Bispecific antibodies for treating cancer

Abstract

A bispecific antibody that binds to PD-1 and CTLA4, and dimers thereof, are disclosed. Methods of treating or slowing the growth of tumors or cancers comprising administering the antibody to a subject afflicted with cancer are further disclosed.

Description

DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025BISPECIFIC ANTIBODIES FOR TREATING CANCER

[0001] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.RELATED APPLICATIONS

[0003] This application claims priority from, and the benefit of, U.S. Provisional Application No. 63 / 725,439 filed November 26, 2024, U.S. Provisional Application No. 63 / 771 ,245 filed March 13, 2025, and US Provisional Application No. 63 / 794,910 filed April 25, 2025, the contents of which are incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0004] The present invention is directed to a bispecific antibody that binds to PD-1 and CTLA4.BACKGROUND OF THE INVENTION

[0005] Bispecific antibodies (BsAb) are antibodies or antibody-like molecules having two different binding specificities. BsAbs have broad applications in biomedicine, especially in immunotherapy for tumors. Presently, a focus of immunotherapy research is on how to utilize cell-mediated cytotoxicity of BsAb to kill tumor cells. A BsAb can be designed to target a tumor cell and an effector cell simultaneously, while triggering the effector cell's destruction of the tumor cell.SUMMARY OF THE INVENTION

[0006] Aspects of the invention are drawn towards a bispecific antibody that specifically binds to PD-1 (e.g., P4B3) and CTLA4 (e.g., E1A8). In embodiments, the bispecific antibody can comprise a first antigen binding site specific for PD-1 comprising VH CDR1 comprising theDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 amino acid sequence of GFTFDDFA, a VH CDR2 comprising the amino acid sequence of ISWNSGSI, and a VH CDR3 comprising the amino acid sequence of ASDYGDKYSYYGMDV, and a VL CDR1 comprising the amino acid sequence of SSNIGSNT, a VL CDR2 comprising the amino acid sequence of DDN, and a VL CDR3 comprising the amino acid sequence of AAWDGGLNGRGV; and a second antigen binding site specific for CTLA4 comprising a VH CDR1 comprising the amino acid sequence of GFTFSSYV, a VH CDR2 comprising the amino acid sequence of ISGSGGST, and a VH CDR3 comprising the amino acid sequence of ARGGSAWSLDI, and a VL CDR1 comprising the amino acid sequence of QDISNS, a VL CDR2 comprising the amino acid sequence of GAS, and a VL CDR3 comprising the amino acid sequence of QQGNSFPIT.

[0007] In embodiments, the first antigen binding site specific for PD-1 can comprise a VH comprising the amino acid sequence ofMAQVQLVQSGGGLVQPGRSLRLSCAASGFTFDDFAMHWVRQAPGKGLEWVSGISW NSGSIGYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCASDYGDKYSYYG MDVWGKGTTVTVSS, and a VL comprising the amino acid sequence of QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQFPGKAPKLLIFDDNQRPSG VPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDGGLNGRGVFGGGTKLTVL.

[0008] In embodiments, the second antigen binding site specific for CTLA4 can comprise a VH comprising the amino acid sequence of QLVQSGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSSISGSGGST DYADSVKGRFPISRDNSKNTLYLEMNSLRAEDTAVYYCARGGSAWSLDIWGQGTTV TVSS, and a VL comprising the amino acid sequence of DIQMTQSPSSVSASIGDRVTITCRASQDISNSLAWYQQKPGKAPKLLIYGASNLRSGV PSRFSGGGSGTYFTLTISSLQPEDFATYYCQQGNSFPITFGQGTRLEIKRTVAAPT.

[0009] Aspects of the invention are further drawn towards a tetravalent antibody molecule. In embodiments, the tetravalent antibody molecule is a dimer of a bispecific antibody fragment. In embodiments, the bispecific antibody fragment can comprise a first antigen binding site specific for PD-1 (e.g.. P4B3), a second antigen binding site specific for CTLA4 (e.g., E1A8), and dimerization domain, and a linker-hinge-linker domain. In embodiments, the first binding site and the second binding site are joined together via the dimerization domain.

[0010] In embodiments, the dimerization domain can comprise an immunoglobulin hinge region or fragment thereof. For example, the immunoglobulin hinge region is an IgGl, IgG2, IgG3, or IgG4 hinge region. For example, the immunoglobulin hinge region can comprise an amino acid sequence according toDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPR CPAPELLGGP, or a sequence at least 90% identical thereto.

[0011] In embodiments, the dimerization domain can comprise one or more amino acid linkers. For example, the amino acid linker can comprise a flexible linker. For example, the amino acid linker can comprise (GGGS)xi-e, (GGGGS)xi-s, or GSAGSAAGSGEF.

[0012] In embodiments, the dimerization domain can comprise a hinge region and at least one amino acid linker. For example, the immunoglobulin hinge region can be flanked on one or both sides by the amino acid linker.

[0013] In embodiments, the linker domain does not comprise an immunoglobulin Fc domain or fragment thereof.

[0014] In embodiments, the tetravalent antibody molecule can comprise one, two. three, or four scFv fragments.

[0015] In embodiments, the first antigen binding site specific for PD-1 can comprise a VH CDR1 comprising the amino acid sequence of GFTFDDFA, a VH CDR2 comprising the amino acid sequence of ISWNSGSI, and a VH CDR3 comprising the amino acid sequence of ASDYGDKYSYYGMDV, and a VL CDR1 comprising the amino acid sequence of SSNIGSNT, a VL CDR2 comprising the amino acid sequence of DDN, and a VL CDR3 comprising the amino acid sequence of AAWDGGLNGRGV.

[0016] In embodiments, the first antigen binding site specific for PD-1 can comprise a VH comprising the amino acid sequence ofMAQVQLVQSGGGLVQPGRSLRLSCAASGFTFDDFAMHWVRQAPGKGLEWVSGISW NSGSIGYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCASDYGDKYSYYG MDVWGKGTTVTVSS, and a VL comprising the amino acid sequence of QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQFPGKAPKLLIFDDNQRPSG VPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDGGLNGRGVFGGGTKLTVL.

[0017] In embodiments, the second antigen binding site specific for CTLA4 can comprise a VH CDR1 comprising the amino acid sequence of GFTFSSYV, a VH CDR2 comprising the amino acid sequence of ISGSGGST, and a VH CDR3 comprising the amino acid sequence of ARGGSAWSLDI, and a VL CDR1 comprising the ammo acid sequence of QDISNS, a VL CDR2 comprising the amino acid sequence of GAS, and a VL CDR3 comprising the amino acid sequence of QQGNSFPIT.

[0018] In embodiments, the second antigen binding site specific for CTLA4 can comprise a VH comprising the amino acid sequence of QLVQSGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSSISGSGGSTDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DYADSVKGRFPISRDNSKNTLYLEMNSLRAEDTAVYYCARGGSAWSLDIWGQGTTV TVSS, and a VL comprising the amino acid sequence of DIQMTQSPSSVSASIGDRVTITCRASQDISNSLAWYQQKPGKAPKLLIYGASNLRSGV PSRFSGGGSGTYFTLTISSLQPEDFATYYCQQGNSFPITFGQGTRLEIKRTVAAPT.

[0019] In embodiments, the tetravalent antibody molecule described herein can comprise the amino acid sequence ofMAQVQLVQSGGGLVQPGRSLRLSCAASGFTFDDFAMHWVRQAPGKGLEWVSGISW NSGSIGYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCASDYGDKYSYYG MDVWGKGTTVTVSSGGGGSGGGGSGGGGSQPGLTQPPSASGTPGQRVTISCSGSSS NIGSNTVNWYQQFPGKAPKLLIFDDNQRPSGVPDRFSASKSGTSASLAISGLQSEDEA DYYCAAWDGGLNGRGVFGGGTKLTVLGQPKAAPSAAAELKTPLGDTTHTCPRCPEP KSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPGGGGSGGG GSGGGGSGGGGSGGGGSRTQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYVMSW VRQAPGKGLEWVSSISGSGGSTDYADSVKGRFPISRDNSKNTLYLEMNSLRAEDTAV YYCARGGSAWSLDIWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSASIG DRVTITCRASQDISNSLAWYQQKPGKAPKLLIYGASNLRSGVPSRFSGGGSGTYFTLT ISSLQPEDFATYYCQQGNSFPITFGQGTRLEIKRTVAAPT, or a sequence at least 90% identical thereto.

[0020] Aspects of the invention are further drawn towards a nucleic acid construct encoding the bispecific antibody described herein or the tetravalent antibody described herein.

[0021] Further, aspects of the invention are drawn towards a vector comprising the nucleic acid construct described herein. In embodiments, the vector comprises a nucleic acid encoding a construct comprising P4B3-IgG3H-ElA8.

[0022] Still further, aspects of the invention are drawn towards a host cell comprising the vector described herein. In embodiments, the cell is a T-cell, a B-cell, a follicular T-cell, or an NK-cell.

[0023] Aspects of the invention are directed towards a pharmaceutical composition comprising the bispecific antibody described herein, the tetravalent antibody molecule described herein, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0024] Also, aspects of the invention are drawn towards a method for treating a subject afflicted with cancer. In embodiments, the method can comprise administering to the subject the bispecific antibody described herein or the tetravalent antibody described herein. In embodiments, the cancer can comprise a renal cancer. For example, the renal cancer can comprise clear cell renal cell carcinoma (ccRCC).DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0025] In embodiments, the bispecific antibody described herein or the tetravalent antibody described herein can be for use in the treatment of a subject afflicted with cancer. In embodiments, the cancer can comprise renal cancer. For example, the renal cancer can comprise clear cell renal cell carcinoma.

[0026] Further, aspects of the invention are drawn towards a method for slowing tumor grow th or cellular proliferation in a subject afflicted with cancer. In embodiments, the method can comprise administering to the subject the bispecific antibody described herein or the tetraval ent antibody described herein. In embodiments, the cancer can comprise a renal cancer. For example, the renal cancer can comprise clear cell renal cell carcinoma (ccRCC).

[0027] In embodiments, the bispecific antibody described herein or the tetravalent antibody described herein can be for use in slowing tumor growth or cellular proliferation in a subject afflicted with cancer. In embodiments, the cancer can comprise renal cancer. For example, the renal cancer can comprise clear cell renal cell carcinoma.

