Combination therapy for treating cancer using antibodies targeting ulbp2

Abstract

This disclosure provides anti-ULBP2 antibodies or antigen-binding fragments thereof (including bispecific ones). These antibodies are administered in combination with chemotherapy agents, radiotherapy, and / or immune checkpoint inhibitors for the treatment of basal squamous cell carcinoma. This disclosure also relates to methods and kits for detecting susceptibility to ULBP2-related disorders, determining their risk, and guiding treatment.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 590755, filed October 16, 2023, under 35 U.S.SC § 119(e), the entire contents of which are incorporated herein by reference.

[0003] Reference to the electronic sequence list

[0004] The contents of the electronic serial number (E070170008WO00-SEQ-LJG.xml; size: 509870 bytes; creation date: October 15, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This disclosure relates to pharmaceutical compositions containing antibodies that bind to ULBP2 and their uses. This disclosure relates to combination therapies for diseases associated with cells expressing ULBP2, such as bladder cancer. This disclosure also relates to methods and kits for detecting cancer susceptibility, determining cancer risk, and guiding cancer therapy. Background Technology

[0006] UL16-binding protein 2 (ULBP2) is a cell surface glycoprotein associated with class I MHC. ULBP2 functions as a stress-induced ligand for the NKG2D receptor. The protein encoded by ULBP2 undergoes further processing to produce a mature protein, which is partially anchored to the membrane or secreted via glycosylphosphatidylinositol.

[0007] It has been shown that NKG2D ligand is upregulated in a range of primary tumors, including lung cancer, kidney cancer, prostate cancer, breast cancer, and colon cancer. The ULBP2-NKG2D-induced immune response may play an important role in the eradication of tumors by T and / or NK cells. Summary of the Invention

[0008] This disclosure is based, at least in part, on the finding that in subjects with cancer (e.g., cancer with elevated ULBP2 expression levels), certain combinations of anti-ULBP2 antibodies or antigen-binding fragments thereof (e.g., bispecific antibodies binding ULBP2 and CD3) with chemotherapy, radiation therapy, or immune checkpoint inhibitors (e.g., pembrolizumab) improve efficacy compared to anti-ULBP2 therapy alone. This disclosure provides a method for reducing a population of cancer cells in a subject, the method comprising administering to the subject: a) an anti-ULBP2 antibody or antigen-binding fragment thereof, and b) a chemotherapy agent, radiation therapy, and / or an immune checkpoint inhibitor, wherein multiple cancer cells express UL16-binding protein 2 (ULBP2), and wherein the subject has cancer (e.g., squamous cell carcinoma).

[0009] In some embodiments, the method results in a reduction of the number of ULBP2-expressing cancer cells in the subject by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% relative to the number of ULBP2-expressing cancer cells in the subject prior to administration of the anti-ULBP2 antibody or its antigen-binding fragment and the chemotherapy agent and / or radiotherapy.

[0010] This disclosure also provides a method for treating a human subject with cancer (e.g., basal squamous cell carcinoma), the method comprising the steps of: a) obtaining a biological sample from the human subject with cancer (e.g., basal squamous cell carcinoma); b) detecting the expression level of ULBP2; c) comparing the detected ULBP2 expression level with a control level of ULBP2 expression; d) identifying the subject as a responder when the detected ULBP2 expression level is greater than the control level of ULBP2 expression; and e) when the subject is identified as a responder, administering an anti-ULBP2 antibody or a fragment thereof in an amount sufficient to alleviate symptoms of the cancer (e.g., basal squamous cell carcinoma).

[0011] In some embodiments, the method includes: i) detecting the expression levels of TROP2, PDL1, HLA-E, NECTIN4, and / or HER2; ii) comparing the detected expression levels of TROP2, PDL1, HLA-E, NECTIN4, and / or HER2 with control levels; iii) identifying the subject as a responder when the detected TROP2 expression level is greater than the control level, the detected PDL1 expression level is greater than the control level, the detected HLA-E expression level is greater than the control level, the detected NECTIN4 expression level is less than the control level, and / or the detected HER2 expression level is less than the control level.

[0012] In some embodiments, the anti-ULBP2 antibody or a fragment thereof comprises: a) a complementarity-determining region 1 (VH2) comprising the amino acid sequence of SEQ ID NO: 428. CDR1 ); containing complementarity-determining region 2 (VH2) of the amino acid sequence of SEQ ID NO: 430 CDR2 ); Complementarity-determining region 3 (VH2) of the amino acid sequence containing SEQ ID NO: 432 CDR3 ); containing complementarity-determining region 1 (VL2) of the amino acid sequence of SEQ ID NO: 433 CDR1 ); Complementarity-determining region 2 (VL2) containing the amino acid sequence of SEQ ID NO: 434CDR2 ); and complementarity-determining region 3 (VL2) containing the amino acid sequence of SEQ ID NO: 435. CDR3 ); or b) containing complementarity-determining region 1 (VH2) of the amino acid sequence of SEQ ID NO: 5. CDR1 ); Complementarity-determining region 2 (VH2) of the amino acid sequence containing SEQ ID NO: 7 CDR2 ); Contains complementarity-determining region 3 (VH2) of the amino acid sequence of SEQ ID NO: 9 CDR3 ); Contains complementarity-determining region 1 (VL2) of the amino acid sequence of SEQ ID NO: 10 CDR1 ); Contains complementarity-determining region 2 (VL2) of the amino acid sequence of SEQ ID NO: 11 CDR2 ); and complementarity-determining region 3 (VL2) containing the amino acid sequence of SEQ ID NO: 12. CDR3 ).

[0013] In some embodiments, the anti-ULBP2 antibody or a fragment thereof is a bispecific antibody that specifically binds to ULBP2 and CD3, or the anti-ULBP2 antibody or a fragment thereof is a bispecific antibody that specifically binds to ULBP2 and PD1. In some embodiments, a CD58 peptide or a fragment thereof is fused to the C-terminus of the heavy chain peptide of the bispecific antibody. In some embodiments, the CD58 comprises the amino acid sequence of SEQ ID NO: 49.

[0014] In some embodiments, the bispecific antibody comprises an anti-ULBP2 antibody or an antigen-binding fragment thereof, which includes a light chain variable region (VL2) containing the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (VH2) containing the amino acid sequence of SEQ ID NO: 629. In some embodiments, the anti-ULBP2 antibody or its antigen-binding fragment comprises a light chain (L2) containing the amino acid sequence of SEQ ID NO: 67 or a heavy chain (H2) containing the amino acid sequence of SEQ ID NO: 621.

[0015] In some embodiments, the bispecific antibody comprises an anti-CD3 antibody or an antigen-binding fragment thereof, which includes a light chain variable region (VL1) containing the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region (VH1) containing the amino acid sequence of SEQ ID NO: 17. In some embodiments, a CD58 polypeptide or a fragment thereof is fused to the C-terminus of the heavy chain of the anti-CD3 antibody or the antigen-binding fragment thereof of the bispecific antibody. In some embodiments, the anti-CD3 antibody or the antigen-binding fragment thereof comprises a light chain (L1) containing the amino acid sequence of SEQ ID NO: 69 and a heavy chain (H1) fused to the CD58 polypeptide containing the amino acid sequence of SEQ ID NO: 622.

[0016] Any suitable chemotherapy can be used in combination with the anti-ULBP2 antibody or antigen-binding fragment described herein. In some embodiments, chemotherapeutic agents suitable for use in this disclosure include, but are not limited to, topoisomerase inhibitors, anthracycline drugs such as doxorubicin, DNA-fracture-inducing antibiotics, pyrimidine antagonists, or platinum alkylating agents.

[0017] In some embodiments, the radiotherapy is selected from 3D conformal radiotherapy (3DCRT), image-guided radiotherapy (IGRT), intensity-modulated radiotherapy (IMRT), volume-modulated arc therapy (VMAT), brachytherapy, intraoperative radiotherapy (IORT), stereotactic radiosurgery (SRS), proton therapy, MRI linear accelerator and / or stereotactic body radiotherapy (SBRT).

[0018] This disclosure also provides a method for identifying human subjects suffering from cancer (e.g., basal squamous cell carcinoma) suitable for treatment with an anti-UL16 binding protein 2 (ULBP2) antibody or an antigen-binding fragment thereof, the method comprising the steps of: a) obtaining a biological sample from a human subject suffering from cancer (e.g., basal squamous cell carcinoma); b) detecting the expression level of ULBP2; c) comparing the detected ULBP2 expression level with a control level of ULBP2 expression; and d) identifying the subject as a responder when the detected ULBP2 expression level is greater than the control level of ULBP2 expression. In some embodiments, the method further comprises: e) administering an anti-ULBP2 antibody or a fragment thereof in an amount sufficient to alleviate symptoms of the cancer (e.g., basal squamous cell carcinoma) when the subject is identified as a responder.

[0019] In some embodiments, the subject has squamous cell carcinoma. In some embodiments, the squamous cell carcinoma is basal squamous cell carcinoma. In some embodiments, the subject has bladder cancer, lung cancer, head and neck cancer, cervical cancer, esophageal cancer, anal cancer, vulvar cancer, penile cancer, thymic cancer, skin cancer cells, or adrenocortical cancer.

[0020] In some embodiments, the subject has adenocarcinoma. In some embodiments, the adenocarcinoma is lung adenocarcinoma, pancreatic adenocarcinoma, bladder adenocarcinoma, esophageal adenocarcinoma, or colon adenocarcinoma.

[0021] In some embodiments, the subject has an EGFR activating mutation or an EGFR copy number alteration. In some embodiments, the subject has previously been treated with a tyrosine kinase inhibitor (TKI). In some embodiments, the TKI is osimertinib. In some embodiments, the TKI does not reduce ULBP2 expression levels in the cancer. In some embodiments, the subject who previously received a TKI experienced cancer recurrence after TKI treatment.

[0022] In some embodiments, the cancer is metastatic cancer. In some embodiments, the subject with metastatic cancer has non-primary lesions in the liver, lungs, bones, brain, or lymph nodes originating from the primary cancer. In some embodiments, the primary cancer is squamous cell carcinoma or adenocarcinoma. In some embodiments, the primary cancer is squamous cell carcinoma, including bladder cancer, lung cancer, head and neck cancer, cervical cancer, esophageal cancer, anal cancer, vulvar cancer, penile cancer, thymic cancer, skin cancer cells, or adrenocortical cancer. In some embodiments, the primary cancer is adenocarcinoma, including lung adenocarcinoma, pancreatic adenocarcinoma, bladder adenocarcinoma, esophageal adenocarcinoma, or colon adenocarcinoma.

[0023] In some embodiments, the subject has previously been administered an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD1 / PDL1 inhibitor or a CTLA4 inhibitor. In some embodiments, the immune checkpoint inhibitor is pembrolizumab, nivolumab, atezolizumab, ipilimumab, or trimemumab. In some embodiments, the immune checkpoint inhibitor does not reduce ULBP2 expression levels in the cancer.

[0024] In some embodiments, the subject is simultaneously or pre-administered with a therapeutically effective amount of the treatment agent.

[0025] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Any suitable chemotherapy may be used in combination with the anti-ULBP2 antibody or antigen-binding fragment described herein. In some embodiments, chemotherapeutic agents suitable for use in this disclosure include, but are not limited to, topoisomerase inhibitors, anthracycline drugs such as doxorubicin, DNA-fracture-inducing antibiotics, pyrimidine antagonists, or platinum alkylating agents.

[0026] In some embodiments, the therapeutic agent is a radiotherapy selected from 3D conformal radiotherapy (3DCRT), image-guided radiotherapy (IGRT), intensity-modulated radiotherapy (IMRT), volume-modulated arc therapy (VMAT), brachytherapy, intraoperative radiotherapy (IORT), stereotactic radiosurgery (SRS), proton therapy, MRI linear accelerator and / or stereotactic body radiotherapy (SBRT).

[0027] In some embodiments, the therapeutic agent is a CAR-T cell therapy, an immune checkpoint inhibitor, a co-stimulatory ligand, or a cytokine. In some embodiments, the immune checkpoint inhibitor is pembrolizumab, nivolumab, atezolizumab, ipilimumab, or trimemumab.

[0028] In some embodiments, the anti-ULBP2 antibody or its antigen-binding fragment thereof and the chemotherapy agent, radiotherapy, and / or immune checkpoint inhibitor are administered sequentially. In some embodiments, the anti-ULBP2 antibody or its antigen-binding fragment thereof and the chemotherapy agent, radiotherapy, and / or immune checkpoint inhibitor are administered simultaneously. Attached Figure Description

[0029] Figure 1A-1B This is a series of heatmaps showing the average levels of a group of intraluminal / adenocarcinoma genes (top) and basal / squamous genes (bottom) in the bladder and lung TCGA datasets and the Encyclopedia of Cancer Cell Lines (CCLE) when clustered using these same genes and other genes (immune infiltration, neurons). Levels in the cell line subsets are shown and labeled according to their bladder (intraluminal, intraluminal infiltrative, or basal-squamous) or lung (mixed / infiltrative or basal-squamous) categories. Figure 1A A bladder dataset is shown. Figure 1B The lung dataset is shown.

[0030] Figure 2 This is a heatmap depicting quantitative immunoblotting at the protein level of intraluminal (FOXA1, PPARG, GATA3) and basal / squamous (KRT5, KRT6, and SERPIB3) markers in several cell lines (shown on the x-axis). ULBP2 protein levels from flow cytometry antigen-binding density are also shown for the same cell lines.

[0031] Figures 3A-3D It is a series of graphs depicting the gene levels in the bladder cancer TCGA dataset, which are defined and shown in Figure 1. Figure 3A The target ULBP2 is shown. Figure 3B TROP2 is shown. Figure 3C HER2 is shown. Figure 3D NECTIN4 is shown. p > 0.05 Indicates that p < 0.01. This indicates that p < 0.001 is represented by... This indicates that p < 0.0001 is represented by... express.

[0032] Figures 4A-4D It is a series of graphs depicting the gene levels in the bladder cancer TCGA dataset, which are defined and shown in Figure 1. Figure 4A The intraluminal gene FOXA1 is shown. Figure 4B The intraluminal gene PPARG is shown. Figure 4C The basal / squamous gene KRT5 is shown. Figure 4D SERPINB3 is shown.

[0033] Figures 5A-5D It is a series of graphs depicting the gene levels in the bladder cancer TCGA dataset, which are defined and shown in Figure 1. Figure 5A The immune gene PDL1 is shown. Figure 5B The T cell gene PD1 is shown. Figure 5C The T cell gene CD8A is shown. Figure 5D The T-cell gene granzyme B is shown.

[0034] Figures 6A-6D It is a series of graphs depicting the gene levels in the TCGA dataset of lung cancer, which is divided into adenocarcinoma or squamous cell carcinoma indications. Figure 6A The target ULBP2 is shown. Figure 6B TROP2 is shown. Figure 6C HER2 is shown. Figure 6D NECTIN4 is shown. p>0.05 Indicates that p < 0.01. This indicates that p < 0.001 is represented by... This indicates that p < 0.0001 is represented by... express.

[0035] Figures 7A-7D It is a series of graphs depicting the gene levels in the TCGA dataset of lung cancer, which is divided into adenocarcinoma or squamous cell carcinoma indications. Figure 7A The intraluminal gene FOXA1 is shown. Figure 7B The intraluminal gene PPARG is shown. Figure 7C The basal / squamous gene KRT5 is shown. Figure 7D SERPINB3 is shown.

[0036] Figures 8A-8D It is a series of graphs depicting the gene levels in the TCGA dataset of lung cancer, which is divided into adenocarcinoma or squamous cell carcinoma indications. Figure 8A The immune gene PDL1 is shown. Figure 8B The T cell gene PD1 is shown. Figure 8C The T cell gene CD8A is shown. Figure 8D The T-cell gene granzyme B is shown.

[0037] Figures 9A-9F This is a series of figures depicting T cell-dependent cytotoxicity and cytokine release data in bladder squamous cell line 5637 when used in combination with the anti-ULBP2 antibody EIP0820 and the negative control EIP0607, either in combination with pembrolizumab or an IgG4 isotype control. Figure 9A The tumor killing effect of donor 1A on day 5 is shown. Figure 9B The tumor killing effect of donor 2A on day 5 is shown. Figure 9C The tumor-killing effect of donor 3A on day 5 is shown. Figure 9D The release of IFNg cytokines from donor 1A on day 2 is shown. Figure 9B The release of IFNg cytokines from donor 2A on day 2 is shown. Figure 9C The release of IFNg cytokines from donor 3A on day 2 is shown.

