotherapeutic agents.
• Non-limiting examples of immunotherapeutic agents include sizumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensit
• the invention provides for methods of treating cancer in a patient by administering two antibodies that bind to the same epitope of the CLDN4 protein or, alternatively, two different epitopes of the CLDN4 protein.
• the cancer can be treated by administering a first antibody that binds to CLDN4 and a second antibody that binds to a protein other than CLDN4.
• the cancer can be treated by administering a bispecific antibody that binds to CLDN4 and that binds to a protein other than CLDN4.
• the other protein other than CLDN4 can include, but is not limited to, IL-12, IL-12R, IL-2, IL-2R, IL-15, IL-15R, IL-7, IL-7R, IL-21, or IL-21R.
• the other protein other than CLDN4 is a tumor-associated antigen; the other protein other than CLDN4 can also be a cytokine.
• Non-limiting examples of the other protein other than CLDN4 includes CTLA- 4, CXCR4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47 and CD73.
• the invention provides for the administration of an anti-PD- 1 antibody alone or in combination with an additional antibody that recognizes another protein other than CLDN4, with cells that can effect or augment an immune response.
• these cells can be peripheral blood mononuclear cells (PBMC), or any cell type that is found in PBMC, e.g., cytotoxic T cells, macrophages, and natural killer (NK) cells.
• PBMC peripheral blood mononuclear cells
• NK natural killer cells
• the invention provides administration of an antibody that binds to the CLDN4 protein and an anti-neoplastic agent, such as a small molecule, a growth factor, a cytokine or other therapeutic including biomolecules such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases.
• Small molecules include, but are not limited to, inorganic molecules and small organic molecules.
• Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
• biological sample is intended to include tissues, cells and biological fluids isolated from a subject (such as a biopsy), as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect cells that express CLDN4 in a biological sample in vitro as well as in vivo.
• in vitro techniques for detection of CLDN4 include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
• a conjugate administered by intravenous injection can achieve localization within minutes of injection.
• a conjugate administered orally can take hours to achieve localization.
• localization can simply refer to the location of the entity within the subject or animal at selected time periods after the entity is administered.
• 5031461-133-WO1 Date of Filing August 15, 2023 of another example, localization is achieved when an moiety becomes distributed following administration. [00278] A reasonable estimate of the time to achieve localization can be made by one skilled in the art.
• a voltage applied across the array results in the release of many electrons from each photon collision.
• the electrons from such a collision exit their channel of origin in a "shotgun" pattern and are detected by the camera.
• Even greater sensitivity can be achieved by placing intensifying microchannel arrays in series, so that electrons generated in the first stage in turn result in an amplified signal of electrons at the second stage. Increases in sensitivity, however, are achieved at the expense of spatial resolution, which decreases with each additional stage of amplification.
• An exemplary microchannel intensifier-based single-photon detection device is the C2400 series, available from Hamamatsu.
• Image processors process signals generated by photodetector devices which count photons to construct an image which can be, for example, displayed on a monitor or printed on a video printer. Such image processors are sold as part of systems which include the sensitive photon-counting cameras described herein, and accordingly, are available from the same sources.
• the image processors are connected to a personal computer, such as an IBM- compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif), which can or cannot be included as part of a purchased imaging system.
• a personal computer such as an IBM- compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif), which can or cannot be included as part of a purchased imaging system.
• image processing programs such as "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mt. View, Calif.
• EXAMPLE 1 – Claudin 4 as a therapeutic target [00291]
• EXAMPLE 2 [00294] – We have transiently transfected 293T cells with CLDN-3 plasmid Docket No.: 5031461-133-WO1 Date of Filing: August 15, 2023 [00295] - Low to no background binding of antibodies to 293T without CLDN-3 or CLDN- 4 [00296] – a CLDN-3 antibody specifically recognized only CLDN-3 cell line [00297] – a CDLN-4 antibodies bound CLDN-3 cell line weakly [00298] - Some 293T CLDN-4 cells can have lost CLDN-4 expression [00299] - We can perform assay of binding of a CLDN-4 minibodies to CLDN-3 expressing cell line ***** EQUIVALENTS [00300] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

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