[0028] Other objects and advantages of this invention will become readily apparent from the ensuing description.BRIEF DESCRIPTION OF THE FIGURES

[0029] FIG. 1 shows one aspect of the disclosure as described herein drawn to CAR-T Therapy in ccRCC. For example, the dual-targeted, fine-tuned CAR mitigates tumor heterogeneity and antigen escape, and further only recognizes high density tumor associated antigens (e.g., anti- CAIX / CD70 CAR). Further, embodiments can secrete immune checkpoint inhibitors (ICIs) at the tumor site to restore active antitumor immunity.

[0030] FIG. 2 shows establishment of humanized ccRCC orthotopic mouse model. See also, Wang et al., iScience 2024.

[0031] FIG. 3 shows humanized ccRCC orthotopic mouse model recapitulates human TME. See also, Wang et al., iScience 2024.

[0032] FIG. 4 shows design of immune restoring (IR) CAR that secrete immune checkpoint inhibitors (ICIs at the tumor site to restore active antitumor immunity.

[0033] FIG. 5 shows immune restoring anti-CAIX CAR-T cells exhibited superior efficacy in humanized ccRCC orthotopic mouse model. See also. Wang et al., iScience 2024.

[0034] FIG. 6 shows tumor microenvironment profile using scRNAseq and scTCRseq. See also, Wang et al., iScience 2024.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0035] FIG. 7 shows G36-PDL1 reshaped the immunosuppressive tumor microenvironment. See also, Wang et al., iScience 2024.

[0036] FIG. 8 shows enhanced B-Tfh and decreased myeloid crosstalk were observed in G36- PDL1 TME. See also, Wang et al., iScience 2024.

[0037] FIG. 9 shows cell lines recapitulate CIX density in human samples. See also, Wang et al., Mol. Cancer 2024.

[0038] FIG. 10 shows affinity fine-tuned G9-41BB CAR-T cells mitigated cytotoxicity on CAIX low expression cholangiocytes. See also, Wang et al.. Mol Cancer 2024.

[0039] FIG. 11 shows G9 has high avidity on skrc-59 tumor cells but low avidity on MMNK- 1 cholangiocytes. See also, Wang et al., Mol Cancer 2024.

[0040] FIG. 12 shows ccRCC PDOTS recapitulates ccRCC tumor microenvironment.

[0041] FIG. 13 shows G9 exhibited superior efficacy on ccRCC PDOTS.

[0042] FIG. 14 shows G9 exhibited superior efficacy in ccRCC NSG-SGM3 mouse model. See also, Wang et al., Mol Cancer 2024.

[0043] FIG. 15 shows ccRCC tumor heterogeneity. For example, left panel shows expression profiling of ccRCC human samples stages I-IV, indicating heterogenous expression of CAIX and CD70 among individual tumors. Tumor heterogeneity is one of the hurdles which prevent CAR-T cell therapy from translating to solid tumors. By IHC staining of both CAIX and CD70 on ccRCC patient samples, we found that both CD70 and CAIX are heterogeneously expressed in ccRCC. CAIX has an overall higher expression level compared to CD70 among different stages ccRCC patients. If we only target CAIX, circa 0-30% cells will be missed. The same is true for CD70. If we only target CD70, roughly 0-90% cells will be missed. To achieve a better therapeutic efficacy, targeting both CAIX and CD70 on the cell surface could lead to a larger targeted population. In patient 477503, the CAIX and CD70 are both highly expressed and coexpressed. However, to treat patients like 477486, targeting CAIX and CD70 would benefit the treatment outcomes greatly.

[0044] FIG. 16 shows CAIX / CD70 targeted CAR-T cells killed CAIX or CD70 positive ccRCC. Based on the clinical sample study, we established CAIX+ CD70+, CAIX+, CD70+, and CAIX- CD70- skrc-59 ccRCC tumor cell lines in vitro. We then assessed anti-CAIX / CD70 CAR-T cells on the cell mixtures. On Day 0, we mixed CAIX+ CD70+ cells with CAIX+ cells as shown in the left column, with CD70+ cells in the middle column, and CAIX- CD70- cells in the right column with 1: 1 ratios of CAR-T to tumor cells. 2 days after co-incubation, there were no CAIX+CD70+. CAIX+ or CD70+ cells left. However the CAR-T cells did not kill CAIX- CD70- cells. These results demonstrate that dual -targeted CAR-T cells are able to killDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025CAIX or CD70 tumor population. The efficacy of dual -targeted CAR-T cells was also validated In vivo.

[0045] FIG. 17 shows design of dual-targeted fine-tuned immune restoring (DFIR) CAR. For example, shown here is a schematic of DFIR CAR-T showing CAR-T cell expresses anti-CAIX and anti-CD70 BsAb antibody to prevent immune checkpoint mediated T cell exhaustion. anti- CAIX CAR-T cells with 4 IBB costimulatory domain exhibit a superior efficacy and long-term persistence in orthotopic ccRCC tumor bearing mouse model. Further, anti-CAIX / CD70 dualtargeted CAR-T cells address tumor heterogeneity and potential antigen escape. Affinity / avidity fine-tuned CAR-T cells only kill TAA high expressing tumor cells but not normal tissues with low expression of the same antigen. Accordingly, CAR-T cells secreting an immune checkpoint inhibitor (ICI) antibody at the TME are able to restore the active anti-tumor immunity. Taking all together, a dual-targeted fine-tuned immune restoring aka (DFIR) CAR was designed as shown in the right panel.

[0046] FIG. 18 shows that advanced RCC patients benefit from anti-PD-1 antibody and anti- CTLA4 Ab combination therapy. See also, Motzer et al.. N Engl J Med. 2018, and Tannir et al., Ann Oncol, 2024. Overall survival and objective response rates were significantly higher with nivolumab plus ipilimumab than with sunitinib among intermediate- and poor-risk patients with previously untreated advanced renal-cell carcinoma.

[0047] FIG. 19 shows BsAb P4B3-IgG3H-ElA8 exhibited superior efficacy compared to Nivolumab + Ipilimumab. As described herein. BsAb P4B3-IgG3H-ElA8 exhibited superior efficacy compared to mono therapy El A8, P4B3, and combination therapy of El A8 and P4B3. In the right plot, BsAb outperformed Ipi+ Nivo combination therapy.

[0048] FIG. 20 shows DFIR CAR-T cells exhibited superior tumor killing compared to B7-3- G9 without an ICI payload after multiple challenges.

[0049] FIG. 21 shows CAIX expression in ccRCC patients and CX70 expression in RCC patients, establishing CAIX and CD70 as tumor associated antigens in ccRCC.

[0050] FIG. 22 show s exemplary P4B3-IgG3H-El A8 bispecific, tetravalent antibody formats.

[0051] FIG. 23 shows P4B3 and E1A8 exhibit superior stimulatory capacity in comparison to clinical standards.

[0052] FIG. 24 shows P4B3-IgG3H-El A8 binds to PD1+CTLA4+ Jurkat cells with higher affinity than E1A8.

[0053] FIG. 25 shows P4B3-IgG3H-ElA8 stimulates PBMCs better than a cocktail of Ipilimumab and Nivolumab. We demonstrated that BsAb P4B3-IgG3H-ElA8 exhibitedDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 superior efficacy compared to mono therapy E1A8, P4B3, and combination therapy of E1A8 and P4B3. In the right plot, BsAb outperformed Ipi+ Nivo combination therapy.

[0054] FIG. 26 shows P4B3-IgG3H-El A8 secreting CAR-T cell eradicates tumor better than CAR-T cell secreting an irrelevant antibody.

[0055] FIG. 27 shows dual anti-CAIX / anti-CD70 CAR-T cells secreting BsAb payload to overcome CAR-T solid tumor challenges.

[0056] FIG. 28A and FIG. 28B ] shows bispecific secreting DFIR CAR-T cells perform better than mono CAR-T cells in cytotoxicity assays. Bispecific secreting DFIR CAR-T cells perform better than mono CAR-T cells in cytotoxicity assays.

[0057] FIG. 29 shows P4B3-IgG3H-ElA8 pay load exhibited superior efficacy in a multichallenge assay. Specifically, CAR-T cells with bispecific antibody payload outperformed the ones with either El A8 or P4B3 mAb.

[0058] FIG. 30A and FIG. 30B shows DFIR CAR-T cells secrete immune-checkpoint inhibitor payload In the plot of FIG. 30A, we quantified the BsAb payload secretion in four different healthy donors. In the plot of FIG. 30B, we performed multi-challenge assays, showing that DFIR CAR-T with BsAb payload outperformed CAR-T cells with irrelevant payload and maintained the killing capacity after multiple rounds of challenges.

[0059] FIG. 31 shows exemplary amino acid sequence of anti-CAIX / anti-CD70 CAR-T cells secreting anti-PDl / anti-CTLA4 bsAb.DETAILED DESCRIPTION OF THE INVENTION

[0060] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0061] The singular forms “a”, "an" and "the" include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0062] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.DOCKET NO: 5031461-000173-W01 DATE OF FILING: November 26, 2025

[0063] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0064] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0065] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0066] The present invention provides bispecific antibodies, bispecific antibody molecules, or antigen binding fragments thereof, that are specific for PD1 (e.g., P4B3) and CTLA4 (e.g., E1A8), and uses thereof.

[0067] For example, the amino acids of the CDRs of the anti-PDl antibodies are provided in Table 1 :DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0068] For example, the amino acid sequences of the VH and VL regions of the anti-PDl antibodies are provided in Table 2:DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0069] For example, the amino acids of the CDRs of the anti-CTLA4 antibodies are provided in Table 3:DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0070] For example, the amino acid sequences of the VH and VL regions of the anti-CTLA4 antibodies are provided in Table 4:DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0071] Antibodies and antigen-binding molecules

[0072] The term "antibody" can refer to an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids primarily responsible for antigen recognition via the complementary determining region (CDRs) contained therein.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0073] The phrase “complementary determining means'’ as used herein describes the six complimentary determining regions (CDRs) that collectively form specific interactions with antigens described herein. CDRs within the scope of complementary determining means are the disclosed CDRs and functional equivalents thereto. Functional equivalent CDRs comprise different specific amino acid residues but bind an antigen within binding affinity range of the antibodies described herein. Functional equivalent CDRs would differ insubstantially to bind an antigen and have a therapeutic effect.

[0074] The CDRs are interspersed with regions that are well-known and generally conserved among and between species (e.g., mouse and human), which are termed framework regions (FRs). 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.