[0038] Figures 10A-10B It is a series of graphs depicting how a range of chemotherapeutic agents (bleomycin, cisplatin, docetaxel, doxorubicin, gemcitabine, irinotecan, and vincristine) upregulate ULBP2 and other biomarkers (HER2, CD58, PDL1, TROP2, and B7H4) at their EC50 concentrations. Figure 10A The bladder squamous cell line 5637 is shown. Figure 10B The lung adenocarcinoma squamous cell line A549 is shown.

[0039] Figure 11 This is a graph depicting T cell-dependent cytotoxicity data in 5637 cells when using EIP0820 in combination with irinotecan chemotherapy and the negative control EIP0607.

[0040] Figure 12 This is a graph depicting T cell-dependent cytotoxicity in squamous bladder cancer 5637 cells when using EIP0820 in combination with gemcitabine / cisplatin (“Cis / Gem”) and the negative control EIP0614.

[0041] Figure 13 It is a graph depicting the gene levels from the Cancer Genome Atlas (TCGA) dataset, which is divided into adenocarcinoma or squamous cell carcinoma indications.

[0042] Figure 14A-C depicts the association between ULBP2 expression, basal-squamous molecular subtypes, and CD8 T cell infiltration, as outlined in Table 14. Figure 14A The relationship between ULBP2 expression and the basal / squamous subtype is shown. Figure 14B The relationship between ULBP2 expression and CD8 T cell infiltration was shown. Figure 14C The relationship between the basal / squamous subtype and CD8 T cell infiltration is shown.

[0043] Figure 15 The expression of ULBP2 in primary and non-primary tumor sites in lung cancer from GSE248249 is shown.

[0044] Figures 16A-16E The genetic levels were depicted in five different datasets, each of which was divided into two groups based on whether the patient was HPV negative or HPV positive. Figure 16A TCGA is shown. Figure 16B GSE65858 is shown. Figure 16C GSE39366 is shown. Figure 16D GSE40774 is shown. Figure 16E GSE6791 is shown.

[0045] Figures 17A-17D ULBP2 expression was depicted in lung or bladder datasets. Samples were stratified by cancer grade and stage, as well as subtype (squamous cell carcinoma or adenocarcinoma). Figure 17A GSE42127 is shown. Figure 17B GSE14814 is shown. Figure 17C TCGA lung squamous cell carcinoma was shown. Figure 17D The TCGA bladder, as shown in Figure 3, was subtyped by molecular characterization of only the basal / squamous structure.

[0046] Figures 18A-18H ULBP2 expression in head and neck cancer, lung cancer, or bladder cancer is depicted. Before / after checkpoint inhibitor treatment in paired or unpaired samples is shown, with lung cancer stratified according to basal and squamous layers. Figure 18A GSE179730 (head and neck) is shown. Figure 18B GSE195832 (head and neck) is shown. Figure 18C and 18D GES248249 (lung) is shown using paired or unpaired samples. Figure 18D and 18E GSE248378 (lung) with adenocarcinoma or squamous cell carcinoma dissection is shown. Figure 18G GSE248249 (lung) with results data from checkpoint inhibitors is shown. Figure 18H GSE212810 (bladder) with result data is shown.

[0047] Figures 19A-19D Depicting the situation in individuals with EGFR mutations ( Figure 19A ), the highest and lowest quartiles of EGFR expression ( Figure 19B EGFR copy number change ( Figure 19C Patients with activating mutations who are most likely to be prescribed the tyrosine kinase inhibitor (TKI) osimertinib ( Figure 19D In ), ULBP2 is expressed from TCGA.

[0048] Figure 20 The graph shows ULBP2 expression in a bladder cancer dataset that stratifies patients from GSE186691 into non-squamous disease and pure squamous cell carcinoma.

[0049] Figures 21A-21C ULBP2 expression in tumors from non-TCGA squamous cell carcinoma datasets is shown. Figure 21A The anal cancer dataset GSE253560 is shown. LGSIL = low-grade squamous intraepithelial lesion; HGSIL = high-grade squamous intraepithelial lesion; ASCC = anal squamous cell carcinoma. Figure 21B The dataset GSE63678, which includes data on cervical cancer, endometrial cancer, and vulvar cancer, is shown. Figure 21C This illustrates vulvar cancer stratified according to the patient's HPV status. Detailed Implementation

[0050] This article provides a method for identifying and treating human subjects with ULBP2-expressing cancers (e.g., squamous cell carcinoma such as basal squamous cell carcinoma including bladder cancer, lung cancer, esophageal cancer, head and neck cancer, etc.; adenocarcinoma including lung adenocarcinoma, pancreatic adenocarcinoma, bladder adenocarcinoma, esophageal adenocarcinoma, or colon adenocarcinoma, or metastatic cancer) suitable for treatment with anti-ULBP2 antibodies or their antigen-binding fragments. This is based in part on the observation that tumors in patients with certain cancers (e.g., basal squamous cell carcinoma, adenocarcinoma, or metastatic cancer) show high levels of ULBP2 in the tumor. Furthermore, ULBP2-positive cancers, such as basal squamous cell carcinoma (particularly bladder cancer), have been observed to show high T-cell infiltration (as seen through the expression of PDL1, PD1, and granzyme B). Without being bound by theory, these combined suggest that T-cell adaptor therapy is likely to be effective in this subset of patients. In addition, combination therapy with anti-ULBP2 antibodies or their antigen-binding fragments in conjunction with immune checkpoint inhibitors (e.g., anti-PD-1 antibodies) has been observed to be beneficial in the treatment of basal squamous cell carcinoma. Unbound by theory, the use of PD1 checkpoint inhibitors can further increase immune infiltration in subtypes with lower immune infiltration, leading to greater therapeutic potential.

[0051] Specific chemotherapy or radiation therapy will induce ULBP2 in any subtype (even and especially in the adenocarcinoma / luminal subtype, where ULBP2 is lower). Therefore, combination therapy and / or treatment with anti-ULBP2 antibodies or their antigen-binding fragments shortly after chemotherapy or radiation therapy will enhance the potency of anti-ULBP2 antibodies or their antigen-binding fragments.

[0052] This article also provides methods for treating subjects with anti-ULBP2 antibodies or their antigen-binding fragments, along with radiotherapy, chemotherapy, and / or immune checkpoint inhibitors. Unbound by theory, the use of chemotherapy can increase tumor ULBP2 levels on cancer cells (e.g., basal squamous cell bladder and lung cancer cells, adenocarcinoma cells, metastatic cancers) (e.g., triggered by chemotherapy-induced DNA damage). Therefore, this presents high therapeutic potential for the use of anti-ULBP2 antibodies in cancers such as squamous cell carcinomas like bladder and lung cancer, adenocarcinomas, or metastatic cancers.

[0053] Therefore, this disclosure provides a method for treating cancers expressing ULBP2. In some embodiments, this disclosure provides a method for reducing a population of cancer cells in a subject. In some embodiments, the method of treating cancer includes administering an effective amount of an anti-ULBP2 antibody or an antigen-binding fragment thereof. In some embodiments, the method of treating cancer includes administering an effective amount of an anti-ULBP2 antibody or an antigen-binding fragment thereof, along with a chemotherapy agent, radiotherapy, and / or an immune checkpoint inhibitor. In some embodiments, the cancer expresses UL16-binding protein 2 (ULBP2). In some embodiments, the cancer expresses KRT5 and / or SERPINB3. In some embodiments, the cancer is squamous cell carcinoma (e.g., basal squamous cell carcinoma). In some embodiments, the basal squamous cell carcinoma is bladder cancer. In some embodiments, the basal squamous cell carcinoma is lung cancer. In some embodiments, the basal squamous cell carcinoma is esophageal cancer. In some embodiments, the basal squamous cell carcinoma is head and neck cancer. In some embodiments, the cancer is adenocarcinoma (e.g., lung adenocarcinoma, pancreatic adenocarcinoma, bladder adenocarcinoma, esophageal adenocarcinoma, or colonic adenocarcinoma). In some embodiments, the cancer is metastatic cancer.

[0054] ULBP2 binding protein

[0055] This disclosure provides a bispecific antibody comprising a first antigen-binding domain that binds to CD3 or PD1 expressed on T cells and a second antigen-binding domain that binds to the ULBP2 antigen on the surface of cancer cells. The antibody of this disclosure may, for example, be used to treat cell proliferation disorders (e.g., cancers expressing ULBP2) or to delay their progression.

[0056] UL16-binding protein 2 (ULBP2) is a class I major histocompatibility complex (MHC)-associated molecule that binds to the NKG2D receptor on natural killer (NK) cells to trigger the release of various cytokines and chemokines, thereby leading to NK cell recruitment and activation. The encoded protein undergoes further processing to produce a mature protein, which is partially anchored to the membrane via glycosylphosphatidylinositol or secreted. Many malignant cells secrete the encoded protein to evade NK cell immune surveillance.

[0057] The terms “ULBP2” and “ULBP2 antigen” are used interchangeably herein and include any variant, isoform, and species homolog of human ULBP2 expressed naturally in cells or on cells transfected with the ULBP2 gene. Binding of the antibodies disclosed herein to the ULBP2 antigen mediates the killing of ULBP2-expressing cells (e.g., tumor cells) through ULBP2 inactivation. The killing of ULBP2-expressing cells may occur through one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0058] According to this disclosure, the term "anti-ULBP2 antibody" is an antibody that specifically binds to the ULBP2 antigen. According to Cragg, MS et al., Blood 103 (2004) 2738-2743 and Cragg, MS et al., Blood 101 (2003) 1045-1052, two types of anti-ULBP2 antibodies (type I and type II anti-ULBP2 antibodies) can be distinguished based on their binding characteristics and biological activity to the ULBP2 antigen.

[0059] In some cases, ULBP2 can be expressed at low copy numbers on target cells (e.g., tumor cells). For example, in some cases, ULBP2 is expressed or present at fewer than 35,000 copies per target cell. In some embodiments, the low copy number of cell surface ULBP2 is present at 100 to 35,000 copies per target cell, 100 to 30,000 copies per target cell, 100 to 25,000 copies per target cell, 100 to 20,000 copies per target cell, 100 to 15,000 copies per target cell, 100 to 10,000 copies per target cell, 100 to 5,000 copies per target cell, 100 to 2,000 copies per target cell, 100 to 1,000 copies per target cell, or 100 to 500 copies per target cell. For example, the copy number of cell surface ULBP2 can be determined using a standard Scratchcard plot.

[0060] ULBP2 is widely and differentially expressed across a variety of solid tumor indications. ULBP2 expression in melanoma and breast cancer is associated with poor prognosis and advanced disease. This disclosure is the first to demonstrate high expression of ULBP2 in bladder cancers, including but not limited to transitional cell carcinoma, basal cell carcinoma, squamous cell carcinoma, basal-squamous cell carcinoma, adenocarcinoma, metastatic cancer, and carcinosarcoma. Specifically, ULBP2 is highly expressed in both the basal and squamous molecular lineages of bladder cancer.

[0061] Senescence is a stress-induced cellular state that limits tumorigenesis by inducing senescence-associated secretory phenotypes (SASP) to prevent cell proliferation and promote immune-mediated clearance of damaged cells (Rodier, F. et al., Persistent DNA damage signaling triggers senescence-associated inflammatory cytokine secretion). Nat. Cell Biol. 11, 973–979 (2009). Aging is also associated with age-related histopathology, in which the accumulation of SASP-positive cells can induce tissue inflammation, leading to tissue dysfunction, manifested in elderly patients as arthritis, autoimmunity, diabetes, fibrosis, and delayed wound healing (Childs, BG et al., Senescent cells: an emerging target for diseases of ageing). Nat. Rev. Drug Discov. 16,718–735 (2017)). SASP-positive cells express a complex and diverse array of secreted and cell surface proteins, including immune-activating cytokines, tissue-remodeling matrix metalloproteinases, and cell surface proteins, including the MHC1-like NKG2D ligand, which mediates the recognition and activation of NK and T cell effectors via the NKG2D co-stimulatory receptor. These proteins collectively promote the elimination of senescent cells. However, age-related decline in immune cell activity and other factors such as chemotherapy for cancer accelerate the induction of SASP-positive cells in tissues and limit the immune system's clearance of senescent cells (Jackola, DR, Ruger, JK, and Miller, RA, Age-associated changes in human T cell phenotype and function). Aging Clin. Exp. Res.6, 25–34 (1994); Demaria, M. et al., Cellular senescence promotes adverse effects of chemotherapy and cancer relapse. Cancer Discov. 7, 165–176 (2017)). Therapeutic strategies to eliminate SASP-positive cells offer opportunities to supplement senescent immune surveillance and alleviate the underlying causes of many age-related diseases and the persistent side effects of chemotherapy. Furthermore, the elimination of senescent cells not only alleviates age-related diseases, but these drugs that eliminate senescent cells also have the potential to prolong lifespan (Baker, DJ, et al., naturally occurring p16Ink4a-positive cells shorten healthy lifespan). Nature 530, 184–189 (2016); Baker, DJ, et al., Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232–236 (2011). ULBP2, an MHCI-like ligand, has emerged as a cell surface protein associated with stress-induced SASP-positive fibroblasts and cancer cells (Sagiv, A. et al., NKG2D ligands mediate immune surveillance of senescent cells). Aging 8, 328–344 (2016); Ruscetti, M. et al., NK cell-mediated cytotoxicity contributes to tumor control by a combination of cytostatic drugs. Science362, 1416–1422 (2018); Muñoz, DP et al., Targetable mechanisms driving immunoevasion of persistent senescent cells link chemotherapy-resistant cancer to aging. JCI Insight 5, e124716, 124716 (2019). ULBP2 or ULBP2 / 5 / 6 targeted drugs, in addition to eradicating cancer cells, have the potential to eliminate SASP-positive cells from tissues and improve tissue function, and may prevent age-related diseases and prolong life.

[0062] ULBP2 encompasses naturally occurring ULBP2 variants, including, for example, splice variants or allelic variants. ULBP2 includes, for example, the human ULBP2 protein (UniProt ID: Q9BZM5), which is 246 amino acids in length.

[0063] In one aspect, this disclosure provides isolated antibodies that bind to ULBP2. In some cases, the anti-ULBP2 antibody binds to a human ULBP2 polypeptide or a portion thereof. In some embodiments, the human ULBP2 polypeptide comprises the amino acid sequence of SEQ ID NO: 421.

[0064]

[0065] ULBP5 (RAET1G) encompasses naturally occurring ULBP5 variants, including, for example, splice variants or allelic variants. ULBP5 includes, for example, the human ULBP5 protein (UniProt ID: Q6H3X3), which is 334 amino acids in length.

[0066] In one aspect, this disclosure provides isolated antibodies that bind to ULBP5 (RAET1G). In some cases, the anti-ULBP5 antibody binds to a human ULBP5 polypeptide or a portion thereof. In some embodiments, the human ULBP5 polypeptide comprises the amino acid sequence of SEQ ID NO: 422.

[0067]

[0068] ULBP6 (RAET1L) encompasses naturally occurring ULBP6 variants, including, for example, splice variants or allelic variants. ULBP6 includes, for example, the human ULBP6 protein (UniProt ID: Q5VY80), which is 246 amino acids in length.

[0069] In one aspect, this disclosure provides isolated antibodies that bind to ULBP6 (RAET1L). In some cases, the anti-ULBP6 antibody binds to a human ULBP6 polypeptide or a portion thereof. In some embodiments, the human ULBP6 polypeptide comprises the amino acid sequence of SEQ ID NO: 423.

[0070]

[0071] Anti-ULBP2 / 5 / 6 antibodies

[0072] This document provides antibodies that bind to anti-ULBP2 / 5 / 6. In some embodiments, an alanine scan mutation can be performed on the "E12" anti-ULBP2 / 5 / 6 antibody to produce an affinity-modified anti-ULBP2 / 5 / 6 antibody. This document also provides antibodies that bind to anti-ULBP2. In some embodiments, an alanine scan mutation can be performed on the "A06" anti-ULBP2 antibody to produce an affinity-modified anti-ULBP2 antibody.