[0075] In embodiments, the CDRs are interspersed with FRs. Antibodies disclosed herein have four FRs, termed FR1, FR2, FR3, and FR4. In embodiments, the FRs are human FRs (e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0076] The three CDRs of the light chain (LC) are referred to as "LCDR1, LCDR2, and LCDR3," and the three CDRs of the heavy chain (HC) are referred to as "HCDR1, HCDR2, and HCDR3." The functional ability of an antibody to bind a particular antigen is largely determined by the six CDRs. Assignment of amino acids to CDR domains within the LCVR and HCVR regions of the antibodies of the present disclosure is based on the IGMT numbering conventions.

[0077] The six CDRs in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain 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 f>-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the [3-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, sinceDOCKET NO: 5031461-000173-W01 DATE OF FILING: November 26, 2025 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)).

[0078] Where there are two or more definitions of a term which is used and / or accepted within the art, the definition of the term as used herein is intended to include such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This region has been described by Kabat et al., U.S. Dept, of Health and Human Sendees, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. 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 herein 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.

[0079] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. The skilled artisan can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering systemDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 set forth by Kabat et al., U.S. Dept, of Health and Human Services, ‘‘Sequence of Proteins of Immunological Interest” (1983). As used herein, “Chothia numbering” refers to the location of the structural loops (see, e.g., Chothia and Lesk, 1987, J. Mol. Biol. 196:901-17). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. Another universal numbering system that has been developed and widely adopted is ImMunoGeneTics (IMGT) Information System® (Lafranc et al., 2003, Dev. Comp. Immunol. 27(l):55-77). “IMGT numbering” refers to an integrated information system specializing in immunoglobulins (IG), T-cell receptors (TCR), and major histocompatibility' complex (MHC) of human and other vertebrates. Correspondence between the numbering system, including, for example, the Kabat numbering and the IMGT unique numbering system, is well known to one skilled in the art. The CDRs of the antibody sequences described herein were identified and numbered according to the IMGT unique numbering scheme as defined by the international ImMunoGeneTics information system (IMGT).

[0080] As used herein, the term "epitope" can include any protein determinant capable of specific binding 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. For example, 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 antigenbinding site. This quaternary antibody structure forms the antigen-binding site at the end of each arm of the Y. Epitopic determinants can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- terminal or C-terminal peptides of a polypeptide. In some embodiments, the antibodies are directed to the EC domain of a tumor-associated antigen. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3).

[0081] The constant region of the antibody defines the isotype of an antibody. The antibodies of the present disclosure include IgG. IgG antibodies can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4. In an embodiment, the antibodies of the present disclosure are IgGl. In an embodiment, the antibodies of the present disclosure are IgG4. The carboxy-terminal portion of each HC defines a constant region primarily responsible for effector function. In a particular embodiment, the antibodies of the present disclosure have one or more modifications in the constant region of each HC that reduces effector function.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0082] The term “antigen binding molecule” is used herein in the broadest sense and can refer to a molecule specifically binding to one or more antigens.

[0083] An "antibody fragment" or “antigen binding fragment” can be a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, scFv, Fab, Fab', Fab'-SH, F(ab)2, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

[0084] Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” can include aptamers (such as spiegelmers), minibodies, and diabodies. The term “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. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, epitopebinding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv). single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti- idiotypic (anti-Id) antibodies.

[0085] Further disclosed herein are antibody fragments, such as well-characterized Fabs (e.g.. Fab, Fab', F(ab')2, F(ab)2.), Fvs (the variable region of the light chain and the variable region of the heavy chain expressed as two chains), and single-chain fragments (e.g., singlechain variable region fragments, scFv, scFv-Fc, and single chain Fabs, scFab), which also bind to VEGF. Antibody fragments can include aptamers, minibodies, and diabodies. Methods of making these fragments are routine (see, e.g.. Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0086] The antibody or antigen-binding fragment or variant thereof can comprise a scFv. A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. A single chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide-encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can connect the N- terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains theDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. A number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent Nos. 5,091,513; 5,892,019; 5,132,405; and 4,946,778, each of which are incorporated by reference in their entireties.

[0087] Very large naive human scFv libraries have been and can be created to offer a large source of rearranged antibody genes against a plethora of target molecules. Smaller libraries can be constructed from individuals with infectious diseases in order to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al, Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)).

[0088] The term ‘‘bispecific antibody” or ‘'bsAb” can refer to an antibody or fragment thereof that has two different binding specificities (making it bispecific). BsAbs have broad applications in biomedicine, especially in immunotherapy for tumors. Presently, a focus of immunotherapy research is on how to utilize cell-mediated cytotoxicity of BsAb to kill tumor cells. A BsAb can be designed to target a tumor cell and an effector cell simultaneously, while triggering the effector cell's destruction of the tumor cell. In embodiments, the bispecific antibody can be specific for PD-1 and CTLA4._BsAb can be prepared by methods such as chemical engineering, cell engineering and genetic engineering.

[0089] The term “tetravalent antibody” or “tetravalent bispecific antibody” or “tetravalent BsAb” or “tBsAb” can refer to an antibody that has two distinct binding sites (making it bispecific) and four antigen-binding domains (making it tetravalent). In embodiments, the tetravalent bispecific antibody secreted by the antibody molecule of the present invention is specific for PD-1 and CTLA4.

[0090] Tetravalency has advantages, such as higher avidity resulting from multiple binding sites, improved simultaneous targeting in trans, and reduced drug quantity.

[0091] In embodiments, the tetravalent antibody is a dimer of a bispecific scFv fragment having a first binding site for a first antigen, a second binding site for a second antigen. The scFv is preferably a tandem scFv. The variable domains of the two binding sites are joined together via a linker domain. In preferred embodiments the linker domain includes an immunoglobulin hinge region amino acid sequence. The hinge region is an IgGl, an IgG2, an IgG3, or an IgG4 hinge region. Exemplary hinge region amino acids sequences include EPKSCDKTHTCPPCP (SEQ ID NO: [ ]); ERKCCVECPPCP (SEQ ID NO: [ ]);DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO: [ ]), and ESKYGPPCPSCP (SEQ ID NO: [ ]). The IgG3 hinge, for example, provides greater flexibility and mobility for better engagement of antigens, and is less susceptible to proteolytic cleavage than other hinges.

[0092] In some embodiments the linker domain, optionally, includes at least a portion of an immunoglobulin Fc domain. The at least a portion of an immunoglobulin Fc domain is an IgGl, an IgG2. an IgG3, or an IgG4 Fc domain. The at least a portion of an immunoglobulin Fc domain is linked to the C-termmus of the hinge region. By at least a portion of an immunoglobulin Fc domain is meant for example, an immunoglobulin CH2 domain amino acid sequence, CH3 domain amino acid sequence, CH4 domain amino acid sequence or any combination thereof.

[0093] Inclusion of at least a portion of an immunoglobulin Fc domain (e.g. CH2 domain) provides a third functional binding site (i.e. Fc effector function) resulting in a trifunctional bispecific antibody. Accordingly, it can be desirable to modify the at least a portion of with respect to effector function, so as to enhance, e.g., the effectiveness of the tBsAb. For example, amino acids substitution, insertion or deletion can be introduced into the at least a portion of the immunoglobulin Fc domain to generate tBsAbs having improved internalization capability and / or increased complement mediated cell killing and antibody dependent cellular cytotoxicity7(ADCC). Alternatively, the at least a portion of the immunoglobulin Fc domain is glycosylated as to improve the stability and solubility of the tBsAbs. For example, the at least a portion of the immunoglobulin Fc domain is glycosylated at the amino acid corresponding to asparagine at amino acid position 297. While glycosylation is important for stability7defucosylation of the CH2 carbohydrate can also increase the binding affinity to FcyRs and lead to further enhancement of ADCC.

[0094] In certain embodiments, the antibody molecule may comprise an Fc variant comprising an amino acid substitution which alters the antigen-independent effector functions of the antibody, in particular the circulating half-life of the antibody. Such antibodies exhibit either increased or decreased binding to FcRn yvhen 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 yvhere long half-life of the administered antibody is desired, e.g., to treat a chronic disease or disorder. In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for in vivo diagnostic imaging or in situations yvhereDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 the starting antibody has toxic side effects when present in the circulation for prolonged periods. In one embodiment, 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 which altered FcRn binding activity' are disclosed in International PCT Publication No. WO05 / 047327 which is incorporated by reference herein. In certain exemplary embodiments, 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).

[0095] Preferably the at least a portion of the Fc domain is a CH2 domain amino acid sequence. An exemplary’ CH2 domain amino acid sequence includes APELLGGPDVFLF.

[0096] In other aspects the immunoglobulin hinge region amino acid sequence or the immunoglobulin hinge region / Fc domain amino acid sequence is flanked by a flexible linker amino acid sequence. Flexible linker amino acid sequences include for example is (GGGS)x=i- 6. (GGGGS)X=I-6, or GSAGSAAGSGEF.

[0097] Increasing the linker by adding multiple repeats (e.g., four or more) will predominantly result in a monomeric scFv, thus it can increase the accessibility to an epitope. Length and composition of the linkers can be chosen to optimize the stability’ and functional activity and takes into account the topography of the epitope on the target protein.

[0098] Also included in the invention is a nucleic acid construct including nucleic acids molecules encoding: a light chain and a heavy chain variable region of an antibody specifically binding to a first antigen; a light chain and heavy chain variable region of an antibody specifically binding to a second antigen; and a linker domain.

[0099] In embodiments, the tetravalent antibody molecule can comprise a dimer of a bispecific scFv fragment. In embodiments, the bispecific antibody fragment can comprise a first antigen binding site specific for PD-1, a second antigen binding site specific for CTLA-4, and a dimerization domain. In embodiments, CTLA-4 mediates immunosuppression in the tumor immune micro-environment (TIME). Accordingly, CTLA4 expression is a reliable prognostic marker for ccRCC. PD-1 / PD-L1 also mediates immunosuppression in the TIME. PD1+ CD8+ / Treg ration is an indicator of treatment efficacy in ccRCC.

[0100] The term “dimerization domain” can refer to region of a protein or polypeptide that facilitates the interaction and binding of two identical or different molecules, resulting in the formation of a dimer. In embodiments, the dimerization domain can comprise an immunoglobulin hinge region or fragment thereof. For example, the immunoglobulin hingeDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 region can comprise IgGl, IgG2, IgG3, or IgG4 hinge region. For example, the immunoglobulin hinge region comprises an amino acid sequence according to ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPR CPAPELLGGP or a sequence at least 90% identical thereto.

[0101] In embodiments, the dimerization domain can comprise one or more amino acid linkers. For example, the amino acid linker can comprise (GGGS)xi-e, (GGGGS)xi-6. or GSAGSAAGSGEF.

[0102] In embodiments, the amino acid linker can comprise a flexible linker. The term “flexible linker” can refer to polypeptide sequence or molecular structure that connects two or more functional domains or proteins, allowing them to retain their individual activities while providing sufficient flexibility for optimal spatial orientation and interaction.

[0103] In embodiments, the dimerization domain can comprise a hinge region and at least one amino acid linker. In embodiments, the immunoglobulin hinge region can be flanked on one or both sides by the amino acid linker. In embodiments, the dimerization domain does not comprise an immunoglobulin Fc domain or fragment thereof.

[0104] Embodiments of the invention can also refer to a fusion antibody. The term “fusion antibody” can refer to an antibody in which one or more functional domains, such as proteins, peptides, or other molecules, are genetically or chemically fused to the antibody structure. This fusion enhances the antibody's therapeutic, diagnostic, or functional properties by combining the targeting ability of the antibody with the specific activity of the fused component. For example, the fusion antibody can be a heavy chain (HC) fusion antibody or a light chain (LC) fusion antibody. For example, the fusion antibody described herein can target PD1 and CTLA4.

[0105] The term “heavy chain fusion antibody” or “HC-fusion antibody” can refer to an antibody where a functional protein or domain is fused to the heavy chain (HC) of the antibody. For example, in an HC-fusion antibody, the anti-PDl antibody can be fused to the light chain and the anti-CTLA4 antibody can be fused to the heavy chain. In another example, the anti- CTLA4 antibody can be fused to the light chain and the anti-PDl antibody can be fused to the heavy chain.

[0106] The term “light chain fusion antibody” or “LC-fusion antibody” can refer to an antibody where a functional protein or domain is fused to the light chain (LC) of the antibody. For example, the fusion antibody described herein can target PD1 and CTLA4. For example, in a LC-fusion antibody, both the anti-PDl antibody and the anti-CTLA4 antibody can be fused to the light chain.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0107] 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 in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (y, q, a, 5, £) with some subclasses among them (e.g., yl-y4). Certain classes have subclasses as well, such as IgGl, IgG2, IgG3 and IgG4 and others. The immunoglobulin subclasses (isotypes) e.g., IgGl, IgG2, IgG3, IgG4, IgG5, etc. are well characterized and are known to confer functional specialization. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides 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., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of an immunoglobulin molecule.

[0108] Light chains are classified as kappa or lambda (K, X). Each heavy chain class can be bound with a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0109] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. The variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. The term "antigen-binding site," or "binding portion" can refer to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" can refer to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibodyDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 molecule, 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." VH and VL regions, which contain the CDRs, as well as frameworks (FRs) of the antibodies (e.g., scFvs) are shown in Tables 2-3.

[0110] Aspects of the invention provide isolated monoclonal antibodies. The term "monoclonal antibody" or “mAb” or "MAb" or "monoclonal antibody composition", as used herein, can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. The complementarity determining regions (CDRs) of the monoclonal antibody are identical in the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with an epitope of the antigen characterized by a unique binding affinity for it. The term "‘isolated"’ as used herein with respect to cells, nucleic acids, such as DNA or RNA, can refer to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolated” can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. For example, an “isolated nucleic acid” can include nucleic acid fragments which are not naturally occurring as fragments and cannot be found in the natural state. “Isolated” can also refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides can include both purified and recombinant polypeptides. “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.

[0111] Some embodiments feature antibodies that have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the antibodies described herein. For example, “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 theDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 number of matching or homologous positions shared by the sequences. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%. 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, or higher identity when compared a specified region or the full length of any one of the antibodies described herein. For example, 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 nucleic acid sequences encoding 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 know n 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 identify or similarity for the nucleic acids and proteins of the invention.

[0112] Polypeptide as used herein can encompass a singular “polypeptide’' as well as plural “polypeptides.” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of tw o 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.

[0113] As to 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 “variant” or "conservatively modified variant". In some embodiments the alteration results in the substitution of an amino acid with a chemically similar amino acid.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0114] Contemplated herein are conservative variants of the disclosed antibodies and fragments thereof. A protein is a conserv ative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein. For example, an antibody that binds a target can include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, in a constant domain, and bind the target. Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art. The following groups are examples of amino acids that are considered conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0115] In some embodiments 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 know n in the art. Such conser atively modified variants of the antibodies disclosed herein can exhibit increased cross-reactivity to an antigen in comparison to an unmodified antibody.

[0116] For example, 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, try ptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0117] Herein, a "degenerate variant" can refer to a polynucleotide encoding a polypeptide (such as an antibody or fragment thereof) that includes a sequence that is degenerate based on the genetic code (i.e., the 20 natural amino acids can be specified by more than one codon). All degenerate nucleotide sequences encoding the disclosed antibody and fragment polypeptide sequences are included.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0118] Further contemplated are variants of the disclosed antibodies and fragments thereof with a sequence identity of at least 80%, at least 85%, at least 90%. at least 95%, or at least 98% of the antibody sequences as described herein. Herein, “sequence identity’’ can referred to as the similarity between amino acid or nucleic acid sequences, which is expressed as the similarity between the sequences. Sequence identity is frequently measured as percent identity, in which two sequences are considered more similar the higher the percentage. Homologs or variants of a polypeptide or nucleic acid molecule possess a relatively high degree of sequence identity when aligned using standard methods, which are well-known. Ceslovas Venclovas, Methods for Sequence-Structure Alignment in Homology Modeling: Methods and Protocols, 55-82 (Andrew Orry and Ruben Abagyan, eds., 2012)).

[0119] The term "binding” (or “binds”) can refer to the well understood interaction between an antibody molecule and a target protein, peptide, or polysaccharide. Binding can be measured in a variety7of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)). A particular antibody or protein binds to a particular target protein, peptide, or polysaccharide and does not bind in a significant amount to other proteins or polysaccharides present in a sample or subject disclosed herein. Binding occurs between the disclosed antibodies and fragments thereof and an epitope of an antigen. By "specifically binds" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the antigen and does not react with other polypeptides. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, antigen-binding fragments (Fab), Fab' and F(ab')2 fragments, scFvs, and Fab expression libraries.

[0120] The terms "immunological binding," and "immunological binding properties" can refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the equilibrium binding constant (KD) of the interaction, wherein a smaller KD represents a greater affinity7. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen- binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361: 186-87 (1993)). The ratioDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 of Koff / Kon enables the cancellation of parameters not related to affinity and is equal to the equilibrium binding constant (KD). (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the invention can specifically bind to a tumor-associated antigen epitope when the equilibrium binding constant (KD) is <10 pM, < 10 nM, < 10 pM, or < 100 pM to about 1 pM, as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art, such as BIAcore.

[0121] “Specifically binds” or “has specificity to,” can refer to an antibody molecule that binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. For example, an antibody molecule is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it can bind to a random, unrelated epitope. For example, the antibody molecule can be monovalent or bivalent, and comprises a single or double chain. Functionally, the binding affinity of the antibody molecule is within the range of 105M to 1012M. For example, the binding affinity of the antibody molecule is from 106M to 1012M. from 107M to 10"12M, from 10SM to 10"12M. from 10"9M to 1012M. from 105M to 10 " M. from 10’6M to 10 " M. from 10’7M to 10"11M, from 10’8M to 10 " M. from 10"9M to 101 1M. from 10’ "' M to 101 1M. from I O5M to 10 "'M. from 106M to 10l0M. from 10’7M to 10 "' M. from 10"8M to 10IOM. from 10’9M to 10 "' M. from 10"5M to 109M. from 10’6M to 10’9M, from 10’7M to 10’9M, from 108M to 10’9M, from 105M to 108M, from 106M to 108M, from 107M to 108M. from 105M to 107M. from 106M to 107M, or from 105M to 106M.

[0122] A target protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.

[0123] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by ascertaining whether the former prevents the latter from binding to a target protein. For example, if the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, as shown by a decrease in binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies bind to the same, or to a closely related, epitope.

[0124] Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with the target protein, with which it is normally reactive, and thenDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind the target protein. If the human monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention. Screening of human monoclonal antibodies of the invention can be also carried out by utilizing a target protein and determining whether the test monoclonal antibody is able to neutralize the target protein.

[0125] A first binding protein (e.g., antibody or ligand) '‘binds to the same epitope” as a second binding protein (e.g., antibody or ligand) if the first binding protein binds to the same site on a target compound that the second binding protein binds, or binds to a site that overlaps (e.g., 50%. 60%. 70%. 80%. 90%, or 100% overlap, e.g.. in terms of amino acid sequence or other molecular feature (e.g., glycosyl group, phosphate group, or sulfate group)) with the site that the second binding protein binds.

[0126] A first binding protein (e.g., antibody or ligand) “competes for binding” with a second binding protein (e.g.. antibody) if the binding of the first binding protein to its epitope decreases (e.g., by 10%. 20%, 30%, 40%, 50%, 60%, 70%. 80%. 90%. 100%. or more) the amount of the second binding protein that binds to its epitope. The competition can be direct (e.g., the first binding protein binds to an epitope that is the same as, or overlaps with, the epitope bound by the second binding protein), or indirect (e.g., the binding of the first binding protein to its epitope causes a steric change in the target compound that decreases the ability of the second binding protein to bind to its epitope).

[0127] V arious procedures know n within the art can be used for the production of antibody molecules described herein, fragments, analogs homologs or orthologs thereof. (See, for example. Antibodies: A Laboratory Manual. Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0128] Antibody molecules 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 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 Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0129] Further antibody molecules having a complementary binding means can be prepared and screened by well-known methods, such as hybridoma, transgenic animals, and phage orDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 yeast display (see, e.g., Monoclonal Antibodies: Methods and Protocols (Vincent Ossipow and Nicolas Fischer, eds., 2d ed. 2014)). Antibody molecules having equivalent complementary binding means differ in their amino acid sequence but perform the same function of binding the target through CDR-target interaction acting as (inhibitor / agonist / antagonist) to achieve the same result (inhibiting tumor growth). Preferably, the complementary binding means functions through the same epitope as the disclosed antibodies.