[0073] In some embodiments, the anti-ULBP2 / 5 / 6 antibody of this disclosure comprises either the VH or VL sequences listed in Table 1. In Table 1, the underlined sequences are CDR sequences according to Kabat, and the bold sequences are CDR sequences according to Chothia.

[0074] In some embodiments, the anti-ULBP2 antibody of this disclosure comprises: a) a heavy chain variable region (VH) including a VH complementarity-determining region 1 (VH2). CDR1 VH complementarity determination region 2 (VH CDR2 ) and VH complementarity determinant 3 (VH CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL CDR1 VL Complementary Determinant Region 2 (VL) CDR2 ) and VL complementarity determinant 3 (VL CDR3 Tables 2 and 3 provide exemplary CDR sequences for the anti-ULBP2 antibodies presented herein.

[0075] Table 1. Exemplary ULBP2 / 5 / 6 Variable Heavy Chain and Variable Light Chain Domains

[0076] Table 2. Exemplary resistance to ULBP2 / 5 / 6 heavy chain CDR

[0077] Table 3. Exemplary CDRs against ULBP2 / 5 / 6 light chains

[0078] In some embodiments, this disclosure provides an antibody that binds to ULBP2 (e.g., comprising an antibody fragment, such as a single-chain variable fragment (scFv)), wherein the antibody comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 5 or 6. CDR1 ), ii) VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 7 or 8 CDR2 ),iii) the VH complementarity-determining region 3 (VH) containing the amino acid sequence of SEQ ID NO: 9. CDR3 ); and b) light chain variable region (VL), which includes i) VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 10. CDR1 ), ii) containing the VL complementarity-determining region 2 (VL) of the amino acid sequence of SEQ ID NO: 11 CDR2 ),iii) the VL complementarity-determining region 3 (VL) containing the amino acid sequence of SEQ ID NO: 12. CDR3 ).

[0079] Exemplary anti-ULBP2 antibodies disclosed herein include ULBP2-01, ULBP2-02, E12, and A06.

[0080] In some embodiments, the anti-ULBP2 antibody ULBP2-01 has a VH region comprising the amino acid sequence of SEQ ID NO: 5. CDR1 VH containing the amino acid sequence of SEQ ID NO: 7 CDR2 , and VH containing the amino acid sequence of SEQ ID NO: 9 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 10. CDR1 VL containing the amino acid sequence of SEQ ID NO: 11 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0081] In some embodiments, the anti-ULBP2 antibody ULBP2-01 has a VH region comprising the amino acid sequence shown in SEQ ID NO: 2 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 1.

[0082] In some embodiments, the anti-ULBP2 antibody ULBP2-02 has a VH region comprising the amino acid sequence of SEQ ID NO: 5. CDR1VH containing the amino acid sequence of SEQ ID NO: 7 CDR2 , and VH containing the amino acid sequence of SEQ ID NO: 9 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 10. CDR1 VL containing the amino acid sequence of SEQ ID NO: 11 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0083] In some embodiments, the anti-ULBP2 antibody ULBP2-02 has a VH region comprising the amino acid sequence shown in SEQ ID NO: 4 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 3.

[0084] In some embodiments, the anti-ULBP2 antibody E12 has a VH region comprising the amino acid sequence of SEQ ID NO: 5. CDR1 VH containing the amino acid sequence of SEQ ID NO: 7 CDR2 , and VH containing the amino acid sequence of SEQ ID NO: 9 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 10. CDR1 VL containing the amino acid sequence of SEQ ID NO: 11 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0085] In some embodiments, the anti-ULBP2 antibody E12 has a VH region comprising the amino acid sequence shown in SEQ ID NO: 425 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 424.

[0086] In some embodiments, the anti-ULBP2 antibody A06 has a VH region comprising the amino acid sequence of SEQ ID NO: 428. CDR1 VH containing the amino acid sequence of SEQ ID NO: 430 CDR2 , and VH containing the amino acid sequence SEQ ID NO: 432 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 433. CDR1 VL containing the amino acid sequence SEQ ID NO: 434 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 435 CDR3 .

[0087] In some embodiments, the anti-ULBP2 antibody A06 has a VH region comprising the amino acid sequence shown in SEQ ID NO: 427 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 426.

[0088] Bispecific anti-ULBP2 / 5 / 6 antibody

[0089] This article provides bispecific antibodies comprising a first antigen-binding domain that binds to a first antigen (e.g., cell surface antigen, CD3ε) and a second antigen-binding domain that binds to a second antigen (e.g., ULBP2 / 5 / 6).

[0090] In some embodiments, the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), the constant region having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2), and a constant region 3 domain (CH1H3); a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1); a second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2), the constant region having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2), and a constant region 3 domain (CH2H3); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0091] In some embodiments, the bispecific antibody of this disclosure includes a second antigen-binding domain (e.g., binding to ULBP2 / 5 / 6) comprising either the VH2 or VL2 sequences listed in Table 1. In Table 1, underlined sequences are CDR sequences according to Kabat, and bold sequences are CDR sequences according to Chothia.

[0092] In some embodiments, the bispecific antibody of this disclosure includes a second antigen-binding domain (e.g., binding to ULBP2), which includes: a) a heavy chain variable region (VH2) containing a VH complementarity-determining region 1 (VH2). CDR1 VH complementarity determinant region 2 (VH2) CDR2 ) and VH2 complementarity determinant 3 (VH2 CDR3 ); and b) the light chain variable region (VL2), which contains the VL complementarity determination region 1 (VL2). CDR1 VL Complementary Determinant Region 2 (VL2) CDR2 ) and VL complementarity determinant 3 (VL2) CDR3 Tables 11 and 12 provide exemplary CDR sequences for the anti-ULBP2 antibodies presented herein.

[0093] In some embodiments, the bispecific antibody includes any of the anti-ULBP2 / 5 / 6 antibodies of this disclosure. Exemplary anti-ULBP2 / 5 / 6 antibodies of this disclosure include ULBP2-01, ULBP2-02, and E12. In some embodiments, the bispecific antibody includes any of the anti-ULBP2 antibodies of this disclosure. Exemplary anti-ULBP2 antibody of this disclosure includes A06.

[0094] In some embodiments, this disclosure provides an isolated antibody (e.g., a monospecific antibody or a bispecific antibody) that specifically binds to ULPB2 / 5 / 6 and competes with any of the aforementioned antibodies.

[0095] In some embodiments, this disclosure provides an antibody (e.g., a monospecific or bispecific antibody) that binds to ULBP2 / 5 / 6 and competes with the antibodies described herein (including ULBP2-01, ULBP2-02, A06, and E12).

[0096] In some embodiments, this disclosure also provides a CDR portion based on the CDR contact region of the ULBP2 / 5 / 6 antibody. The CDR contact region is the region of the antibody that confers specificity to the antigen. Generally, the CDR contact region includes residue sites in the CDR and Vernier region that are restricted to maintain the appropriate loop structure for antibody binding to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. The determination of the CDR contact region is entirely within the scope of the art.

[0097] The binding affinity (KD) of the ULBP2 / 5 / 6 antibodies (e.g., monospecific or bispecific antibodies) described herein to ULBP2 / 5 / 6 (e.g., human ULBP2 (e.g., SEQ ID NO: 421), ULBP5 (SEQ ID NO: 422), ULBP6 (SEQ ID NO: 423)) can be from about 0.001 to about 5000 nM.

[0098] In some embodiments, the binding affinity is approximately 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 nM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM, 400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM, 207 nM, 200 nM, 186 nM, 172 nM, 136 nM, 113 nM, 104 nM, 101 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74 nM, 54 nM, 50 nM, 45 nM, 42nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 Any of nM, 16 nM, 15nM, 12 nM, 10 nM, 9 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM or 0.001 nM.

[0099] In some embodiments, the binding affinity is approximately less than any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0100] In some embodiments, this disclosure provides a nucleic acid encoding any of the isolated anti-ULBP2 / 5 / 6 antibodies (e.g., monospecific or bispecific antibodies). In some embodiments, this disclosure provides a vector comprising such a nucleic acid. In some embodiments, this disclosure provides a host cell comprising such a nucleic acid.

[0101] CD3 antibody

[0102] In some embodiments, this disclosure provides anti-CD3 antibodies. In some embodiments, the anti-CD3 antibodies are multispecific (e.g., bispecific) and bind to a second biomolecule (e.g., a cell surface antigen, such as a disease-associated antigen) in addition to CD3 or fragments thereof. The antibodies of this disclosure can be used, for example, to treat or delay the progression of cell proliferation disorders (e.g., cancer) or autoimmune disorders, or to enhance immune function in subjects with such disorders.

[0103] Unless otherwise stated, the term “differentiation antigen cluster 3” or “CD3” as used herein refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), including, for example, CD3ε, CD3γ, CD3α, and CD3β chains. CD3 is a cell surface complex expressed on T cells and binding to the T cell receptor. The CD3 complex is required for the activation of CD8+ and CD4+ T lymphocytes. It is formed by three distinct but highly related chains: one CD3γ chain, one CD3δ chain, and two CD3ε chains, which bind together to form CD3ε / γ heterodimers and CD3ε / δ heterodimers. These two CD3 heterodimers, together with the T cell receptor (TCR) and the signal transduction ζ chain homodimer, form the T cell receptor complex.

[0104] The term encompasses “full-length” unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 produced in cells through processing. The term also encompasses naturally occurring CD3 variants, including, for example, splice variants or allelic variants. For example, CD3 includes the human CD3ε protein (NCBI RefSeqNo. NP_000724), which is 207 amino acids in length.

[0105] In some embodiments, this disclosure provides isolated antibodies that bind to CD3. In some embodiments, this disclosure provides antibodies that bind to CD3ε. In certain cases, the anti-CD3ε antibody binds to a human CD3ε polypeptide or a cynomolgus monkey (SEQ ID NO: 419) CD3ε polypeptide. In certain cases, the human CD3 polypeptide or the cynomolgus monkey CD3 polypeptide is, respectively, a human CD3ε polypeptide (SEQ ID NO: 420). In certain cases, the anti-CD3 antibody binds to an epitope within the CD3ε (e.g., human CD3ε) fragment consisting of amino acid residues 1-26 or 1-27 of human CD3ε (SEQ ID NO: 419).

[0106] A useful method for identifying antibodies that can be targeted for mutated residues or regions is called "alanine scanning mutation," as described by Cunningham and Wells (1989). Science As described in 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at the amino acid position to confirm functional sensitivity to the initial substitution. Optionally or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0107] Anti-CD3ε antibody

[0108] This document provides anti-CD3ε antibodies. In some embodiments, the "SP34" anti-CD3ε antibody is subjected to an alanine scan mutation to produce the affinity-modified anti-CD3ε antibody of this disclosure.

[0109] In some embodiments, the anti-CD3ε antibody of this disclosure comprises either the VH or VL sequences listed in Table 4. In Table 4, the underlined sequences are CDR sequences according to Kabat, and the bold sequences are CDR sequences according to Chothia.

[0110] In some embodiments, the anti-CD3 antibody of this disclosure comprises: a) a heavy chain variable region (VH) including a VH complementarity-determining region 1 (VH... CDR1 VH complementarity determination region 2 (VH CDR2 ) and VH complementarity determinant 3 (VH CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL CDR1 VL Complementary Determinant Region 2 (VL) CDR2 ) and VL complementarity determinant 3 (VL CDR3 Tables 8 and 9 provide exemplary CDR sequences for the anti-CD3 antibodies presented herein.

[0111] Table 4. Anti-CD3 variable heavy chain and variable light chain domains

[0112] Table 5. Anti-CD3 heavy chain CDR

[0113] Table 6. Anti-CD3 light chain CDR

[0114] In some embodiments, this disclosure provides an antibody that specifically binds to CD3ε (e.g., comprising an antibody fragment, such as a single-chain variable fragment (scFv)), wherein the antibody comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 29, 30, 31, 32 or 33. CDR1 ), ii) VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 34, 35 or 36 CDR2 ),iii) VH complementarity-determining region 3 (VH) containing the amino acid sequence of SEQ ID NO: 37, 38, 39, 40 or 41 CDR3 ); and b) light chain variable region (VL), which includes i) VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 42. CDR1 ), ii) VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 43 or 44 CDR2 ),iii) VL complementarity-determining region 3 (VL) containing the amino acid sequence of SEQ ID NO: 45, 46, 47 or 48 CDR3 ).

[0115] Exemplary anti-CD3 antibodies disclosed herein include CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0116] In some embodiments, the anti-CD3 antibody CD3-A1 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 37 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 46 CDR3 .

[0117] In some embodiments, the anti-CD3 antibody CD3-A1 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 13 and a VL region containing the amino acid sequence shown in SEQ ID NO: 25.

[0118] In some embodiments, the anti-CD3 antibody CD3-A2 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 37 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 44 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0119] In some embodiments, the anti-CD3 antibody CD3-A2 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 13 and a VL region containing the amino acid sequence shown in SEQ ID NO: 27.

[0120] In some embodiments, the anti-CD3 antibody CD3-A3 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 37 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0121] In some embodiments, the anti-CD3 antibody CD3-A3 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 14 and a VL region containing the amino acid sequence shown in SEQ ID NO: 23.

[0122] In some embodiments, the anti-CD3 antibody CD3-A4 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2and VH containing the amino acid sequence of SEQ ID NO: 38 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0123] In some embodiments, the anti-CD3 antibody CD3-A4 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 15 and a VL region containing the amino acid sequence shown in SEQ ID NO: 26.

[0124] In some embodiments, the anti-CD3 antibody CD3-A5 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 39 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0125] In some embodiments, the anti-CD3 antibody CD3-A5 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 16 and a VL region containing the amino acid sequence shown in SEQ ID NO: 26.

[0126] In some embodiments, the anti-CD3 antibody CD3-A6 comprises a VH region containing the amino acid sequence of SEQ ID NO: 30. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 37 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0127] In some embodiments, the anti-CD3 antibody CD3-A6 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 17 and a VL region containing the amino acid sequence shown in SEQ ID NO: 22.

[0128] In some embodiments, the anti-CD3 antibody CD3-A7 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 35 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 38 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0129] In some embodiments, the anti-CD3 antibody CD3-A7 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 18 and a VL region containing the amino acid sequence shown in SEQ ID NO: 22.

[0130] In some embodiments, the anti-CD3 antibody CD3-A8 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 35 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 38 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0131] In some embodiments, the anti-CD3 antibody CD3-A8 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 18 and a VL region containing the amino acid sequence shown in SEQ ID NO: 26.

[0132] In some embodiments, the anti-CD3 antibody CD3-A9 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2and VH containing the amino acid sequence of SEQ ID NO: 40 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0133] In some embodiments, the anti-CD3 antibody CD3-A9 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 19 and a VL region containing the amino acid sequence shown in SEQ ID NO: 26.

[0134] In some embodiments, the anti-CD3 antibody CD3-A10 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 41 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0135] In some embodiments, the anti-CD3 antibody CD3-A10 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 20 and a VL region containing the amino acid sequence shown in SEQ ID NO: 26.

[0136] In some embodiments, the anti-CD3 antibody CD3-A11 comprises a VH region containing the amino acid sequence of SEQ ID NO: 31. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 37 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0137] In some embodiments, the anti-CD3 antibody CD3-A11 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 21 and a VL region containing the amino acid sequence shown in SEQ ID NO: 22.

[0138] In some embodiments, the anti-CD3 antibody CD3-A12 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 37 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0139] In some embodiments, the anti-CD3 antibody CD3-A12 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 13 and a VL region containing the amino acid sequence shown in SEQ ID NO: 24.

[0140] In some embodiments, the anti-CD3 antibody CD3-A13 comprises a VH region containing the amino acid sequence of SEQ ID NO: 29. CDR1 VH containing the amino acid sequence of SEQ ID NO: 34 CDR2 and VH containing the amino acid sequence of SEQ ID NO: 39 CDR3 ; and the VL region, which contains the VL region containing the amino acid sequence of SEQ ID NO: 42. CDR1 VL containing the amino acid sequence of SEQ ID NO: 43 CDR2 and VL containing the amino acid sequence of SEQ ID NO: 48 CDR3 .

[0141] In some embodiments, the anti-CD3 antibody CD3-A13 comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 16 and a VL region containing the amino acid sequence shown in SEQ ID NO: 28.