[0130] Fully human antibodies, for example, are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies", or "fully human antibodies" herein. “Humanized antibodies’" can be antibodies from non-human species whose light chain and heavy’ chain protein sequences have been modified to increase their similarity to antibody variants produced in humans. Humanized antibodies are antibody molecules derived from a non-human species antibody that bind the antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen-binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen-binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g.. Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) For example, the non-human part of the antibody (such as the CDR(s) of a light chain and / or heavy chain) can bind to the target antigen. A humanized monoclonal antibody can also be referred to a “human monoclonal antibody” herein.

[0131] Antibody molecules can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5): 489-498 (1991); Studnicka et ., Protein Engineering 7 (6) 805-814 (1994); Roguska. et al . Croc. Natl. Sei. USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which is incorporated by’ reference in its entirety’). “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 (i.e.. 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, WangDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025H, 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).

[0132] 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. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0133] In addition, antibody molecules can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al.. J. Mol. Biol, 222:581 (1991)). Similarly, 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. 5.545,807; 5,545,806; 5,569,825; 5,625, 126; 5,633,425; 5,661,016, and in Marks et al, Bio / Technology 10, 779-783 (1992); Lonberg et al, Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

[0134] Human antibodies can additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See PCT publication no. W094 / 02602 and U.S. Patent No. 6,673,986). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides the modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. A non-limiting example of such a nonhuman animalDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 is a mouse, and is termed the Xenomouse™ as disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules. Thus, using such a technique, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. For an overview of this technology for producing human antibodies, see Lonberg and Huszar / Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98 / 24893; WO 96 / 34096; WO 96 / 33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545.806; 5,814,318; and 5,939.598, which are incorporated by reference herein in their entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can be engaged to provide human antibodies directed against a selected antigen using technology7similar to that described herein.

[0135] An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method, which includes deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embry onic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

[0136] One method for producing 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 culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the t o cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

[0137] As used herein, the terms "cell." "cell line." and "cell culture" can be used interchangeably. All of these terms also include their progeny, which is any and allDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 subsequent generations. For example, all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, "host cell" can refer to a eukaryotic cell that is can replicate a vector and / or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors. A host cell can be "transfected" or "transformed," which can refer to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" cells or host cells can refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced. Therefore, recombinant cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced nucleic acid. In embodiments of the invention, a host cell is a T cell, including a cytotoxic T cell (also known as TC, Cytotoxic T Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell); NK cells and NKT cells are also encompassed in the disclosure.

[0138] In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, are disclosed in PCT publication No. WO 99 / 53049.

[0139] The antibody molecule of the present invention can be expressed from an expression vector. Recombinant techniques to generate such expression vectors are well known in the art.

[0140] DNA constructs, which can also be referred to as "DNA vectors", as described herein, can be cloned into a vector which will be used to transduce and produce chimeric- antigen receptor T-cells that secrete polypeptides and / or fragments thereof. For example, DNA constructs can be cloned into a lenti viral vector for production of lentivirus, which will be used to transduce and produce chimeric-antigen receptor T-cells that secrete a mono, bi- or tri- specific immune-modulating antibody / minibody and / or antibody-fusion protein at the tumor site.

[0141] The antibody molecule can be expressed by a vector containing a DNA segment encoding the antibody or portions thereof 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 vector (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 antibodyDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, viral vectors, etc. 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. I. et al. Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al, Proc Natl. Acad. Sci USA 87: 1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al, Science, 259:988 (1993); Davidson, et al, Nat. Genet 3 :219 (1993); Yang, et al, J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G.. et al, Nat. Genet. 8: 148 (1994).

[0142] Pox viral vectors introduce the gene into the cell’s cytoplasm. Avipox virus vectors result in only a short-term expression of the nucleic acid. Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors can be used for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaP04 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection. cell microinjection, and viral vectors.

[0143] The vector can be employed to target essentially any target cell. For example, stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a location. Additionally, 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. (See Bobo et al, Proc. Natl. Acad. Sci. USA 91 :2076-2080 (1994); Mornson et al, Am. J. Physiol. 266:292- 305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.

[0144] 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 a target protein in a sample. The antibody can also be used to try to bind to and disrupt an activity of a target protein.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0145] Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (see e.g., U.S. Patent No. 4.946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the specificity for a protein or derivatives, fragments, analogs or homologs thereof. 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 Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fvfragments.

[0146] The antibody molecule 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. For example, 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). (See Caron et al, J. Exp Med., 176: 1 191-1 195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al, Anti-Cancer Drug Design, 3 : 219-230 (1989)).

[0147] In certain embodiments, an antibody molecule of the invention can comprise an Fc variant comprising an amino acid substitution which alters the antigen-independent effector functions of the antibody, in particular 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 desired, e.g., to treat a chronic disease or disorder. In contrast, Fc variants with decreased FcRn binding affinity have shorter halt-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. In addition, other applications in which reduced FcRn binding affinity can be desired include thoseDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 applications in which localization to the brain, kidney, and / or liver is desired. In one embodiment, the Fc-variant containing antibodies can exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the altered antibodies of the invention exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, 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 which altered FcRn binding activity are disclosed in PCT Publication No. WO 05 / 047327 which is incorporated by reference herein.

[0148] Expression vectors that encode the antibody molecule can be introduced as one or more DNA molecules or constructs, where there may be at least one marker that will allow for selection of host cells that contain the construct(s).

[0149] The constructs can be prepared in conventional ways, where the genes and regulatory regions can be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restnction or sequencing, or other convenient means. Particularly, using PCR, individual fragments including all or portions of a functional unit can be isolated, where one or more mutations may be introduced using "primer repair", ligation, in vitro mutagenesis, etc., as appropriate. The construct(s) once completed and demonstrated to have the appropriate sequences may then be introduced into the CTL by any convenient means. The constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors or lentiviral vectors, for infection or transduction into cells. The constructs may include viral sequences for transfection. Alternatively, the construct may be introduced by fusion, electroporation, biolistics, transfection, hpofection, or the like. The host cells can be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct(s). The cells are then expanded and screened by virtue of a marker present in the construct. Various markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.

[0150] In some instances, one can have a target site for homologous recombination, where a construct be integrated at a particular locus. For example,) can knock-out an endogenous gene and replace it (at the same locus or elsewhere) with the gene encoded for by the construct using materials and methods as are known in the art for homologousDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 recombination. For homologous recombination, one may use either .OMEGA, or O-vectors. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al.. Nature (1989) 338, 153-156.

[0151] The constructs can be introduced as a single DNA molecule encoding at least the CAR and optionally another gene, or different DNA molecules having one or more genes. Other genes include genes that encode therapeutic molecules or suicide genes, for example. The constructs may be introduced simultaneously or consecutively, each with the same or different markers.

[0152] Vectors containing useful elements such as bacterial or yeast origins of replication, selectable and / or amplifiable markers, promoter / enhancer elements for expression in prokaryotes or eukaryotes, etc. that may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art, and many are commercially available.

[0153] Antibody molecules of the invention, including bispecific, fusion, polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents will be employed to treat or prevent 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 target protein(s).

[0154] A specific dosage and treatment regimen for any patient will depend upon a variety of factors, including the antibody molecule, variant or derivative thereof used, the patient's age, 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 desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0155] Antibody molecules of the invention specifically binding a target protein(s) or fragment thereof can be administered for the treatment of a cancer in the form of pharmaceutical compositions. Principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising the antibody, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R. Gennaro, et al, editors) Mack Pub. Co., Easton, Pa., 2000; DrugDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers. Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0156] A therapeutically effective amount of an antibody molecule 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 can be 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.

[0157] Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, 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. USA, 90: 7889-7893 (1993).

[0158] The antibody molecules to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0159] An antibody molecule according to the invention can be used as an agent for detecting the presence of a target protein (or a protein fragment thereof) in a sample. For example, 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 term "labeled", with regard to the probe or antibody, can encompass direct labeling of theDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. 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. The term "biological sample" can include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, 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. For example, 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.

[0160] Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0161] Antibody molecules directed against a target protein (or a fragment thereof) can be used in methods known within the art relating to the localization and / or quantitation of a target protein (e.g., for use in measuring levels of the target protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to a target 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").

[0162] An antibody specific for a target protein of the invention can be used to isolate a polypeptide by standard techniques, such as immunoaffmity, chromatography or immunoprecipitation. Antibodies directed against a target protein (or a fragment thereof) canDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 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.

[0163] Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-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 include1251,1311,35S,32P or3H.

[0164] Aspects of the invention are draw n to methods of treating a subject afflicted with cancer (e.g., renal cancer), comprising administering the antibody molecule described herein to the subject. As used herein, 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 undesired physiological change or disorder, such as the progression of cancer. Beneficial 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. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

[0165] The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a cancer, or other cell proliferation-related diseases or disorders.

[0166] The terms "cancer" and "cancerous" can refer to or describe, for example, the physiological condition in mammals that is characterized by unregulated cell growth. For example, the methods are used to treat, prevent or alleviate a symptom cancer. In an embodiment, the methods are used to treat, prevent or alleviate a symptom of a solid tumor such as a renal cell carcinoma (e.g., clear cell renal cell carcinoma). Renal cell carcinoma represents 3% of all adult cancers, and clear cell renal cell carcinoma (ccRCC) is the majorDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 subtype of RCC (representing about 80% of all renal tumors). A hallmark of ccRCC is loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene. About 50% of patients develop metastatic disease despite treatment with targeted and immune therapies. Patients with metastatic ccRCC still have poor outcomes with a medial progression-free survival of 15.1 months. See, for example, Bini et al., Lancet 2009, and Bini et al. NEJM, 2019. Standard Clinical Treatments for ccRCC include partial / radical nephrectomy, targeted therapy (e.g., tyrosine kinase inhibitors (TKI) (VEGFR blockers), cytokine therapy (e.g., IL2, IFNa), or immune checkpoint inhibitors (e g., anti-CTLA4, anti-PDl, anti-PDLl). Non-limiting examples of other tumors that can be treated by embodiments herein comprise breast cancer, glioblastoma, colorectal cancer, lung cancer, cervical cancer, ovarian cancer, pancreatic cancer, and bladder cancer, or a combination thereof. Alternatively, the methods can be used to treat, prevent or alleviate a symptom of a cancer that has metastasized.

[0167] In cancer, the normal intercellular interactions in tissues are disrupted, and the tumor microenvironment evolves to accommodate the growing tumor. The tumor microenvironment (TME) can refer to the cellular environment in which a tumor exists, including components such as surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM). Tumor microenvironment is complex and is heavily influenced by immune system. For example, in the ccRCC tumor immune microenvironment, CD8 T infiltration is associated with a worse prognosis. See also. Fridman et al., Nat Rev Clin Oncol, 2017; Chevrier et al.. Cell, 2017; Giraldo et al., Clin Cancer Res, 2017; and Braun et al., Cancer cell, 2021.

[0168] In embodiments, the method comprises administering to a subject afflicted with a cancer an “effective” or “therapeutically effective” amount of an antibody molecule as described herein. The dosages and dosage regimen to achieve the desired therapeutic result depending on the means of administration and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody molecule to elicit a desired response in the individual. The dosage administered to a subject (e.g., a patient) of the antibody molecule described herein is typically 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. An effective amount is also one in which any toxic or detrimental effects of the monoclonal antibody of the present disclosure are outweighed by the therapeutically beneficial effects.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0169] Therapeutically effective amounts can depend on the severity and course of the cancer, previous therapy, the subject's health status, weight, and response to the drugs, and the judgment of the treating physician.

[0170] The subject can be afflicted with cancer such as liquid cancers (i.e., blood cancers) and / or solid cancers (i.e., tumors). The cancer can be benign or malignant, and can be one that is influenced by the immune system.

[0171] Aspects of the invention are further drawn to methods of reducing progression or promoting regression of a cancer in a subject afflicted with cancer, comprising administering the antibody molecule described herein to the subject. The term “reducing progression” of a cancer can refer to as any measurable decrease in the rate, extent, or severity' of cancer growth, spread, or advancement. This may include but is not limited to slowing tumor growth or reducing tumor size; inhibiting metastasis or the spread of cancer cells to other tissues; delaying the onset of symptoms or clinical manifestations associated with cancer progression, and improving survival rates or extending the time to disease progression. The term “promoting regression” of a cancer can refer to inducing or facilitating a measurable reduction in the size, extent, or severity of a cancerous growth. This may include but is not limited to reduction in tumor size; inhibition of metastasis; improvement in biomarkers; and restoration of normal tissue function.

[0172] Aspects of the invention are further drawn to methods of reducing cellular proliferation of a cancer cell in a subject, comprising administering the antibody molecule described herein to the subject. The term “reducing cellular proliferation of a cancer cell” can refer to any measurable decrease in the rate or extent of cancer cell division and growth. This may include but is not limited to inhibiting cell division; reducing cancer cell numbers; and modulating molecular and / or cellular pathways that drive cancer cell proliferation.

[0173] Embodiments as described herein can modulate the immune system so as to treat the subject afflicted with cancer. The term "modulating" can refer to up-regulation, induction, stimulation, potentiation, and / or relief of inhibition, as w ell as inhibition, attenuation and / or down-regulation or suppression. In embodiments, the activity of the subject's immune system is modulated, the microenvironment surrounds the cancer cell and / or tumor is modulated, or both. For example, embodiments as described herein can alter the immune-repressive tumor microenvironment, reducing the microenvironment-dependent immune suppression, so as to modulate (or allow) the immune system to kill tumor cells.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0174] One embodiment is directed towards methods of treating a subject afflicted with renal cell carcinoma. Antibody molecules, such as those described herein, offer an exciting therapeutic option for RCC.

[0175] Accordingly, in one aspect, 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 an antibody molecule of the invention.

[0176] Herein, "preventing" a disease can refer to inhibiting the full development of a disease, such as cancer. "Treating" can refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. "Ameliorating" can refer to the reduction in the number or severity of signs or symptoms of a disease, such as cancer. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology', such as cancer.

[0177] 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.

[0178] The antibodies and fragments thereof disclosed herein can be administered to subjects or patients. Herein, administration can refer to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether selfadministered or administered by a clinician or other qualified care giver. For example, administration of the antibody molecule disclosed herein can abrogate or inhibit or interfere with an activity of the target protein(s).

[0179] The term therapeutic antibody can refer to an antibody suitable for use in human treatment of cancer. In embodiments, such an antibody can have a KD of less than 10’6molar, such as less than 10‘7molar, less than 10'8molar, less than 10’9molar, or less than 10'10molar and any toxic or detrimental effects of the antibody are outweighed by the therapeutic beneficial effects.

[0180] Herein, a “subject” includes both human patient and veterinary subjects, including human and non-human mammals. In embodiments, the subject or patient has or has a risk of cancer. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats,DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the subject is a human.

[0181] The cells according to the disclosure can be used for treating cancer in a patient in need thereof. In another embodiment, said isolated cell according to the invention can be used in the manufacture of a medicament for treatment of a cancer, viral infections of autoimmune disorders, in a patient in need thereof.

[0182] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the antibody molecules of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0183] Also included in the invention are 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. By 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. For example, 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.

[0184] Alternatively, the immune response is a response induced due to a vaccination.

[0185] Accordingly , in another aspect the invention provides a method of increasing vaccine efficiency by administering to the subject a monoclonal antibody or scFv antibody of the invention and a vaccine. The antibody and the vaccine are administered sequentially or concurrently. The vaccine is a tumor vaccine a bacterial vaccine or a viral vaccine.

[0186]

[0187] Nucleic Acid-Based Expression SystemsDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0188] The bsAb of the disclosure (e.g., P4B3-IgG3H-ElA8) can be expressed from an expression vector. Recombinant techniques to generate such expression vectors are well know n in the art.

[0189] DNA constructs, which can also be referred to as "DNA vectors", as described herein, can be cloned into a vector which will be used to transduce and produce chimeric- antigen receptor T-cells that secrete polypeptides and / or fragments thereof. For example, DNA constructs can be cloned into a lenti viral vector for production of lenti virus, which will be used to transduce and produce the bsAb.

[0190]

[0191] Kits of the Invention

[0192] Any of the compositions described herein may be comprised in a kit. In a nonlimiting example, one or more antibody molecules of the present invention may be comprised in a kit. The kit components are provided in suitable container means.

[0193] Some components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits can include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and suitably aliquoted. Where there are more than one component in the kit, the kit also can contain a second, third or other additional container into which the additional components may be separately placed. How ever, various combinations of components may be comprised in a vial. The kits of the present invention also can include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0194] When the components of the kit are provided in one and / or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may itself be a syringe, pipette, and / or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and / or even applied to and / or mixed with the other components of the kit.

[0195] However, the components of the kit may be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. For example, the solvent may also be provided in another container means. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and / or other diluent.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0196] The kit may comprise reagents and materials to make the desired antibody molecule. In specific embodiments, the reagents and materials include primers for amplifying desired sequences, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases the reagents include vectors and / or DNA that encodes a CAR as described herein and / or regulatory elements therefor.

[0197] In particular embodiments, there are one or more apparatuses in the kit suitable for extracting one or more samples from an individual. The apparatus may be a syringe, scalpel, and so forth.

[0198] Other Embodiments

[0199] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0200] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

[0201] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.EXAMPLES

[0202] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary' modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0203] CAR-T cells secreting anti-PDl / CTLA4 bispecific antibody exhibit superior antitumor efficacy for ccRCC treatment

[0204] Clear cell renal cell carcinoma (ccRCC) has been reported to have high presentation of tumor associated antigens (TAA) carbonic anhydrase IX (CAIX) and cluster of differentiation 70 (CD70) regardless of stage of tumor. Single-cell RNA sequencing (scRNA- seq) studies of patient samples have shown that advanced ccRCC patients have enriched populations of exhausted CD8+ T cells, which present immune checkpoint (IC) molecules such as PD1 and CTLA4 as potential therapeutic targets. Anti-PD-1 and anti-CTLA4 act independently to reinvigorate T cells, with the former directly blocking effector cell exhaustion and the latter preventing Treg-mediated negative regulation. The combination therapy of anti- PD1 Nivolumab and anti-CTLA4 Ipilimumab exhibits synergistic effects and has superior therapeutic effectiveness to mono-therapy in advanced ccRCC treatment. A bispecific antibody (BsAb) has advantages compared to monospecific antibody, including a superior efficacy, reduced production cost, and less systemic side effects. The P4B3-IgGH3-ElA8 is an anti- PDl / anti-CTLA4 tetravalent, bispecific antibody, the four scFvs linked to the IgG3 hinge by G4S linkers. The BsAb is capable of binding to both CTLA4 and PD1 on the surface of T cells preventing ligand binding to CD80 and PD-L1 respectively. In effect, P4B3-IgGH3-ElA8 prevents IC mediated T cell exhaustion and exhibited superior inflammatory cytokine interleukin-2 (IL-2) secretion from PBMCs compared to an equimolar cocktail of its monospecific counterparts and also Ipilimumab and Nivolumab. We designed the dual-targeted fine-tuned immune restoring (DFIR) chimeric antigen receptor (CAR)-T to simultaneously target CAIX and CD70 on the surface of tumour cells and also secrete P4B3-IgGH3-ElA8 as a payload. As label-based sorting of CAR-T cells is not advised for infusion into patients, we sorted DFIR CAR-T cells using the Vision-Sort which combines traditional fluorescence flow cytometry with high-dimensional morphological profiling and Al to enable label-free cell sorting and unbiased single cell profiling. CAR positive T cells were utilized for the downstream assays. Using an MSD multiplex quantification assay, we showed that DFIR CAR-T cells secreted the bispecific antibody in culture, inhibited growth of Skrc-59 ccRCC tumor cells in-vitro and retained greater levels of cytotoxic activity in a multi-challenge assay. P4B3-IgGH3-El A8 secreting CAR-T exhibited greater tumor eradication in humanized NSG- SGM3 mice when compared to the CAR-T with an irrelevant payload. In all, we report DFIR CAR-T cell secreting anti-PDl / anti-CTLA4 bispecific antibody has potential to achieve ccRCC cures.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025EXAMPLE 2

[0205] P4B3-IgG3H-El A8 is a tetravalent, bispecific.anti-PDl / anti-CTLA4 that is linked by an IgG3 hinge

[0206] The BsAb is designed to be secreted as a payload from anti-CAIX / anti-CD70 Dual CAR-T cells.

[0207] The BsAb surpasses the current clinical care - Ipilimumab +Nivolumab.