[0142] Bispecific anti-CD3ε antibody

[0143] This article provides bispecific antibodies comprising a first antigen-binding domain that binds to a first antigen (e.g., CD3ε) and a second antigen-binding domain that binds to a second antigen (e.g., a disease-associated antigen).

[0144] In some embodiments, the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), the constant region having a constant region 1 domain (CH1). H1 ), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 The first light chain polypeptide (L1) comprises a variable region (VL1) and a constant region (CL1); the second heavy chain polypeptide (H2) comprises a variable region (VH2) and a constant region (CH2), wherein the constant region has a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 ); and the second light chain polypeptide (L2), which contains a variable region (VL2) and a constant region (CL2).

[0145] In some embodiments, the bispecific antibody of this disclosure includes a first antigen-binding domain (e.g., CD3-binding) comprising either the VH1 or VL1 sequences listed in Table 4. In Table 4, underlined sequences are CDR sequences according to Kabat, and bold sequences are CDR sequences according to Chothia.

[0146] In some embodiments, the bispecific antibody of this disclosure includes a first antigen-binding domain (e.g., binding to CD3ε), which includes: a) a heavy chain variable region (VH1) containing a VH complementarity-determining region 1 (VH1). CDR1 VH complementarity determinant region 2 (VH1) CDR2 ) and VH complementarity determinant 3 (VH1) CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL1) CDR1 VL Complementary Determinant Region 2 (VL1) CDR2 ) and VL complementarity determinant 3 (VL1) CDR3 Tables 8 and 9 provide exemplary CDR sequences for the anti-CD3 antibodies presented herein.

[0147] In some embodiments, the bispecific antibody comprises any of the anti-CD3 antibodies of this disclosure. Exemplary anti-CD3 antibodies of this disclosure include CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13.

[0148] In some embodiments, this disclosure provides an isolated antibody (e.g., a monospecific antibody or a bispecific antibody) that specifically binds to CD3ε and competes with any of the antibodies described above.

[0149] In some embodiments, this disclosure provides an antibody (e.g., a monospecific or bispecific antibody) that binds to CD3ε and competes with antibodies described herein (including CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12, and CD3-A13).

[0150] In some embodiments, this disclosure also provides a CDR portion of the CD3ε antibody based on the CDR contact region. The CDR contact region is the region of the antibody that confers specificity to the antigen. Generally, the CDR contact region includes residue sites in the CDR and Vernier region that are restricted to maintain the appropriate loop structure for antibody binding to a specific antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. The determination of the CDR contact region is entirely within the scope of the art.

[0151] The binding affinity (Kb) of the disclosed anti-CD3ε antibody (e.g., monospecific or bispecific antibody) to human CD3ε (e.g., human CD3ε, e.g., SEQ ID NO: 419) is [missing information]. D The value can be approximately 0.001 to approximately 5000 nM.

[0152] In some embodiments, the binding affinity is approximately 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 nM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM, 400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM, 207 nM, 200 nM, 186 nM, 172 nM, 136 nM, 113 nM, 104 nM, 101 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74 nM, 54 nM, 50 nM, 45 nM, 42nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 Any of nM, 16 nM, 15nM, 12 nM, 10 nM, 9 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM or 0.001 nM.

[0153] In some embodiments, the binding affinity is approximately less than any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0154] The binding affinity (Kb) of the disclosed anti-CD3ε antibody (e.g., monospecific or bispecific antibody) to cynomolgus monkey CD3ε (e.g., cynomolgus monkey CD3ε, e.g., SEQ ID NO: 422) is [missing information]. D The value can be approximately 0.001 to approximately 5000 nM.

[0155] In some embodiments, the binding affinity is approximately 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM, 1000 nM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM, 400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM, 207 nM, 200 nM, 186 nM, 172 nM, 136 nM, 113 nM, 104 nM, 101 nM, 100 nM, 90 nM, 83 nM, 79 nM, 74 nM, 54 nM, 50 nM, 45 nM, 42nM, 40 nM, 35 nM, 32 nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 Any of nM, 16 nM, 15nM, 12 nM, 10 nM, 9 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM or 0.001 nM.

[0156] In some embodiments, the binding affinity is approximately less than any of 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0157] In some embodiments, this disclosure provides a nucleic acid encoding any of the isolated anti-CD3ε antibodies (e.g., monospecific or bispecific antibodies). In some embodiments, this disclosure provides a vector comprising such a nucleic acid. In some embodiments, this disclosure provides a host cell comprising such a nucleic acid.

[0158] Exemplary bispecific antibodies binding to CD3ε and ULBP2 / 5 / 6

[0159] This article provides bispecific antibodies comprising a first antigen-binding domain that binds to a first antigen (e.g., CD3ε) and a second antigen-binding domain that binds to a second antigen (e.g., ULBP2 / 5 / 6).

[0160] In some embodiments, the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), the constant region having a constant region 1 domain (CH1). H1 ), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 The first light chain polypeptide (L1) comprises a variable region (VL1) and a constant region (CL1); the second heavy chain polypeptide (H2) comprises a variable region (VH2) and a constant region (CH2), wherein the constant region has a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 ); and the second light chain polypeptide (L2), which contains a variable region (VL2) and a constant region (CL2).

[0161] In some embodiments, the bispecific antibody of this disclosure includes a first antigen-binding domain (e.g., binding to CD3ε), which includes: a) a heavy chain variable region (VH1) containing a VH complementarity-determining region 1 (VH1). CDR1 VH complementarity determinant region 2 (VH1) CDR2 ) and VH complementarity determinant 3 (VH1) CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL1) CDR1 VL Complementary Determinant Region 2 (VL1) CDR2 ) and VL complementarity determinant 3 (VL1) CDR3 ); and a second antigen-binding domain (e.g., binding to ULBP2 / 5 / 6), which includes: a) a heavy chain variable region (VH2) containing VH complementarity-determining region 1 (VH2 CDR1 VH complementarity determinant region 2 (VH2) CDR2 ) and VH2 complementarity determinant 3 (VH2 CDR3 ); and b) the light chain variable region (VL2), which contains the VL complementarity determination region 1 (VL2). CDR1 VL Complementary Determinant Region 2 (VL2) CDR2 ) and VL complementarity determinant 3 (VL2) CDR3 Tables 5 and 6 provide exemplary CDR sequences for the anti-CD3ε antibodies provided herein. Tables 2 and 3 provide exemplary CDR sequences for the anti-ULBP2 / 5 / 6 antibodies provided herein.

[0162] In some embodiments, the bispecific antibody of this disclosure comprises a first antigen-binding domain (e.g., binding to CD3ε) containing either of the VH1 and VL1 sequences listed in Table 4 and a second antigen-binding domain (e.g., binding to ULBP2 / 5 / 6) containing either of the VH2 and VL2 sequences listed in Table 1.

[0163] In some embodiments, the bispecific antibody of this disclosure comprises a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), comprising any of the sequences listed in Tables 7 and 8. Italicized sequences are heavy chain variable regions and light chain variable regions. Underlined sequences are according to Kabat's CDR, and bold sequences are according to Chothia's CDR.

[0164] In some embodiments, the bispecific antibody provided herein comprises an H1 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the H1 sequence listed in Tables 7 and 8.

[0165] In some embodiments, the bispecific antibody provided herein comprises an L1 sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the L1 sequence listed in Tables 7 and 8.

[0166] In some embodiments, the bispecific antibody provided herein comprises an H2 having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the H2 sequence listed in Tables 7 and 8.

[0167] In some embodiments, the bispecific antibody provided herein comprises an L2 sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the L2 sequence listed in Tables 7 and 8.

[0168] In some embodiments, the amino acid sequence of H1 is numbered according to SEQ ID NO: 439. In some embodiments, the amino acid sequence of L1 is numbered according to SEQ ID NO: 438. In some embodiments, the amino acid sequence of H2 is numbered according to SEQ ID NO: 437. In some embodiments, the amino acid sequence of L2 is numbered according to SEQ ID NO: 436.

[0169] Table 7. Exemplary bispecific antibodies binding CD3ε and ULBP2 / 5 / 6

[0170] The exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibodies disclosed herein include EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598.

[0171] Bispecific antibodies EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598 contain a first antigen-binding domain that binds to CD3ε, which contains VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0172] Bispecific antibodies EIP0174, EIP0175, EIP0187, EIP0205, EIP0206, EIP0207, EIP0208, EIP0294, EIP0295, EIP0306, EIP0307, ​​EIP0318, EIP0340, EIP0342, EIP0354, EIP0356, EIP0367, EIP0377, EIP0404, EIP0406, EIP0473, and EIP0598 contain a second antigen-binding domain that binds to ULBP2 / 5 / 6, which contains VH2, said VH2 containing the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 having the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, said VL2 comprising the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0173] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0174 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) of CL1 is K, and the amino acid at position 137 (EU number) of CH1 is D; H1 The amino acid at position 128 (EU number) is C, and the amino acid at position 118 (EU number) of CL1 is C; the amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acid at position CH1 H3 The amino acid at position 349 (EU number) is C; the CH1H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0174] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0174 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) is D, and the amino acid at position 180 (EU number) of CL2 is R; the amino acids at positions 234, 235, and 237 (EU number) of H2H are A; the amino acids at positions CH2 H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0175] In some embodiments, the bispecific antibody EIP0174 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 2, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0176] In some embodiments, the bispecific antibody EIP0174 comprises H1 containing the amino acid sequence of SEQ ID NO: 58, L1 containing the amino acid sequence of SEQ ID NO: 57, H2 containing the amino acid sequence of SEQ ID NO: 56, and L2 containing the amino acid sequence of SEQ ID NO: 55.

[0177] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0175 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0178] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0175 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R; H1 The amino acid at position 134 (EU number) of the CL2 is C, and the amino acid at position 116 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0179] In some embodiments, the bispecific antibody EIP0175 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0180] In some embodiments, the bispecific antibody EIP0175 comprises H1 containing the amino acid sequence of SEQ ID NO: 62, H2 containing the amino acid sequence of SEQ ID NO: 60, L1 containing the amino acid sequence of SEQ ID NO: 61, and L2 containing the amino acid sequence of SEQ ID NO: 59.

[0181] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0187 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0182] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0187 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R; H1The amino acid at position 136 (EU number) of the CL2 is C, and the amino acid at position 114 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0183] In some embodiments, the bispecific antibody EIP0187 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0184] In some embodiments, the bispecific antibody EIP0187 comprises H1 containing the amino acid sequence of SEQ ID NO: 66, H2 containing the amino acid sequence of SEQ ID NO: 64, L1 containing the amino acid sequence of SEQ ID NO: 65, and L2 containing the amino acid sequence of SEQ ID NO: 63.

[0185] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0205 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) of CL1 is K, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; the amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acid at position CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0186] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0205 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) is D, and the amino acid at position 180 (EU number) of CL2 is R; the amino acids at positions 234, 235, and 237 (EU number) of H2H are A; the amino acids at positions CH2 H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0187] In some embodiments, the bispecific antibody EIP0205 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0188] In some embodiments, the bispecific antibody EIP0205 comprises H1 containing the amino acid sequence of SEQ ID NO: 70, H2 containing the amino acid sequence of SEQ ID NO: 68, L1 containing the amino acid sequence of SEQ ID NO: 69, and L2 containing the amino acid sequence of SEQ ID NO: 67.

[0189] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0206 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1...H1 The amino acid at position 185 (EU number) of CL1 is K, and the amino acid at position 137 (EU number) of CH1 is D; H1 The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; the amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 of CL1 is S; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acid at position CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0190] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0206 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R; H1 The amino acid at position 134 (EU number) is C, and the amino acid at position 116 (EU number) of CL2 is C; the amino acids at positions 234, 235, and 237 (EU number) of H2H are A; the amino acids at positions CH2 H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0191] In some embodiments, the bispecific antibody EIP0206 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0192] In some embodiments, the bispecific antibody EIP0206 comprises H1 containing the amino acid sequence of SEQ ID NO: 74, H2 containing the amino acid sequence of SEQ ID NO: 72, L1 containing the amino acid sequence of SEQ ID NO: 73, and L2 containing the amino acid sequence of SEQ ID NO: 71.

[0193] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0207 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0194] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0207 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R; H1The amino acid at position 131 (EU number) of the CL2 is C, and the amino acid at position 114 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0195] In some embodiments, the bispecific antibody EIP0207 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0196] In some embodiments, the bispecific antibody EIP0207 comprises H1 containing the amino acid sequence of SEQ ID NO: 78, H2 containing the amino acid sequence of SEQ ID NO: 76, L1 containing the amino acid sequence of SEQ ID NO: 77, and L2 containing the amino acid sequence of SEQ ID NO: 75.

[0197] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0208 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0198] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0208 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 170 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0199] In some embodiments, the bispecific antibody EIP0208 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0200] In some embodiments, the bispecific antibody EIP0208 comprises H1 containing the amino acid sequence of SEQ ID NO: 82, H2 containing the amino acid sequence of SEQ ID NO: 80, L1 containing the amino acid sequence of SEQ ID NO: 81, and L2 containing the amino acid sequence of SEQ ID NO: 79.

[0201] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0294 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1...H1 The amino acid at position 185 (EU number) is E; the amino acid at position 137 (EU number) of CL1 is K; and the amino acid at position 179 (EU number) of CL1 is E; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1... H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0202] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0294 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0203] In some embodiments, the bispecific antibody EIP0294 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0204] In some embodiments, the bispecific antibody EIP0294 comprises H1 containing the amino acid sequence of SEQ ID NO: 86, H2 containing the amino acid sequence of SEQ ID NO: 84, L1 containing the amino acid sequence of SEQ ID NO: 85, and L2 containing the amino acid sequence of SEQ ID NO: 83.

[0205] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0295 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0206] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0295 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0207] In some embodiments, the bispecific antibody EIP0295 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0208] In some embodiments, the bispecific antibody EIP0295 comprises H1 containing the amino acid sequence of SEQ ID NO: 90, H2 containing the amino acid sequence of SEQ ID NO: 88, L1 containing the amino acid sequence of SEQ ID NO: 89, and L2 containing the amino acid sequence of SEQ ID NO: 87.

[0209] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0306 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) of CL1 is E, the amino acid at position 137 (EU number) of CL1 is K, and the amino acid at position 179 (EU number) of CL1 is E; the amino acid at position CH1 H1 The amino acid at position 185 (EU number) is E, and the amino acid at position 179 (EU number) of CL1 is E; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0210] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0306 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R; H1 The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0211] In some embodiments, the bispecific antibody EIP0306 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0212] In some embodiments, the bispecific antibody EIP0306 comprises H1 containing the amino acid sequence of SEQ ID NO: 94, H2 containing the amino acid sequence of SEQ ID NO: 92, L1 containing the amino acid sequence of SEQ ID NO: 93, and L2 containing the amino acid sequence of SEQ ID NO: 91.

[0213] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0307 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0214] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0307 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R; H1 The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0215] In some embodiments, the bispecific antibody EIP0307 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0216] In some embodiments, the bispecific antibody EIP0307 comprises H1 containing the amino acid sequence of SEQ ID NO: 98, H2 containing the amino acid sequence of SEQ ID NO: 96, L1 containing the amino acid sequence of SEQ ID NO: 97, and L2 containing the amino acid sequence of SEQ ID NO: 95.

[0217] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0318 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) of CL1 is K, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0218] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0318 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R;H1 The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0219] In some embodiments, the bispecific antibody EIP0318 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0220] In some embodiments, the bispecific antibody EIP0318 comprises H1 containing the amino acid sequence of SEQ ID NO: 102, H2 containing the amino acid sequence of SEQ ID NO: 100, L1 containing the amino acid sequence of SEQ ID NO: 101, and L2 containing the amino acid sequence of SEQ ID NO: 99.

[0221] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0340 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is D, and the amino acid at position 38 (Kabat number) of VL1 is K; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is E; the amino acid at position 137 (EU number) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1... H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0222] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0340 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is K, and the amino acid at position 38 (Kabat number) of VL2 is D; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 136 (EU number) of the CL2 is C, and the amino acid at position 114 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0223] In some embodiments, the bispecific antibody EIP0340 comprises VH1 containing the amino acid sequence of SEQ ID NO: 14, VH2 containing the amino acid sequence of SEQ ID NO: 4, VL1 containing the amino acid sequence of SEQ ID NO: 23, and VL2 containing the amino acid sequence of SEQ ID NO: 3.