[0208] BsAb secreting anti-CAIX CAR-T cells eradicate tumor better in-vivo than CAR-T without ICI.

[0209] BsAb secretion enhances CAR-T cell activity in-vivo and delays exhaustion.

[0210] Dual anti-CAIX / anti-CD70 CAR-T cells secreting BsAb show enhanced cytotoxic activity in-vitro than CAR-T without ICI.

[0211] The combination of CAR-T cell therapy and dual ICIs could elicit a stronger and longer lasting anti-tumor response and achieve ccRCC cures.EXAMPLE 3

[0212] See FIG. 21-28 CAR-T cells secreting anti-PDl / anti-CTLA4 bispecific antibody exhibited superior antitumor efficacy for ccRCC treatment

[0213] FIG. 21 : Renal Cell Carcinomas (RCC) are among the most prevalent genitourinary cancers in records. Clear cell renal cell carcinoma (ccRCC) alone makes up 80% of documented RCC cases, and most of these cancers are sporadic in nature with a spontaneous loss of function mutation in the VHL protein. This mutation disrupts the degradation of HIF1 a, which then forms a complex with HIFlb and activates transcription of several proteins including CAIX and CD70. These proteins are established to be overexpressed in ccRCC patient samples. There are several first line treatment options for ccRCC. including nephrectomy, TKIs, ICIs, etc, but, over 50% of the patients diagnosed, even when treated early, end up developing metastatic disease. When treated with anti-PDl and TKI combined, the five- year progression free survival of advanced ccRCC patients is around 15%, and there is still room for improvement in efficacy and safety of treatments.

[0214] The immune microenvironment of ccRCC has been studied through several techniques including scRNAseq, IHC and mass spectrometry. The consensus is that over 70% of cells in the ccRCC tumor microenvironment (TME) are CD45+ immune cells. Among these, are mostly CD3+ T cells, which show a high prevalence of exhaustion markers such as CTLA4, PD1, TIM3, and FoxP3. Thus. ccRCC is highly immune infiltrated but immunologically cold. Additionally, ccRCC tumor cells have been documented to overexpress PD-L1, which bindsDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 to PD-1 on the surface of T cells. Accordingly, ccRCC falls among the cohort of solid tumors that would benefit from immunotherapeutic approaches, and due to the prevalence of T cells in the TME, CAR-T cell therapy is one approach.

[0215] A CAR-T cell is a T cell engineered to express a CAR domain that is directed to a tumor associated antigen. The CAR domain has evolved in complexity over the years, with each new generation adding a costimulatory domain. These CAR-T cells home into the TME and are activated by antigen binding, thereby directly killing tumor cells and also recruiting systemic immune cells through cytokines. Historically, CAR-T cells for solid tumors have failed in clinical settings even when they show promise in the lab due to poor persistence, high toxicity and low effectiveness. This is in part due to tumor heterogeneity, on-target off-tumor effects and CAR-T exhaustion.

[0216] One way to overcome poor CAR-T performance in solid tumors is to add an immune checkpoint inhibitor (ICI) payload. This can result in enhanced CAR-T cell persistence, reversal of endogenous T cell exhaustion and immune recruitment, ultimately acting synergistically with CAR-T cells to eliminate tumors better than non-payload secreting CAR- T cells as we already showed with an anti-PDLl antibody in-vivo experiment in our lab previously. As disclosed herein, we decided to target PD-1 and CTLA4 with our ICI payload.

[0217] CTLA4 and PD1 are the two primarily studied immune checkpoint molecules in several tumors. CTLA4, which is expressed on activated T cells and Treg cells has a similar binding motif and higher ligand binding affinity to the costimulatory protein CD28. On CTLs, preferential ligand binding of CTLA4 to CD80 / 86 on APCs results in T cell exhaustion and repression of inflammatory cytokines. In Treg cells, CTLA4 binding increases its immune regulatory' activity by anti-inflammatory cytokine secretion and T and B cell repression. This is why. CTLA4 overexpression is a very’ reliable prognostic marker in ccRCC. PD1, similarly expressed on CTLs, when it binds to PD-L1 initiates multiple signaling pathways that result in T cell inactivity, exhaustion and decreased cytokine secretion. PD1 is an indirect treatment marker because when expressed on Treg cells, it reduces ICI treatment efficacy, but, when predominantly expressed on exhausted CD8+ cells, is an indicator of ICI efficacy.

[0218] Ipilimumab (anti-CTLA4) and nivolumab (anti-PDl) are two FDA approved ICIs with the former being the only anti-CTLA4 in the clinic and the latter being an anti-PDl that is approved for first line treatment of ccRCC. Although both of these antibodies have been used in several carcinomas, including ccRCC, to great therapeutic success, anti-CTLA4 treatment is notorious for its adverse side effects and anti-PDl therapy has been linked to treatment resistant relapse. Thus, CTLA4 and PD1 blockade have been combined to both elicit stronger immuneDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025 response and also lessen the likelihood of treatment resistant relapse by targeting two ICs instead of one.

[0219] A cocktail of nivolumab and ipilimumab has been approved for first line treatment in ccRCC due to its obvious advantages and higher OS and PFS in comparison to sunitinib. The combination of the two works synergistically by targeting ligand binding between T cells and APCs or tumor cells and also enhancing T cell activation signaling via the PI3K pathway by dual-blockade thus preventing T cell exhaustion. Using the combination also prevents treatment resistant relapse. The same combination has also shown effectiveness in other cancers, such as melanoma and mesothelioma, but the common pattern here seems to be the consistently high rate of incidence of severe side effects and is too bulky for a pay load.

[0220] A bispecific combination of the two ICIs might just be the answer to this problem as these could lower dosage, encourage cooperative binding, possibly reduce treatment related side effects and also treatment cost. There are four anti-CTLA4 / anti-PDl bispecifics in testing right now . tw o of which, XmAb20717 and MEDI5752 being bivalent in nature with the latter having a knob-in-hole mutation to drive heterodimerization. The other two are tetravalent, AK104 is a heavy chain fusion antibody and MGD019 is a DART. All these antibodies are in preliminary clinical testing by systemic administration but the bulkiness of the Fc region makes them unfit as payload.

[0221] FIG. 22: As described herein, our payload, P4B3-IgG3H-El A8, is tetravalent, lacks an Fc region and is connected by an IgG3 hinge. This makes the antibody capable of binding to four antigens at once with high avidity and greater flexibility while being less susceptible to proteolytic cleavage. The lack of the Fc region makes the antibody less bulky and easier to transduce. Also, without the Fc region, there is reduced off target effects on innate immune cells. Lacking an Fc region has advantages, such as reduced antibody size which helps with tumor penetration, lower risk of anti-drug antibodies (ADA), and beneficial for neutralizing therapeutics like ICIs.

[0222] FIG. 23: Here, we used phage display to pan a 27 billion member library and used multiple rounds of selection to narrow down to five - ten candidates and tested their kinetic, binding and functional properties to land on one candidate each for anti-CTLA4 and anti-PDl. P4B3 (anti-PDl) and E1A8 (anti-CTLA4) exhibited superior stimulatory capacity in comparison to clinical standards. These candidates outperformed ipilimumab in a PBMC stimulation assay by measuring IL-2 secretion and nivolumab in a commercial bioassay by measuring the induction of luciferase upon blockade of ligand binding. We then cloned these into the bispecific format as described herein.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025

[0223] FIG. 24: We saw that the P4B3-IgG3H-ElA8 bispecific antibody blocks CD80 binding to CTLA4+PD1+ and CTLA4+ single positive cells with lower EC50 that its monospecific counterpart which showed us that the addition of the anti-PDl in this format somehow increases antibody affinity to antigen. Without wishing to be bound by theory, this finding promises synergistic effect of the combination of the two antibodies.

[0224] FIG. 25: We see this synergistic effect functionally when we used a PBMC stimulation assay to test inflammatory cytokine secretion induced by addition of the antibodies. The PBMCs were baseline stimulated with anti-CD3 and SEB and then the antibodies were added to the culture for further stimulation. The IL-2 secretion by the PBMCs was calculated using an ELISA after 3 days of culture and normalized for the baseline secretion. We can see in these assays the P4B3-IgG3H-ElA8 bispecific antibody shows better PBMC stimulation than a monospecific cocktail telling us that the bispecific format does indeed have a synergistic advantage. Further, the bispecific antibody is markedly better at PBMC stimulation than the FDA approved clinical cocktail Ipi+Nivo. This finding prompted us to incorporate the BsAb into our anti-CAIX CAR-T.

[0225] FIG. 26: To test the P4B3-IgG3H-ElA8 BsAb as a payload secreted by our anti- CAIX CAR-T cells in-vivo, we used humanized mice and treated them with CAR-T cells secreting the BsAb, an anti-PDLl and an anti-SARS antibody. After 4 weeks of treatment, we can clearly see by tumor weight that the presence of the BsAb results in higher tumor eradication. This shows us that the BsAb effectively compliments CAR-T activity.

[0226] FIG. 27: We then incorporated the P4B3-IgG3H-El A8 BsAb as a payload into our Dual anti-CAIX / anti-CD70 CAR-T cell giving us a dual targeted, immune restoring CAR-T that was also fine-tuned for CAIX binding, hence the DFIR CAR-T. The aim of this complex combinatorial approach is to prevent antigen escapes, delay CAR-T exhaustion, re-activate exhausted T cells and also hopefully elicit a stronger systemic immune response.

[0227] FIG. 28: Once we generated these DFIR CAR-T cells, we checked for BsAb secretion using an MSD assay and found that across four donors, the CAR-T cell secreted detectable amounts of the payload. We tested these CAR-T cells in-vitro in a multi-challenge killing assay assessed in real time by transferring the same CAR-T cells to three sets of freshly seeded tumor cells and found that the DFIR CAR-T retained cytotoxic activity longer than the dual CAR-T cell secreting an anti-SARS antibody. This shows in-vitro that our BsAb is capable of preventing CAR-T exhaustion even when rechallenged with fresh tumor cells every 48 hours.DOCKET NO: 5031461-000173-W01 DATE OF FILING: November 26, 2025

[0228] In summary, P4B3-IgG3H-ElA8 is a tetravalent, bispecific, anti-PDl / anti-CTLA4 that is linked by an IgG3 hinge, and is designed to be secreted as a payload from anti- CAIX / anti-CD70 Dual CAR-T cells. The P4B3-IgG3H-ElA8 BsAb surpasses the cunent clinical care - Ipilimumab +Nivolumab. BsAb secreting anti-CAIX CAR-T cells eradicate tumor better in-vivo than CAR-T without ICI. BsAb secretion enhances CAR-T cell activity in-vivo and delays exhaustion. Dual anti-CAIX / anti-CD70 CAR-T cells secreting P4B3- IgG3H-ElA8 BsAb show enhanced cytotoxic activity in-vitro than CAR-T without ICI.