[0224] In some embodiments, the bispecific antibody EIP0340 comprises H1 containing the amino acid sequence of SEQ ID NO: 106, H2 containing the amino acid sequence of SEQ ID NO: 104, L1 containing the amino acid sequence of SEQ ID NO: 105, and L2 containing the amino acid sequence of SEQ ID NO: 103.

[0225] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0342 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is D, and the amino acid at position 38 (Kabat number) of VL1 is K; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is E; the amino acid at position 137 (EU number) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1... H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0226] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0342 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is K, and the amino acid at position 38 (Kabat number) of VL2 is D; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0227] In some embodiments, the bispecific antibody EIP0342 comprises VH1 containing the amino acid sequence of SEQ ID NO: 14, VH2 containing the amino acid sequence of SEQ ID NO: 4, VL1 containing the amino acid sequence of SEQ ID NO: 23, and VL2 containing the amino acid sequence of SEQ ID NO: 3.

[0228] In some embodiments, the bispecific antibody EIP0342 comprises H1 containing the amino acid sequence of SEQ ID NO: 110, H2 containing the amino acid sequence of SEQ ID NO: 108, L1 containing the amino acid sequence of SEQ ID NO: 109, and L2 containing the amino acid sequence of SEQ ID NO: 107.

[0229] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0354 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is D, and the amino acid at position 38 (Kabat number) of VL1 is K; the amino acid at position CH1... H1 The amino acid at position 185 (EU number) is K, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A, and the amino acid at position CH1 is... H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0230] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0354 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is K, and the amino acid at position 38 (Kabat number) of VL2 is D; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0231] In some embodiments, the bispecific antibody EIP0354 comprises VH1 containing the amino acid sequence of SEQ ID NO: 14, VH2 containing the amino acid sequence of SEQ ID NO: 4, VL1 containing the amino acid sequence of SEQ ID NO: 23, and VL2 containing the amino acid sequence of SEQ ID NO: 3.

[0232] In some embodiments, the bispecific antibody EIP0354 comprises H1 containing the amino acid sequence of SEQ ID NO: 114, H2 containing the amino acid sequence of SEQ ID NO: 112, L1 containing the amino acid sequence of SEQ ID NO: 113, and L2 containing the amino acid sequence of SEQ ID NO: 111.

[0233] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0356 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is D, and the amino acid at position 38 (Kabat number) of VL1 is K; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) of CL1 is K, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 185 (EU number) of CL1 is E, and the amino acid at position 137 (EU number) of CH1 is K; H1 The amino acid at position 145 (EU number) is S, and the amino acid at position 180 (EU number) of CL1 is E; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0234] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0356 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is K, and the amino acid at position 38 (Kabat number) of VL2 is D; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 170 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0235] In some embodiments, the bispecific antibody EIP0356 comprises VH1 containing the amino acid sequence of SEQ ID NO: 14, VH2 containing the amino acid sequence of SEQ ID NO: 4, VL1 containing the amino acid sequence of SEQ ID NO: 23, and VL2 containing the amino acid sequence of SEQ ID NO: 3.

[0236] In some embodiments, the bispecific antibody EIP0356 comprises H1 containing the amino acid sequence of SEQ ID NO: 118, H2 containing the amino acid sequence of SEQ ID NO: 116, L1 containing the amino acid sequence of SEQ ID NO: 117, and L2 containing the amino acid sequence of SEQ ID NO: 115.

[0237] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0367 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) of CL1 is D, and the amino acid at position 131 (EU number) of CH1 is K; H1 The amino acid at position 185 (EU number) is E, and the amino acid at position 137 (EU number) of CL1 is D; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0238] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0367 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, the amino acid at position 137 (EU number) of CL2 is K, and the amino acid at position 138 (EU number) of CL2 is R; the amino acid at position CH2 H1 The amino acid at position 170 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0239] In some embodiments, the bispecific antibody EIP0367 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0240] In some embodiments, the bispecific antibody EIP0367 comprises H1 containing the amino acid sequence of SEQ ID NO: 122, H2 containing the amino acid sequence of SEQ ID NO: 120, L1 containing the amino acid sequence of SEQ ID NO: 121, and L2 containing the amino acid sequence of SEQ ID NO: 119.

[0241] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0377 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) is D, and the CH1 H1 The amino acid at position 145 (EU number) is S, and the amino acid at position 131 (EU number) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0242] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0377 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2...H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 170 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0243] In some embodiments, the bispecific antibody EIP0377 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0244] In some embodiments, the bispecific antibody EIP0377 comprises H1 containing the amino acid sequence of SEQ ID NO: 126, H2 containing the amino acid sequence of SEQ ID NO: 124, L1 containing the amino acid sequence of SEQ ID NO: 125, and L2 containing the amino acid sequence of SEQ ID NO: 123.

[0245] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0404 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is D, and the amino acid at position 38 (Kabat number) of VL1 is K; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) of CL1 is K, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 185 (EU number) is E; the amino acid at position 137 (EU number) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1... H3The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0246] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0404 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is K, and the amino acid at position 38 (Kabat number) of VL2 is D; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) of CL2 is D, and the amino acid at position 180 (EU number) of CH2 is R; H1 The amino acid at position 136 (EU number) of the CL2 is C, and the amino acid at position 114 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0247] In some embodiments, the bispecific antibody EIP0404 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 24, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0248] In some embodiments, the bispecific antibody EIP0404 comprises H1 containing the amino acid sequence of SEQ ID NO: 130, H2 containing the amino acid sequence of SEQ ID NO: 128, L1 containing the amino acid sequence of SEQ ID NO: 129, and L2 containing the amino acid sequence of SEQ ID NO: 127.

[0249] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0406 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is E; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) of CL1 is K, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 185 (EU number) is E; the amino acid at position 137 (EU number) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1... H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0250] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0406 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 136 (EU number) of the CL2 is C, and the amino acid at position 114 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A.H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0251] In some embodiments, the bispecific antibody EIP0406 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 24, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0252] In some embodiments, the bispecific antibody EIP0406 comprises H1 containing the amino acid sequence of SEQ ID NO: 134, H2 containing the amino acid sequence of SEQ ID NO: 132, L1 containing the amino acid sequence of SEQ ID NO: 133, and L2 containing the amino acid sequence of SEQ ID NO: 131.

[0253] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0473 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 147 (EU number) of CL1 is D, and the amino acid at position 131 (EU number) of CH1 is K; H1 The amino acid at position 185 (EU number) is D; the amino acid at position 137 (EU number) of CL1 is K; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acids at positions CH1... H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0254] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0473 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 187 (EU number) of CL2 is D, and the amino acid at position 138 (EU number) of CH2 is K; H1 The amino acid at position 171 (EU number) of the CL2 is C, and the amino acid at position 162 (EU number) of the CL2 is C; the amino acid at position 220 (EU number) of the H2H is S, and the amino acid at position 214 (EU number) of the CL2 is S; the amino acids at positions 234, 235, and 237 (EU number) of the H2H are A; the amino acid at position 234, 235, and 237 of the CH2 is A. H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0255] In some embodiments, the bispecific antibody EIP0473 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 4, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 3.

[0256] In some embodiments, the bispecific antibody EIP0473 comprises H1 containing the amino acid sequence of SEQ ID NO: 138, H2 containing the amino acid sequence of SEQ ID NO: 136, L1 containing the amino acid sequence of SEQ ID NO: 137, and L2 containing the amino acid sequence of SEQ ID NO: 135.

[0257] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0598 also contains the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 170 (EU number) of CL1 is S, and the amino acid at position 131 (EU number) of CH1 is D; H1The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; the amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acid at position CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0258] In addition to the specific complementarity-determining regions mentioned above, the bispecific antibody EIP0598 also contains the following amino acid substitutions in H2 and L2: the amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; the amino acid at position CH2... H1 The amino acid at position 147 (EU number) is D, and the amino acid at position 180 (EU number) of CL2 is R; the amino acids at positions 234, 235, and 237 (EU number) of H2H are A; the amino acids at positions CH2 H3 The amino acid at position 354 (EU number) is C; the CH2 H3 The amino acid at position 366 (EU number) is W; and the CH2 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H2 amino acid sequence of SEQ NO: 437 and the L2 amino acid sequence of SEQ NO: 436.

[0259] In some embodiments, the bispecific antibody EIP0598 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VH2 containing the amino acid sequence of SEQ ID NO: 2, VL1 containing the amino acid sequence of SEQ ID NO: 22, and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0260] In some embodiments, the bispecific antibody EIP0598 comprises H1 containing the amino acid sequence of SEQ ID NO: 142, H2 containing the amino acid sequence of SEQ ID NO: 140, L1 containing the amino acid sequence of SEQ ID NO: 141, and L2 containing the amino acid sequence of SEQ ID NO: 139.

[0261] Any of the bispecific antibodies shown in Table 7 above can be further modified by replacing any of the anti-CD3ε antigen-binding regions with any of the anti-CD3ε binding regions shown in Tables 7-9. For example, the anti-CD3ε antigen-binding region of the bispecific antibody “EIP0205” can be replaced with any of the anti-CD3ε binding regions shown in Tables 7-9 to produce the bispecific antibody of this disclosure. Exemplary antibodies are shown in Table 8. The underlined sequences are based on the CDR sequences of Kabat, and the bold sequences are based on the CDR sequences of Chothia.

[0262] Table 8. Exemplary bispecific antibodies binding to CD3ε and ULBP2 / 5 / 6

[0263] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibodies of this disclosure include EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525.

[0264] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 contain the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1The amino acid at position 147 (EU number) of CL1 is K, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; the amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acid at position CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0265] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622, and EIP0525 have a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, wherein VH2 comprises the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0266] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622 and EIP0525 contain VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0267] Bispecific antibodies EIP0527, EIP0624, EIP0486, EIP0626, EIP0483, EIP0623, EIP0621, EIP0625, EIP0622 and EIP0525 have H2 containing the amino acid sequence of SEQ ID NO: 68 and L2 containing the amino acid sequence of SEQ ID NO: 67.

[0268] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0527 also includes a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 comprises the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 46 CDR3 .

[0269] In some embodiments, the bispecific antibody EIP0527 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the bispecific antibody EIP0527 comprises H1 having the amino acid sequence of SEQ ID NO: 146 and L1 having the amino acid sequence of SEQ ID NO: 145.

[0270] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0624 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47CDR3 .

[0271] In some embodiments, the bispecific antibody EIP0624 comprises VH1 having the amino acid sequence of SEQ ID NO: 15 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0624 comprises H1 having the amino acid sequence of SEQ ID NO: 150 and L1 having the amino acid sequence of SEQ ID NO: 149.

[0272] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0486 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 44 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0273] In some embodiments, the bispecific antibody EIP0486 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody EIP0486 comprises H1 having the amino acid sequence of SEQ ID NO: 154 and L1 having the amino acid sequence of SEQ ID NO: 153.

[0274] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0626 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 39 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0275] In some embodiments, the bispecific antibody EIP0626 comprises VH1 having the amino acid sequence of SEQ ID NO: 16 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0626 comprises H1 having the amino acid sequence of SEQ ID NO: 158 and L1 having the amino acid sequence of SEQ ID NO: 157.

[0276] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0483 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 30. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0277] In some embodiments, the bispecific antibody EIP0483 comprises VH1 having the amino acid sequence of SEQ ID NO: 17 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0483 comprises H1 having the amino acid sequence of SEQ ID NO: 162 and L1 having the amino acid sequence of SEQ ID NO: 161.

[0278] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0623 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42.CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0279] In some embodiments, the bispecific antibody EIP0623 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0623 comprises H1 having the amino acid sequence of SEQ ID NO: 166 and L1 having the amino acid sequence of SEQ ID NO: 165.

[0280] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0621 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0281] In some embodiments, the bispecific antibody EIP0621 comprises VH1 having the amino acid sequence of SEQ ID NO: 19 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0621 comprises H1 having the amino acid sequence of SEQ ID NO: 170 and L1 having the amino acid sequence of SEQ ID NO: 169.

[0282] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0625 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0283] In some embodiments, the bispecific antibody EIP0625 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0625 comprises H1 having the amino acid sequence of SEQ ID NO: 174 and L1 having the amino acid sequence of SEQ ID NO: 173.

[0284] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0622 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0285] In some embodiments, the bispecific antibody EIP0622 comprises VH1 having the amino acid sequence of SEQ ID NO: 20 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0622 comprises H1 having the amino acid sequence of SEQ ID NO: 178 and L1 having the amino acid sequence of SEQ ID NO: 177.

[0286] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0525 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 31. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0287] In some embodiments, the bispecific antibody EIP0525 comprises VH1 having the amino acid sequence of SEQ ID NO: 21 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0525 comprises H1 having the amino acid sequence of SEQ ID NO: 182 and L1 having the amino acid sequence of SEQ ID NO: 181.

[0288] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibodies of this disclosure include EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629.

[0289] Bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1... H1 The amino acid at position 170 (EU number) of CL1 is S, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; the amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acid at position CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3 The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0290] Bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513, and EIP0629 have a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, wherein VH2 comprises the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0291] Bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513 and EIP0629 comprise VH2 having the amino acid sequence of SEQ ID NO: 2 and VL2 having the amino acid sequence of SEQ ID NO: 1.

[0292] Bispecific antibodies EIP0630, EIP0540, EIP0628, EIP0542, EIP0627, EIP0515, EIP0477, EIP0541, EIP0513 and EIP0629 contain H2 with the amino acid sequence of SEQ ID NO: 140 and L2 with the amino acid sequence of SEQ ID NO: 139.

[0293] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0630 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 46 CDR3 .

[0294] In some embodiments, the bispecific antibody EIP0630 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the bispecific antibody EIP0630 comprises H1 having the amino acid sequence of SEQ ID NO: 186 and L1 having the amino acid sequence of SEQ ID NO: 185.

[0295] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0540 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0296] In some embodiments, the bispecific antibody EIP0540 comprises VH1 having the amino acid sequence of SEQ ID NO: 15 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0540 comprises H1 having the amino acid sequence of SEQ ID NO: 190 and L1 having the amino acid sequence of SEQ ID NO: 189.

[0297] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0628 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 44 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0298] In some embodiments, the bispecific antibody EIP0628 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, the bispecific antibody EIP0628 comprises H1 having the amino acid sequence of SEQ ID NO: 194 and L1 having the amino acid sequence of SEQ ID NO: 193.

[0299] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0542 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 39 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0300] In some embodiments, the bispecific antibody EIP0542 comprises VH1 having the amino acid sequence of SEQ ID NO: 16 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0542 comprises H1 having the amino acid sequence of SEQ ID NO: 198 and L1 having the amino acid sequence of SEQ ID NO: 197.

[0301] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0627 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 30. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0302] In some embodiments, the bispecific antibody EIP0627 comprises VH1 having the amino acid sequence of SEQ ID NO: 17 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0627 comprises H1 having the amino acid sequence of SEQ ID NO: 202 and L1 having the amino acid sequence of SEQ ID NO: 201.

[0303] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0515 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47CDR3 .

[0304] In some embodiments, the bispecific antibody EIP0515 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0515 comprises H1 having the amino acid sequence of SEQ ID NO: 206 and L1 having the amino acid sequence of SEQ ID NO: 205.

[0305] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0477 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0306] In some embodiments, the bispecific antibody EIP0477 comprises VH1 having the amino acid sequence of SEQ ID NO: 19 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0477 comprises H1 having the amino acid sequence of SEQ ID NO: 210 and L1 having the amino acid sequence of SEQ ID NO: 209.

[0307] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0541 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0308] In some embodiments, the bispecific antibody EIP0541 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0541 comprises H1 having the amino acid sequence of SEQ ID NO: 214 and L1 having the amino acid sequence of SEQ ID NO: 213.

[0309] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0513 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0310] In some embodiments, the bispecific antibody EIP0513 comprises VH1 having the amino acid sequence of SEQ ID NO: 20 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, the bispecific antibody EIP0513 comprises H1 having the amino acid sequence of SEQ ID NO: 218 and L1 having the amino acid sequence of SEQ ID NO: 217.

[0311] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0629 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 31. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42.CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0312] In some embodiments, the bispecific antibody EIP0629 comprises VH1 having the amino acid sequence of SEQ ID NO: 21 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the bispecific antibody EIP0629 comprises H1 having the amino acid sequence of SEQ ID NO: 222 and L1 having the amino acid sequence of SEQ ID NO: 221.

[0313] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure comprises the CH1 H3 and / or CH2 H3 The amino acid at position 446 (EU number) and / or position 447 (EU number). In some embodiments, the exemplary bispecific antibody comprises the CH1. H3 and / or CH2 H3 The amino acid at position 446 (EU number) is G. In some embodiments, the exemplary bispecific antibody comprises CH1. H3 and / or CH2 H3 The bispecific antibody also contains the amino acid at position 446 (EU number), and further comprises the CH1 amino acid. H3 and / or CH2 H3 The amino acid at position 447 (EU number). In some embodiments, the exemplary bispecific antibody comprises the CH1. H3 and / or CH2 H3 The amino acid at position 447 (EU number) is K.

[0314] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure comprises the CH1 H3 or CH2 H3 The amino acid at position 446 (EU number). In some embodiments, the bispecific antibody comprises CH1. H3 and CH2 H3 The amino acid at position 446 (EU number). In some embodiments, the bispecific antibody comprises CH1. H3The amino acid at position 446 (EU number). In some embodiments, the bispecific antibody comprises the CH2. H3 The amino acid at position 446 (EU number).

[0315] In some embodiments, the bispecific antibody comprises CH1. H3 and / or CH2 H3 The amino acid at position 446 (EU number) is G. In some embodiments, the bispecific antibody comprises CH1. H3 The amino acid at position 446 (EU number) is G. In some embodiments, the bispecific antibody comprises CH2. H3 The amino acid at position 446 (EU number) is G. In some embodiments, the bispecific antibody comprises CH1. H3 and CH2 H3 The amino acid at position 446 (EU number), CH1 H3 and CH2 H3 The amino acid at position 446 (EU number) is G.

[0316] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure comprises the CH1 H3 and / or CH2 H3 The amino acid at position 447 (EU number). In some embodiments, the bispecific antibody comprises CH1. H3 or CH2 H3 The amino acid at position 447 (EU number). In some embodiments, the bispecific antibody comprises CH1. H3 and CH2 H3 The amino acid at position 447 (EU number). In some embodiments, the bispecific antibody comprises CH1. H3 The amino acid at position 447 (EU number). In some embodiments, the bispecific antibody comprises the CH2. H3 The amino acid at position 447 (EU number).

[0317] In some embodiments, the CD3ε x ULBP2 / 5 / 6 bispecific antibody comprises the CH1 H3 and / or CH2 H3The amino acid at position 447 (EU number) is K. In some embodiments, the bispecific antibody comprises CH1. H3 The amino acid at position 447 (EU number) is K. In some embodiments, the bispecific antibody comprises CH2. H3 The amino acid at position 447 (EU number) is K.

[0318] Fusion peptide

[0319] This article provides an antibody (e.g., a monospecific antibody or a bispecific antibody) having a fusion peptide fused to the N-terminus or C-terminus of a first heavy chain polypeptide or a second heavy chain polypeptide.

[0320] Key to the initial T cell response is the ability of T cells to detect foreign and mutated proteins via their T cell receptors. This response, commonly referred to as Signal 1 (S1) of T cell activation, occurs when the T cell receptor binds to a cell displaying a foreign or mutated protein fragment or antigen in a specific protein complex called major histocompatibility complex I (MHCI). Activation of the T cell receptor itself is an activation and autoregulation of the T cell. Strong binding of the TCR to the MHCI complex leads to chronic activation of the TCR. This form of signaling is associated with T cells that respond to self-antigens. T cells are programmed to inactivate upon experiencing this form of activation. T cells with a weaker but sufficiently activated TCR undergo acute signal transduction and have the potential to remain active and differentiate into memory T cells. This is becoming an important consideration in the design of T cell therapeutics.

[0321] T cell cytokine activation, commonly referred to as signal 3, is important in the transition of T cells from a non-dividing state to a rapidly dividing state or from one phenotypic state to another. T cell cytokine receptors bind to cytokines produced by both immune and non-immune cells, and depending on the cytokines and the state of the T cell upon receiving cytokine signals, can induce cell proliferation, maintain viability, or induce T cell differentiation into specific cellular states suitable for sustained activation or inactivation after infection.

[0322] One example is the transformation that naive cells undergo through cytokines, which can induce the proliferation of naive T cells and promote their differentiation into memory T cells. Exemplary cytokines include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21.

[0323] Activation of co-stimulatory receptors, known as signal 2, provides the background-specific intercellular enhancement for T activation. The most recognized form of co-stimulation occurs when T cells interact with activated antigen-presenting cells via the T cell co-stimulatory receptor CD28 with CD80 and CD86 ligands present on APCs. These interactions can “elicit” specific T cells carrying T cell receptors that are responsive to pathogens or oncoproteins.

[0324] Less understood are the co-stimulatory responses induced at sites of infection and malignancy. This includes co-stimulation via CD2 and NKG2D receptors, which are responsive to ligands such as CD58 and UL16-binding proteins (e.g., ULBP2 / 5 / 6) induced in immune cells and epithelial cells after viral infection. These signals not only provide enhancement of T cell activation but also demonstrate that the lethal effector activity of T cells is targeted at single-cell precision. Although many co-stimulatory receptors have been identified, the specific contextual importance of each receptor and the impact of multiple co-stimulatory receptors signaling simultaneously remain largely unknown. This is an area that greatly advances our understanding of T cell biology and opens up possibilities for the development of novel tumor-targeting T cell therapies.

[0325] Co-stimulatory ligands include, but are not limited to, CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, or ICOSLG.

[0326] CD58 is superior to other costimulatory ligands because it is the primary costimulatory pathway available at the tumor site. This is because tumor-infiltrating T lymphocytes often lose the expression of other costimulatory receptors such as CD28, or because tumor cells do not adequately activate T cells due to their low immunogenicity, thus limiting the potential of inducible costimulatory receptors such as 41BB.

[0327] As previously described, the anti-CD3ε antibody of this disclosure induces varying levels of T cell receptor activation, leading to changes in T cell viability and cytokine production. Therefore, the fusion of the co-stimulatory ligand CD58 with the anti-CD3ε bispecific antibody provides integrated co-stimulatory T cell activation to achieve optimal T cell activation.

[0328] In some embodiments, the bispecific antibody has a peptide fused to the N-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a polypeptide fused to the N-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the second heavy chain polypeptide (H2). Exemplary peptides include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, or portions thereof. Exemplary peptides include, but are not limited to, CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG, or portions thereof. Exemplary peptide sequences fused to the bispecific antibody include, but are not limited to, those listed in Tables 9 and 10.

[0329] Table 9. Exemplary fusion peptide sequences

[0330] Table 10. Exemplary fusion peptide sequences

[0331] In some embodiments, the peptide is directly fused to the bispecific antibody. In some embodiments, the peptide is indirectly fused via a linker. In some embodiments, the bispecific antibody fused to the peptide includes a linker sequence. Exemplary linker sequences include, but are not limited to, those listed in Tables 11 and 12.

[0332] Table 11. Exemplary connector sequences

[0333] Table 12. Exemplary connector sequences

[0334] Bispecific antibodies containing fusion peptides

[0335] In some embodiments, this disclosure provides a bispecific antibody (e.g., a bispecific antibody targeting ULBP2 and CD3) fused to a peptide (e.g., the co-stimulatory CD58 peptide described herein). In some embodiments, the bispecific antibody described herein (e.g., a bispecific antibody targeting ULBP2 and CD3) comprises a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the C-terminus of the heavy chain of the CD3 targeting arm. In some embodiments, the bispecific antibody described herein (e.g., a bispecific antibody targeting ULBP2 and CD3) comprises a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the N-terminus of the heavy chain of the CD3 targeting arm. In some embodiments, the bispecific antibody described herein (e.g., a bispecific antibody targeting ULBP2 and CD3) comprises a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the C-terminus of the light chain of the CD3 targeting arm. In some embodiments, the bispecific antibodies described herein (e.g., bispecific antibodies targeting ULBP2 and CD3) comprise a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the N-terminus of the light chain of the CD3 targeting arm. In some embodiments, the bispecific antibodies described herein (e.g., bispecific antibodies targeting ULBP2 and CD3) comprise a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the C-terminus of the heavy chain of the ULBP2 targeting arm. In some embodiments, the bispecific antibodies described herein (e.g., bispecific antibodies targeting ULBP2 and CD3) comprise a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the N-terminus of the heavy chain of the ULBP2 targeting arm. In some embodiments, the bispecific antibodies described herein (e.g., bispecific antibodies targeting ULBP2 and CD3) comprise a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the C-terminus of the light chain of the ULBP2 targeting arm. In some embodiments, the bispecific antibodies described herein (e.g., bispecific antibodies targeting ULBP2 and CD3) comprise a peptide (e.g., the co-stimulatory CD58 peptide described herein) fused to the N-terminus of the light chain of the ULBP2 targeting arm. Table 13 illustrates exemplary bispecific antibodies targeting ULBP2 and CD3 fused with the CD58 peptide.

[0336] Table 13. Exemplary bispecific antibodies fused with peptides

[0337] (When applicable:) italics -Variable region, italics and underlines – Kabat CDR definition italics and bold –Chothia CDR definition underline – CD58 or IL-7 fusion underline – Cysteine ​​mutation)

[0338] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0820.

[0339] In some embodiments, the bispecific antibody EIP0820 further comprises the following amino acid substitutions in H1 and L1: the amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; the amino acid at position CH1 H1 The amino acid at position 147 (EU number) of CL1 is K, and the amino acid at position 131 (EU number) of CH1 is D; H1 The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; the amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; the amino acids at positions 234, 235, and 237 (EU number) of H1H are A; the amino acid at position CH1 H3 The amino acid at position 349 (EU number) is C; the CH1 H3 The amino acid at position 366 (EU number) is S; the CH1 H3 The amino acid at position 368 (EU number) is A; the CH1 H3 The amino acid at position 407 (EU number) is V; and the CH1 H3The amino acid at position 447 (EU number) is missing, where the number is based on the H1 amino acid sequence of SEQ NO: 439 and the L1 amino acid sequence of SEQ NO: 438.

[0340] In some embodiments, the bispecific antibody EIP0820 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0341] In some embodiments, the bispecific antibody EIP0820 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0342] In some embodiments, the bispecific antibody EIP0820 comprises VH2 containing the amino acid sequence of SEQ ID NO: 629 and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0343] In some embodiments, the bispecific antibody EIP0820 comprises H2 containing the amino acid sequence of SEQ ID NO: 621 and L2 containing the amino acid sequence of SEQ ID NO: 67.

[0344] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0820 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 30. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0345] In some embodiments, the bispecific antibody EIP0820 comprises VH1 having the amino acid sequence of SEQ ID NO: 17 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0820 comprises H1 having the amino acid sequence of SEQ ID NO: 622 and L1 having the amino acid sequence of SEQ ID NO: 69.

[0346] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0359.

[0347] In some embodiments, the bispecific antibody EIP0359 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0348] In some embodiments, the bispecific antibody EIP0359 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0349] In some embodiments, the bispecific antibody EIP0359 comprises H2 containing the amino acid sequence of SEQ ID NO: 224 and L2 containing the amino acid sequence of SEQ ID NO: 223.

[0350] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0359 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 32.CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0351] In some embodiments, the bispecific antibody EIP0359 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0359 comprises H1 having the amino acid sequence of SEQ ID NO: 226 and L1 having the amino acid sequence of SEQ ID NO: 225.

[0352] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0566.

[0353] In some embodiments, the bispecific antibody EIP0566 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0566 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0566 comprises H2 containing the amino acid sequence of SEQ ID NO: 228 and L2 containing the amino acid sequence of SEQ ID NO: 227.

[0354] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0566 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 32. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0566 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0566 comprises H1 having the amino acid sequence of SEQ ID NO: 230 and L1 having the amino acid sequence of SEQ ID NO: 229.

[0355] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0634.

[0356] In some embodiments, the bispecific antibody EIP0634 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3In some embodiments, the bispecific antibody EIP0634 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0634 comprises H2 containing the amino acid sequence of SEQ ID NO: 232 and L2 containing the amino acid sequence of SEQ ID NO: 231.

[0357] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0634 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 32. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0634 comprises VH1 having the amino acid sequence of SEQ ID NO: 15 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0634 comprises H1 having the amino acid sequence of SEQ ID NO: 234 and L1 having the amino acid sequence of SEQ ID NO: 233.

[0358] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0562.

[0359] In some embodiments, the bispecific antibody EIP0562 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0562 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0562 comprises H2 containing the amino acid sequence of SEQ ID NO: 236 and L2 containing the amino acid sequence of SEQ ID NO: 235.

[0360] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0562 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 32. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 44 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0562 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0562 comprises H1 having the amino acid sequence of SEQ ID NO: 238 and L1 having the amino acid sequence of SEQ ID NO: 237.

[0361] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0636.

[0362] In some embodiments, the bispecific antibody EIP0636 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0636 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0636 comprises H2 containing the amino acid sequence of SEQ ID NO: 240 and L2 containing the amino acid sequence of SEQ ID NO: 239.

[0363] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0636 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 32. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 39 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0636 comprises VH1 having the amino acid sequence of SEQ ID NO: 16 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0636 comprises H1 having the amino acid sequence of SEQ ID NO: 242 and L1 having the amino acid sequence of SEQ ID NO: 241.

[0364] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0561.

[0365] In some embodiments, the bispecific antibody EIP0561 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0561 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0561 comprises H2 containing the amino acid sequence of SEQ ID NO: 244 and L2 containing the amino acid sequence of SEQ ID NO: 243.

[0366] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0561 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 30. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3In some embodiments, the bispecific antibody EIP0561 comprises VH1 having the amino acid sequence of SEQ ID NO: 17 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0561 comprises H1 having the amino acid sequence of SEQ ID NO: 246 and L1 having the amino acid sequence of SEQ ID NO: 245.

[0367] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0633.

[0368] In some embodiments, the bispecific antibody EIP0633 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0633 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0633 comprises H2 containing the amino acid sequence of SEQ ID NO: 248 and L2 containing the amino acid sequence of SEQ ID NO: 247.

[0369] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0633 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0633 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0633 comprises H1 having the amino acid sequence of SEQ ID NO: 250 and L1 having the amino acid sequence of SEQ ID NO: 249.

[0370] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0631.

[0371] In some embodiments, the bispecific antibody EIP0631 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0631 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0631 comprises H2 containing the amino acid sequence of SEQ ID NO: 252 and L2 containing the amino acid sequence of SEQ ID NO: 251.

[0372] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0631 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0631 comprises VH1 having the amino acid sequence of SEQ ID NO: 19 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0631 comprises H1 having the amino acid sequence of SEQ ID NO: 254 and L1 having the amino acid sequence of SEQ ID NO: 253.

[0373] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0635.

[0374] In some embodiments, the bispecific antibody EIP0635 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3In some embodiments, the bispecific antibody EIP0635 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0635 comprises H2 containing the amino acid sequence of SEQ ID NO: 256 and L2 containing the amino acid sequence of SEQ ID NO: 255.

[0375] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0635 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0635 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0635 comprises H1 having the amino acid sequence of SEQ ID NO: 258 and L1 having the amino acid sequence of SEQ ID NO: 257.

[0376] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0632.

[0377] In some embodiments, the bispecific antibody EIP0632 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0632 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0632 comprises H2 containing the amino acid sequence of SEQ ID NO: 260 and L2 containing the amino acid sequence of SEQ ID NO: 259.

[0378] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0632 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0632 comprises VH1 having the amino acid sequence of SEQ ID NO: 20 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0632 comprises H1 having the amino acid sequence of SEQ ID NO: 262 and L1 having the amino acid sequence of SEQ ID NO: 261.

[0379] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0565.

[0380] In some embodiments, the bispecific antibody EIP0565 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0565 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0565 comprises H2 containing the amino acid sequence of SEQ ID NO: 264 and L2 containing the amino acid sequence of SEQ ID NO: 263.

[0381] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0565 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 31. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0565 comprises VH1 having the amino acid sequence of SEQ ID NO: 21 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0565 comprises H1 having the amino acid sequence of SEQ ID NO: 266 and L1 having the amino acid sequence of SEQ ID NO: 265.

[0382] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0599.