[0229] Without wishing to be bound by theory, the combination of CAR-T cell therapy and dual ICIs could elicit a stronger and longer lasting anti-tumor response and achieve ccRCC cures.EQUIVALENTS

[0230] 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.

Claims

DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025What is claimed:

1. A bispecific antibody that specifically binds to PD-1 and CTLA4, wherein the bispecific antibody comprises a first antigen binding site specific for PD-1 comprising VH CDR1 comprising the amino acid sequence of GFTFDDFA, a VH CDR2 comprising the amino acid sequence of ISWNSGSL and a VH CDR3 comprising the amino acid sequence of ASDYGDKYSYYGMDV, and a VL CDR1 comprising the amino acid sequence of SSNIGSNT, a VL CDR2 comprising the amino acid sequence of DDN, and a VL CDR3 comprising the amino acid sequence of AAWDGGLNGRGV; and a second antigen binding site specific for CTLA4 comprising a VH CDR1 comprising the amino acid sequence of GFTFSSYV, a VH CDR2 comprising the amino acid sequence of ISGSGGST, and a VH CDR3 comprising the amino acid sequence of ARGGSAWSLDI, and a VL CDR1 comprising the amino acid sequence of QDISNS, a VL CDR2 comprising the amino acid sequence of GAS, and a VL CDR3 comprising the amino acid sequence of QQGNSFPIT.

2. The bispecific antibody of claim 1, wherein the first antigen binding site specific for PD-1 comprises a VH comprising the amino acid sequence of MAQVQLVQSGGGLVQPGRSLRLSCAASGFTFDDFAMHWVRQAPGKG LEWVSGISWNSGSIGYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDT AVYYCASDYGDKYSYYGMDVWGKGTTVTVSS, and a VL comprising the amino acid sequence of QPGLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQFPGKAPKLLI FDDNQRPSGVPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDGGL NGRGVFGGGTKLTVL; and wherein the second antigen binding site specific for CTLA4 comprises a VH comprising the amino acid sequence of QLVQSGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWV SSISGSGGSTDYADSVKGRFPISRDNSKNTLYLEMNSLRAEDTAVYYC ARGGSAWSLDIWGQGTTVTVSS, and a VL comprising the amino acid sequence ofDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025DIQMTQSPSSVSASIGDRVTITCRASQDISNSLAWYQQKPGKAPKLLIY GASNLRSGVPSRFSGGGSGTYFTLTISSLQPEDFATYYCQQGNSFPITFG QGTRLEIKRTVAAPT.

3. A tetraval ent antibody molecule, wherein the tetravalent antibody molecule is a dimer of a bispecific antibody fragment, wherein the bispecific antibody fragment comprises a first antigen binding site specific for PD-1, a second antigen binding site specific for CTLA-4, and dimerization domain, a linker-hinge-linker domain, wherein the first binding site and the second binding site are joined together via the dimerization domain.

4. The tetravalent antibody molecule of claim 3, wherein the dimerization domain comprises an immunoglobulin hinge region or fragment thereof.

5. The tetraval ent antibody molecule of claim 4, wherein the immunoglobulin hinge region is an IgGl, IgG2, IgG3, or IgG4 hinge region.

6. The tetravalent antibody molecule of claim 5, wherein the immunoglobulin hinge region comprises an amino acid sequence according toELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGP or a sequence at least 90% identical thereto.

7. The tetravalent antibody molecule of claim 3, wherein the dimerization domain comprises one or more amino acid linkers.

8. The tetravalent antibody molecule of claim 7, wherein the amino acid linker comprises a flexible linker.

9. The tetravalent antibody molecule of claim 8, wherein the amino acid linker comprises (GGGS)xi-6, (GGGGS)XI-6, or GSAGSAAGSGEF.

10. The tetraval ent antibody molecule of claim 3, wherein the dimerization domain comprises a hinge region and at least one amino acid linker.

11. The tetraval ent antibody molecule of claim 10, wherein the immunoglobulin hinge region is flanked on one or both sides by the amino acid linker.

12. The tetravalent antibody molecule of claim 3, wherein the linker domain does not comprise an immunoglobulin Fc domain or fragment thereof.

13. The tetraval ent antibody molecule of claim 3, wherein the tetraval ent antibody molecule comprises one, two, three, or four scFv fragments.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 202514. The tetravalent antibody molecule of claim 3, wherein the first antigen binding site specific for PD-1 comprises: a VH CDR1 comprising the amino acid sequence of GFTFDDFA, a VH CDR2 comprising the amino acid sequence of ISWNSGSI, and a VH CDR3 comprising the amino acid sequence of ASDYGDKYSYYGMDV, and a VL CDR1 comprising the amino acid sequence of SSNIGSNT, a VL CDR2 comprising the amino acid sequence of DDN, and a VL CDR3 comprising the amino acid sequence of AAWDGGLNGRGV.

15. The tetraval ent antibody molecule of claim 14, wherein the first antigen binding site specific for PD-1 comprises: a VH comprising the amino acid sequence ofMAQVQLVQSGGGLVQPGRSLRLSCAASGFTFDDFAMHWVRQAPGKGLEW VSGISWNSGSIGYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCAS DYGDKYSYYGMDVWGKGTTVTVSS, and a VL comprising the amino acid sequence ofQPGLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQFPGKAPKLLIFDDN QRPSGVPDRFSASKSGTSASLAISGLQSEDEADYYCAAWDGGLNGRGVFGG GTKLTVL.

16. The tetravalent antibody molecule of claim 3, wherein the second antigen binding site specific for CTLA4 comprises: a VH CDR1 comprising the amino acid sequence of GFTFSSYV, a VH CDR2 comprising the amino acid sequence of ISGSGGST, and a VH CDR3 comprising the amino acid sequence of ARGGSAWSLDI, and a VL CDR1 comprising the amino acid sequence of QDISNS. a VL CDR2 comprising the amino acid sequence of GAS, and a VL CDR3 comprising the amino acid sequence of QQGNSFPIT.

17. The tetravalent antibody molecule of claim 16, wherein the second antigen binding site specific for CTLA4 comprises: a VH compnsing the amino acid sequence ofQLVQSGGGLVQPGGSLRLSCAASGFTFSSYVMSWVRQAPGKGLEWVSSISG SGGSTDYADSVKGRFPISRDNSKNTLYLEMNSLRAEDTAVYYCARGGSAWS LDIWGQGTTVTVSS, and a VL comprising the amino acid sequence ofDIQMTQSPSSVSASIGDRVTITCRASQDISNSLAWYQQKPGKAPKLLIYGASNDOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 2025LRSGVPSRFSGGGSGTYFTLTfSSLQPEDFATYYCQQGNSFPITFGQGTRLEfK RTVAAPT.

18. The tetravalent antibody molecule of claim 3 according to the amino acid sequence of MAQVQLVQSGGGLVQPGRSLRLSCAASGFTFDDFAMHWVRQAPGKGLEWVSGI SWNSGSIGYADSVKGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYCASDYGDKY SYYGMDVWGKGTTVTV S SGGGGSGGGGS GGGGS QPGLTQPP S ASGTPGQRVTIS CSGSSSNIGSNTVNWYQQFPGKAPKLLIFDDNQRPSGVPDRFSASKSGTSASLAIS GLQSEDEADYYCAAWDGGLNGRGVFGGGTKLTVLGQPKAAPSAAAELKTPLGD TTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPEL LGGPGGGGSGGGGSGGGGSGGGGSGGGGSRTQVQLVQSGGGLVQPGGSLRLSC AASGFTFSSYVMSWVRQAPGKGLEWVSSISGSGGSTDYADSVKGRFPISRDNSK NTLYLEMNSLRAEDTAVYYCARGGSAWSLDIWGQGTTVTVSSGGGGSGGGGSG GGGSDIQMTQSPSSVSASIGDRVTITCRASQDISNSLAWYQQKPGKAPKLLIYGAS NLRSGVPSRFSGGGSGTYFTLTISSLQPEDFATYYCQQGNSFPITFGQGTRLEIKRT VAAPT or a sequence at least 90% identical thereto.

19. A nucleic acid construct encoding the bispecific antibody of claim 1 or the tetraval ent antibody molecule of claim 3.

20. A vector comprising the nucleic acid construct of claim 19.

21. A host cell comprising the vector of claim 20.

22. The host cell of claim 21, wherein the cell is a T-cell, a B-cell, a follicular T-cell, or an NK-cell.

23. A pharmaceutical composition comprising the bispecific antibody of claim 1, the tetravalent antibody molecule of claim 3, and a pharmaceutically acceptable carrier, excipient, or diluent.

24. A method for treating a subject afflicted with cancer, the method comprising administering to the subject the bispecific antibody of claim 1 or the tetravalent antibody of claim 3.

25. The method of claim 24, wherein cancer is a renal cancer.

26. The method of claim 25, wherein the renal cancer is clear cell renal cell carcinoma (ccRCC).

27. The bispecific antibody of claim 1 or the tetravalent antibody of claim 3 for use in the treatment of a subject afflicted with cancer.

28. The use of claim 27, wherein the cancer is renal cancer.

29. The use of claim 28, wherein the renal cancer is clear cell renal cell carcinoma.DOCKET NO: 5031461-000173-W01DATE OF FILING: November 26, 202530. A method for slowing tumor growth or cellular proliferation in a subject afflicted with cancer, the method comprising administering to the subject the bispecific antibody of claim 1 or the tetraval ent antibody of claim 3.

31. The method of claim 30, wherein cancer is a renal cancer.

32. The method of claim 31, wherein the renal cancer is clear cell renal cell carcinoma (ccRCC).

33. The bispecific antibody of claim 1 or the tetraval ent antibody of claim 3 for use in slowing tumor growth or cellular proliferation in a subject afflicted with cancer.

34. The use of claim 33, wherein the cancer is renal cancer.

35. The use of claim 34, wherein the renal cancer is clear cell renal cell carcinoma.

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