[0383] In some embodiments, the bispecific antibody EIP0599 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0599 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0599 comprises H2 containing the amino acid sequence of SEQ ID NO: 267 and L2 containing the amino acid sequence of SEQ ID NO: 330.

[0384] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0599 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 32. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3In some embodiments, the bispecific antibody EIP0599 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0599 comprises H1 having the amino acid sequence of SEQ ID NO: 269 and L1 having the amino acid sequence of SEQ ID NO: 268.

[0385] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0640.

[0386] In some embodiments, the bispecific antibody EIP0640 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0640 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0640 comprises H2 containing the amino acid sequence of SEQ ID NO: 271 and L2 containing the amino acid sequence of SEQ ID NO: 270.

[0387] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0640 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 46 CDR3 In some embodiments, the bispecific antibody EIP0640 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0640 comprises H1 having the amino acid sequence of SEQ ID NO: 273 and L1 having the amino acid sequence of SEQ ID NO: 272.

[0388] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0567.

[0389] In some embodiments, the bispecific antibody EIP0567 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0567 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0567 comprises H2 containing the amino acid sequence of SEQ ID NO: 275 and L2 containing the amino acid sequence of SEQ ID NO: 274.

[0390] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0567 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0567 comprises VH1 having the amino acid sequence of SEQ ID NO: 15 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0567 comprises H1 having the amino acid sequence of SEQ ID NO: 277 and L1 having the amino acid sequence of SEQ ID NO: 276.

[0391] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0638.

[0392] In some embodiments, the bispecific antibody EIP0638 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3In some embodiments, the bispecific antibody EIP0638 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0638 comprises H2 containing the amino acid sequence of SEQ ID NO: 279 and L2 containing the amino acid sequence of SEQ ID NO: 278.

[0393] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0638 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 44 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0638 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 27. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0638 comprises H1 having the amino acid sequence of SEQ ID NO: 281 and L1 having the amino acid sequence of SEQ ID NO: 280.

[0394] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0568.

[0395] In some embodiments, the bispecific antibody EIP0568 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0568 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0568 comprises H2 containing the amino acid sequence of SEQ ID NO: 283 and L2 containing the amino acid sequence of SEQ ID NO: 282.

[0396] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0568 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 39 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0568 comprises VH1 having the amino acid sequence of SEQ ID NO: 16 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0568 comprises H1 having the amino acid sequence of SEQ ID NO: 285 and L1 having the amino acid sequence of SEQ ID NO: 284.

[0397] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0637.

[0398] In some embodiments, the bispecific antibody EIP0637 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0637 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0637 comprises H2 containing the amino acid sequence of SEQ ID NO: 287 and L2 containing the amino acid sequence of SEQ ID NO: 286.

[0399] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0637 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 30. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0637 comprises VH1 having the amino acid sequence of SEQ ID NO: 17 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0637 comprises H1 having the amino acid sequence of SEQ ID NO: 289 and L1 having the amino acid sequence of SEQ ID NO: 288.

[0400] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0564.

[0401] In some embodiments, the bispecific antibody EIP0564 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0564 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0564 comprises H2 containing the amino acid sequence of SEQ ID NO: 291 and L2 containing the amino acid sequence of SEQ ID NO: 290.

[0402] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0564 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3In some embodiments, the bispecific antibody EIP0564 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0564 comprises H1 having the amino acid sequence of SEQ ID NO: 293 and L1 having the amino acid sequence of SEQ ID NO: 292.

[0403] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0560.

[0404] In some embodiments, the bispecific antibody EIP0560 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0560 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0560 comprises H2 containing the amino acid sequence of SEQ ID NO: 295 and L2 containing the amino acid sequence of SEQ ID NO: 294.

[0405] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0560 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0560 comprises VH1 having the amino acid sequence of SEQ ID NO: 19 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0560 comprises H1 having the amino acid sequence of SEQ ID NO: 297 and L1 having the amino acid sequence of SEQ ID NO: 296.

[0406] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0662.

[0407] In some embodiments, the bispecific antibody EIP0662 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0662 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0662 comprises H2 containing the amino acid sequence of SEQ ID NO: 299 and L2 containing the amino acid sequence of SEQ ID NO: 298.

[0408] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0662 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0662 comprises VH1 having the amino acid sequence of SEQ ID NO: 18 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0662 comprises H1 having the amino acid sequence of SEQ ID NO: 301 and L1 having the amino acid sequence of SEQ ID NO: 300.

[0409] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0563.

[0410] In some embodiments, the bispecific antibody EIP0563 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3In some embodiments, the bispecific antibody EIP0563 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0563 comprises H2 containing the amino acid sequence of SEQ ID NO: 303 and L2 containing the amino acid sequence of SEQ ID NO: 302.

[0411] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0563 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 In some embodiments, the bispecific antibody EIP0563 comprises VH1 having the amino acid sequence of SEQ ID NO: 20 and VL1 having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0563 comprises H1 having the amino acid sequence of SEQ ID NO: 305 and L1 having the amino acid sequence of SEQ ID NO: 304.

[0412] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0639.

[0413] In some embodiments, the bispecific antibody EIP0639 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0639 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0639 comprises H2 containing the amino acid sequence of SEQ ID NO: 307 and L2 containing the amino acid sequence of SEQ ID NO: 306.

[0414] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0639 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 31. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0639 comprises VH1 having the amino acid sequence of SEQ ID NO: 21 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0639 comprises H1 having the amino acid sequence of SEQ ID NO: 309 and L1 having the amino acid sequence of SEQ ID NO: 308.

[0415] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0547.

[0416] In some embodiments, the bispecific antibody EIP0547 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0547 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0547 comprises H2 containing the amino acid sequence of SEQ ID NO: 311 and L2 containing the amino acid sequence of SEQ ID NO: 310.

[0417] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0547 contains a first antigen-binding domain that binds to CD3ε, which includes: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 In some embodiments, the bispecific antibody EIP0547 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the heavy chain (H1) targeting CD3. Therefore, in some embodiments, the bispecific antibody EIP0547 comprises H1 having the amino acid sequence of SEQ ID NO: 313 and L1 having the amino acid sequence of SEQ ID NO: 312.

[0418] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0641.

[0419] In some embodiments, the bispecific antibody EIP0641 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0641 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0641 comprises H2 containing the amino acid sequence of SEQ ID NO: 315 and L2 containing the amino acid sequence of SEQ ID NO: 314.

[0420] In addition to the VH2, VL2, H2, and L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0641 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3In some embodiments, the bispecific antibody EIP0641 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 49 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0641 comprises H1 having the amino acid sequence of SEQ ID NO: 317 and L1 having the amino acid sequence of SEQ ID NO: 316.

[0421] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0360.

[0422] In some embodiments, the bispecific antibody EIP0360 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 In some embodiments, the bispecific antibody EIP0360 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bispecific antibody EIP0360 comprises H2 containing the amino acid sequence of SEQ ID NO: 319 and L2 containing the amino acid sequence of SEQ ID NO: 318.

[0423] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0360 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0424] In some embodiments, the bispecific antibody EIP0360 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, a CD58 peptide comprising the amino acid sequence of SEQ ID NO: 50 is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0360 comprises H1 having the amino acid sequence of SEQ ID NO: 321 and L1 having the amino acid sequence of SEQ ID NO: 320.

[0425] In some embodiments, the exemplary CD3ε x ULBP2 / 5 / 6 bispecific antibody of this disclosure includes EIP0363.

[0426] In some embodiments, the bispecific antibody EIP0363 has a second antigen-binding domain that binds to ULBP2 / 5 / 6, comprising: VH2, said VH2 comprising the amino acid sequence having SEQ ID NO: 5. CDR1 VH2 with the amino acid sequence SEQ ID NO: 7 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 9 CDR3 ; and VL2, wherein VL2 comprises the amino acid sequence having SEQ ID NO: 10. CDR1 VL2 with the amino acid sequence of SEQ ID NO: 11 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 12 CDR3 .

[0427] In some embodiments, the bispecific antibody EIP0363 comprises VH2 containing the amino acid sequence of SEQ ID NO: 2 and VL2 containing the amino acid sequence of SEQ ID NO: 1.

[0428] In some embodiments, the bispecific antibody EIP0363 comprises H2 containing the amino acid sequence of SEQ ID NO: 323 and L2 containing the amino acid sequence of SEQ ID NO: 322.

[0429] In addition to the VH2, VL2, H2, L2 sequences and amino acid substitutions shown above, the bispecific antibody EIP0363 contains a first antigen-binding domain that binds to CD3ε, comprising: VH1, wherein the VH1 contains the amino acid sequence having SEQ ID NO: 32. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, wherein VL1 comprises the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0430] In some embodiments, the bispecific antibody EIP0363 comprises VH1 having the amino acid sequence of SEQ ID NO: 13 and VL1 having the amino acid sequence of SEQ ID NO: 22. In some embodiments, the IL-7 peptide is fused to the C-terminus of the CD3-targeting heavy chain (H1). Therefore, in some embodiments, the bispecific antibody EIP0363 comprises H1 having the amino acid sequence of SEQ ID NO: 325 and L1 having the amino acid sequence of SEQ ID NO: 324.

[0431] How to use

[0432] Any bispecific antibody disclosed herein can be used in a treatment method. In one aspect, a bispecific antibody can be used as a drug. In a further aspect, the antibody can be used to treat or delay the progression of cell proliferation disorders (e.g., cancer). In some embodiments, an antibody for a treatment method is provided. In some embodiments, this disclosure provides an antibody for a method of treating an individual with a cell proliferation disorder, the method comprising administering an effective amount of the antibody to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as the additional therapeutic agent described below, to the individual. In other embodiments, this disclosure provides an antibody for enhancing immune function in an individual with a cell proliferation disorder. In some embodiments, this disclosure provides an antibody for a method of enhancing immune function in an individual with a cell proliferation disorder, the method comprising administering an effective amount of the antibody to the individual to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) effector cell populations, reduce target cell populations, and / or kill target cells (e.g., target tumor cells). An “individual” according to any of the above embodiments can be a human being.

[0433] In another aspect, this disclosure provides the use of the antibodies of this disclosure in the manufacture or preparation of a pharmaceutical. In one embodiment, the pharmaceutical is used to treat a cell proliferation disorder (e.g., cancer, such as squamous cell carcinoma, adenocarcinoma, or metastatic cancer). In another embodiment, the pharmaceutical is used as a method of treating a cell proliferation disorder or an autoimmune disorder, the method comprising administering an effective amount of the pharmaceutical to an individual suffering from a cell proliferation disorder or an autoimmune disorder. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as the additional therapeutic agent described below, to the individual. In another embodiment, the pharmaceutical is used to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) effector cell populations, reduce target cell populations (e.g., cells expressing ULBP2), and / or kill target cells (e.g., target tumor cells) in the individual. In another embodiment, the drug is used in a method of enhancing immune function in an individual suffering from a cell proliferation disorder or an autoimmune disorder, the method comprising administering an effective amount of the drug to the individual to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) the effector cell population, reduce the target cell population (e.g., cells expressing ULBP2), and / or kill target cells (e.g., target tumor cells). The “individual” according to any of the above embodiments can be a human being.

[0434] In another aspect, this disclosure provides a method for treating cell proliferation disorders (e.g., cancer). In one embodiment, the method includes administering an effective amount of a bispecific antibody to an individual suffering from such a cell proliferation disorder. In one such embodiment, the method further includes administering an effective amount of at least one additional therapeutic agent to the individual, such as the additional therapeutic agent described below. An "individual" according to any of the above embodiments can be a human being. In another aspect, this disclosure provides a method for enhancing immune function in an individual suffering from a cell proliferation disorder or an autoimmune disorder. In one embodiment, the method includes administering an effective amount of a bispecific antibody to the individual to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) the effector cell population, reduce the target cell population (e.g., cells expressing ULBP2), and / or kill target cells (e.g., target tumor cells).

[0435] The method further includes comparing the ULBP2 expression level in the cancer sample with the ULBP2 expression level in healthy / control / reference tissues to determine whether the ULBP2 expression level in the cancer sample is higher. If the ULBP2 expression level in the cancer sample is higher, the subject receives a ULBP2-targeted therapeutic agent. In some embodiments, the ULBP2-targeted therapeutic agent is one or more antibodies provided herein (e.g., a bispecific antibody targeting ULBP2 and CD3). In some embodiments, any method for measuring ULBP2 levels can be used. In some embodiments, this is any amount greater than a negative amount in a staining assay. In some embodiments, this is any amount higher than the level present in surrounding healthy / control / reference tissues or in the corresponding tissue from a healthy / control / reference subject. In some embodiments, a high ULBP2 expression level is defined compared to the ULBP2 expression level in a non-cancer sample.

[0436] In some embodiments, the ULBP2 expression level in the cancer samples is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0% higher than the ULBP2 expression level in the non-cancer samples. In some embodiments, the ULBP2 expression level in the cancer samples is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher than the ULBP2 expression level in the non-cancer samples. In some embodiments, the ULBP2 expression level in the cancer samples is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% higher than the ULBP2 expression level in the non-cancer samples. In some embodiments, the ULBP2 expression level in the cancer samples is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than the ULBP2 expression level in the non-cancer samples.

[0437] The method further includes comparing the expression levels of TROP2, NECTIN4, KRT5, KRT6A, SERPINB3, SERPINB4, and / or HER2 in cancer samples with those in healthy / control / reference tissues to determine whether the expression levels of TROP2, NECTIN4, KRT5, KRT6A, SERPINB3, SERPINB4, and / or HER2 in the cancer samples are higher. If the expression levels of TROP2, NECTIN4, KRT5, KRT6A, SERPINB3, SERPINB4, and / or HER2 in the cancer samples are higher, the subject receives an ULBP2-targeted therapeutic agent. In some embodiments, the ULBP2-targeted therapeutic agent is one or more antibodies provided herein. In some embodiments, any method for measuring TROP2, NECTIN4, KRT5, KRT6A, SERPINB3, SERPINB4, and / or HER2 levels can be used.

[0438] In some embodiments, the healthy / control / reference sample is a sample from normal tissue. In some embodiments, the normal tissue is tissue adjacent to the cancer in the subject. For example, when expression is determined by staining, the protein level is higher than a level that would be considered “negative” by a particular assay. Exemplary assays include, but are not limited to, RNA-based assays, FACS, and IHC, each with its appropriate positive and negative result levels. Any method for detecting protein levels in a sample is contemplated. A person skilled in the art can select an appropriate method based on the type of sample being analyzed and the identity and quantity of the protein being detected. Non-limiting exemplary such methods include immunohistochemistry, ELISA, Western blotting, multiplex analyte detection (using, for example, Luminex technology), mass spectrometry, etc. Similarly, any method for detecting mRNA levels in a sample is contemplated. A person skilled in the art can select an appropriate method based on the type of sample being analyzed and the identity and quantity of the mRNA being detected. Non-limiting exemplary such methods include RT-PCR, quantitative RT-PCR, and microarray-based methods, etc.

[0439] ULBP2-positive cancers according to any of the above embodiments can be basal squamous bladder cancer or lung cancer. In some embodiments, ULBP2-positive cancers are cancers with an anti-ULBP2 immunohistochemical (IHC) or in situ hybridization (ISH) score greater than "0" (which corresponds to very weak or no staining in >90% of tumor cells). In another embodiment, ULBP2-positive cancers express ULBP2 at levels of 1+, 2+, or 3+. In some embodiments, ULBP2-positive cancers are cancers that express ULBP2 according to a reverse transcriptase PCR (RT-PCR) assay for detecting ULBP2 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR.

[0440] In another aspect, this disclosure provides pharmaceutical formulations comprising any bispecific antibody provided herein (e.g., bispecific antibodies for any of the above-described treatment methods). In one embodiment, the pharmaceutical formulation comprises any bispecific antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any bispecific antibody provided herein and at least one additional therapeutic agent, such as the additional therapeutic agent described herein.

[0441] The antibodies disclosed herein can be used alone or in combination with other agents for therapy. For example, the antibodies of this disclosure can be administered co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, growth inhibitor, cytotoxic agent, agent used in radiotherapy, anti-angiogenic agent, apoptotic agent, anti-microtubule agent, or other agent, such as epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva™), platelet-derived growth factor inhibitors (e.g., Gleevec™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferon, cytokines, or antibodies other than the anti-CD3 antibodies of this disclosure (e.g., capillary antibodies). Antibodies targeting one or more of the following: ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMAVEGF or VEGF receptor, TRAIL / Apo2, PD-1 (e.g., nivolumab, pembrolizumab, cimipril), PD-L1 (atezolizumab, avelumab, durvalumab), PD-L2), or another biologically active or organic chemical agent. In some embodiments, this disclosure provides a method in which the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0442] In some respects, the additional therapeutic agent is a checkpoint inhibitor. The term “inhibition” or “inhibitor” includes a reduction in a parameter (e.g., activity) of a given molecule, such as an immune checkpoint inhibitor. For example, the term includes inhibiting the activity of, for example, PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFRβ by at least 5%, 10%, 20%, 30%, 40%, or more. The level of inhibition is not necessarily 100%.

[0443] In some aspects, the checkpoint inhibitor is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor is an anti-PD1 antibody. In some aspects, the PD-1 inhibitor is an anti-PD-1 monoclonal antibody. Exemplary anti-PD-1 monoclonal antibodies include, but are not limited to, cimipril (Libtayo), nivolumab (Opdivo), and pembrolizumab (Keytruda). In some aspects, the checkpoint inhibitor is a PD-L1 inhibitor. Exemplary PD-L1 inhibitors include, but are not limited to, avelumab (Bavencio), durvalumab (Imfinzi), and atezolizumab (Tecentriq).

[0444] The aforementioned combination therapy encompasses both combined administration (where two or more therapeutic agents are contained in the same or separate formulation) and single administration, in which case the administration of the antibody of this disclosure may be performed before, simultaneously with, and / or after the administration of the one or more additional therapeutic agents. In one embodiment, the administration of the bispecific antibody and the administration of the additional therapeutic agent occur within approximately one month, or approximately one week, two weeks, or three weeks, or approximately one day, two days, three days, four days, five days, or six days. The bispecific antibody of this disclosure may also be used in combination with radiotherapy.

[0445] In some aspects, the additional therapeutic agent is a chimeric antigen receptor (CAR) T-cell therapy. In some embodiments, the CAR-T cell therapy specifically binds to CD19. Exemplary CAR-T cell therapies that specifically bind to CD19 include, but are not limited to, BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™ (axicabtagene ciloleucel), ABECMA® (idecabtagene vicleucel), or CARVYKTI™ (ciltacabtagene autoleucel).

[0446] The antibodies (and / or any additional therapeutic agents) disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and may also include intralesional administration if local treatment is required. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies are administered subcutaneously. In some embodiments, anti-CD3ε antibodies administered subcutaneously result in less toxic responses in patients compared to the same anti-CD3ε antibody administered intravenously. Administration may be carried out via any suitable route, such as injection, e.g., intravenous or subcutaneous, depending in part on whether the administration is transient or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulsatile infusion.

[0447] The antibodies disclosed herein will be formulated, dosed, and administered in accordance with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of delivery, the method of administration, the schedule of administration, and other factors known to a medical practitioner. The antibody is not necessarily, but optionally, formulated with one or more agents currently used for the prevention or treatment of the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. They are generally used at the same dose and route of administration as described herein (or approximately 1% to 99% of the dose described herein), or at any dose and route determined empirically / clinically as appropriate.

[0448] For the prevention or treatment of disease, the appropriate dosage of the antibody disclosed herein (whether used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody may be administered to the patient once or appropriately within a series of treatments.

[0449] Generally, whether administered once or multiple times, the therapeutically effective dose of a bispecific antibody administered to a human will be in the range of about 0.01 to about 100 mg / kg of patient body weight. In some embodiments, the antibody used is, for example, administered daily at doses of about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg. In one embodiment, on day 1 of a 21-day cycle, the bispecific antibody described herein is administered to a person at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg. The dose may be administered as a single dose or multiple doses (e.g., 2 or 3 doses), such as by infusion. For repeated administration over several days or longer, treatment typically continues until the desired suppression of disease symptoms is achieved, depending on the condition. An exemplary dose of the antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses can be administered intermittently, for example, once weekly or every three weeks (e.g., giving the patient about 2 to about 20 doses, or for example about 6 doses of the bispecific antibody). An initial higher loading dose can be administered, followed by one or more lower doses. Progression of this therapy can be easily monitored using conventional techniques and assays.

[0450] In some embodiments, the method may further include additional therapies. These additional therapies may be radiotherapy, surgery, chemotherapy, gene therapy, DMA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above therapies. The additional therapies may take the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzyme inhibitors or anti-transfer agents. In some embodiments, the additional therapy is the administration of side effect limiters (e.g., agents designed to reduce the occurrence and / or severity of treatment side effects, such as antinausea agents). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be the separate administration of one or more of the above therapeutic agents.

[0451] It should be understood that the application of the therapeutic entity according to this disclosure will be administered in conjunction with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in all formularies known to medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. Any of the above mixtures may be used for treatments and therapies according to this disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible with and tolerable to the route of administration. For additional information relating to formulations, excipients, and carriers known to medicinal chemists, see also Baldrick P., “Pharmaceutical excipient development: the need for preclinical guidance,” Regul. Toxicol Pharmacol. 32(2):210-8 (2000); Wang W., “Lyophilization and development of solid protein pharmaceuticals,” Int. J. Pharm. 203(1-2):1-60 (2000); Charman WN, “Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts,” J Pharm Sci. 89(8):967-78 (2000); Powell et al., “Compendium of excipients for parenteral formulations,” PDA J Pharm SciTechnol. 52:238-311 (1998), and quotations therein.

[0452] Therapeutic formulations of this disclosure, containing antibodies of the present disclosure, are used to treat or alleviate symptoms associated with cancer, such as, but not limited to, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney and renal pelvis cancer, oral and pharyngeal cancer, endometrial cancer, and / or melanoma. This disclosure also provides methods for treating or alleviating symptoms associated with cancer. Treatment regimens are administered by identifying subjects (e.g., human patients with cancer (or at risk of developing cancer)) using standard methods.

[0453] The efficacy of the treatment is determined in conjunction with any known methods for diagnosing or treating a specific immune-related disorder. Relief of one or more symptoms of the immune-related disorder indicates that the antibody provides clinical benefit.

[0454] Methods for screening antibodies with desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immune-mediated techniques known in the art.

[0455] Antibodies (or fragments thereof) targeting targets such as CD3ε, ULBP2, or combinations thereof can be used in methods known in the art related to the localization and / or quantification of these targets, such as for measuring the levels of these targets in appropriate physiological samples, for diagnostic methods, for protein imaging, etc. In one given embodiment, an antibody containing an antibody-derived antigen-binding domain specifically targeting any of these targets or their derivatives, fragments, analogs, or homologs is used as a pharmacologically active compound (hereinafter referred to as a "therapeutic agent").

[0456] The antibodies disclosed herein can be used to separate specific targets using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. The antibodies (or fragments thereof) disclosed herein can be used diagnostically to monitor protein levels in tissues as part of clinical testing procedures, such as to determine the efficacy of a given treatment regimen. Detection can be facilitated by conjugating (i.e., physically linking) the antibodies to a detectable substance. Examples of detectable substances include various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and jellyfish luminescent protein; examples of suitable radioactive materials include… 125 I, 131 I, 35 S or3 H.

[0457] The antibodies disclosed herein (including polyclonal, monoclonal, humanized, and fully human antibodies) can be used as therapeutic agents. Such agents are commonly used to treat or prevent diseases or pathologies associated with the aberrant expression or activation of a given target in a subject. The antibody preparation (preferably an antibody preparation with high specificity and high affinity for its target antigen) is administered to the subject and is generally effective due to its binding to the target. Administration of the antibody may abolish, inhibit, or interfere with the signal transduction function of the target. Administration of the antibody may abolish, inhibit, or interfere with the binding of the target to its naturally bound endogenous ligands.

[0458] Therapeutic effective doses of the antibodies disclosed herein generally refer to the amount required to achieve the therapeutic target. As mentioned above, this may be due to the binding interaction between the antibody and its target antigen, which in some cases interferes with the function of said target. Furthermore, the amount required to be administered will depend on the binding affinity of the antibody to its specific antigen, and will also depend on the rate at which the administered antibody is depleted from the free volume of the other subject to which it has been administered. As a non-limiting example, a common range of therapeutically effective doses of the antibodies or antibody fragments of this disclosure may be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies may range, for example, from twice daily to once weekly.

[0459] The antibodies or fragments thereof disclosed herein can be administered in the form of pharmaceutical compositions for the treatment of a variety of diseases and disorders. The principles and considerations involved in the preparation of such compositions, as well as guidance on the selection of components, are provided in, for example, the following literature: *Remington: The Science and Practice of Pharmacy*, 19th edition (edited by Alfonso R. Gennaro et al.), Mack Pub. Co., Easton, Pa.: 1995; *Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends*, Harwood Academic Publishers, Langhorne, Pa., 1994; "Peptide and Protein Drug Delivery," *Advances in Parenteral Sciences*, Vol. 4, 1991, M. Dekker, New York.

[0460] When using antibody fragments, minimally inhibitory fragments that specifically bind to the binding domain of the target protein are preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation may also contain more than one active compound as required for a particular indication being treated, preferably those compounds having complementary activities that do not adversely affect each other. Optionally or additionally, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are suitably combined in amounts that effectively achieve the intended purpose.

[0461] The active ingredient can also be encapsulated in microcapsules, for example, prepared by coagulation technology or interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in crude emulsions.

[0462] Formulations intended for internal administration must be sterile. This can be easily achieved through filtration using sterile membrane filters.

[0463] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, said matrices being in the form of molded articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, and degradable lactic-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic-glycolic acid copolymer and leuprolide acetate) and poly(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for a shorter time.

[0464] The antibodies according to this disclosure can be used as reagents to detect the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable marker. The antibody is polyclonal, or more preferably monoclonal. Intact antibodies or fragments thereof (e.g., F) are used. ab ,scFv or F (ab)2Regarding probes or antibodies, the term "labeled" is intended to cover both direct labeling of the probe or antibody by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of the probe or antibody by reactivity with another directly labeled reagent. Examples of indirect labeling include detecting a first antibody using a fluorescently labeled second antibody, and end-labeling a DNA probe with biotin so that it can be detected by fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within the subject's body. Thus, the use of the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of this disclosure can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in the following literature: “ELISA: Theory and Practice,” *Methods in Molecular Biology*, Vol. 42, JR 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 detecting analyte proteins involve introducing labeled anti-analyte protein antibodies into the subject. For example, the antibodies may be labeled with radioactive markers, and their presence and location in the subject can be detected using standard imaging techniques.

[0465] In some embodiments of this disclosure, a method of treating squamous cell carcinoma in a subject is provided, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) as described herein in combination with chemotherapy, radiotherapy, and / or an immune checkpoint inhibitor. Any suitable chemotherapy may be used in the methods described herein, including but not limited to topoisomerase inhibitors, anthracycline drugs such as doxorubicin, antibiotics that induce DNA breaks, pyrimidine antagonists, or platinum alkylating agents. Any suitable radiotherapy may be used in the methods described herein, including but not limited to 3D conformal radiotherapy (3DCRT), image-guided radiotherapy (IGRT), intensity-modulated radiotherapy (IMRT), volume-modulated arc therapy (VMAT), brachytherapy, intraoperative radiotherapy (IORT), stereotactic radiosurgery (SRS), proton therapy, MRI linear accelerator, and / or stereotactic body radiotherapy (SBRT). Any suitable immune checkpoint inhibitor may be used in the methods described herein, including but not limited to avelumumab, durvalumab, atezolizumab, pembrolizumab, nivolumab, atezolizumab, ipilimumab, or trimemumab.

[0466] Squamous cell carcinoma (SCC) is a malignant tumor caused by squamous cells, a subtype of epithelial cells found in the skin and mucous membranes. SCC is commonly associated with skin cancer but can occur in any organ lined with squamous cells, including but not limited to the bladder, lung, esophagus, cervix, anus, vulva, penis, thymus, thyroid, and oral cavity (e.g., in head and neck squamous cell carcinoma or oropharyngeal squamous cell carcinoma). In some embodiments, this disclosure provides a method of treating bladder squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method of treating lung squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method of treating esophageal squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating head and neck squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating cervical squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating cutaneous squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating thymic squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating anal squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating vulvar squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating penile squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein.In some embodiments, this disclosure provides a method for treating adrenocortical squamous cell carcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) provided herein. In some embodiments, this disclosure provides a method for treating squamous cell carcinoma in a subject, the method comprising administering an anti-ULBP2 antibody provided herein and a chemotherapy agent, radiotherapy, and / or an immune checkpoint inhibitor.

[0467] In some embodiments, this disclosure provides a method of treating adenocarcinoma in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) as described herein in combination with chemotherapy, radiotherapy, and / or an immune checkpoint inhibitor. In some embodiments, the adenocarcinoma is lung adenocarcinoma, pancreatic adenocarcinoma, esophageal adenocarcinoma, bladder adenocarcinoma, or colon adenocarcinoma. In some embodiments, a subject with cancer (squamous cell carcinoma or adenocarcinoma) has elevated ULBP2 expression levels in cancer cells relative to non-cancer cells. In some embodiments, ULBP2 expression levels are well correlated with the squamous cell carcinoma portion of the cancerous tissue. In some embodiments, the adenocarcinoma has elevated ULBP2 expression when it has an EGFR activating mutation or an altered EGFR copy number (e.g., an increased EGFP copy number). EGFR activating mutations are known, for example, as described in Gazdar, Oncogene. 2009 Aug; 28 (Suppl 1): S24–S31. Therefore, this disclosure provides a method for treating adenocarcinoma with EGFR activating mutations or EGFR copy number alterations (e.g., EGFR copy number increase), the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) as described herein in combination with chemotherapy or radiotherapy. In some embodiments, a subject with cancer (e.g., adenocarcinoma) having EGFR activating mutations or EGFR copy number alterations (e.g., EGFR copy number increase) has previously been treated with a tyrosine kinase inhibitor (TKI) (e.g., osimertinib), however, the TKI treatment does not reduce ULBP2 expression levels in the cancer. In some embodiments, a subject with cancer (e.g., adenocarcinoma) having EGFR activating mutations or EGFR copy number alterations (e.g., EGFR copy number increase) has previously been treated with a tyrosine kinase inhibitor (TKI) (e.g., osimertinib), but the cancer relapsed after remission. In some embodiments, administration of the anti-ULBP2 antibody (e.g., a bispecific antibody that binds ULBP2 and CD3) provided herein reduces ULBP2 expression levels in cancers with EGFR activating mutations or EGFR copy number alterations (e.g., EGFR copy number increase), and combination therapy with chemotherapy, radiotherapy, and / or immune checkpoint inhibitors enhances the efficacy of the anti-ULBP2 antibody (e.g., a bispecific antibody that binds ULBP2 and CD3).

[0468] In some embodiments, this disclosure provides a method of treating metastatic cancer in a subject, the method comprising administering to the subject an anti-ULBP2 antibody (e.g., a bispecific antibody binding ULBP2 and CD3) as described herein in combination with chemotherapy, radiation therapy, and / or an immune checkpoint inhibitor. Metastatic cancer is caused by tumors (non-primary tumors) that have spread from a primary site to other parts of the body. Non-primary tumors can metastasize via the bloodstream or lymphatic system. Metastatic cancers are typically more advanced and generally more difficult to treat because they can invade organs such as the lungs, liver, bone, or brain. Among squamous cell carcinomas, metastatic squamous cell carcinoma of the lung and cervix are particularly common.

[0469] Non-primary tumors (metastatic tumors) are identified based on their cellular characteristics and location relative to the presumed primary tumor. Methods for identifying non-primary tumors include histopathology, immunohistochemistry, imaging (e.g., CT, MRI, or PET scans), genomic analysis (e.g., DNA sequencing), and medical history. In some embodiments, metastatic cancers with non-primary tumors have ULBP2 expression levels similar to those of their primary tumor cells. In some embodiments, administration of anti-ULBP2 antibodies (e.g., bispecific antibodies binding ULBP2 and CD3 provided herein) reduces ULBP2 expression levels in non-primary metastatic cancers, and combination therapy with chemotherapy, radiotherapy, and / or immune checkpoint inhibitors enhances the efficacy of said anti-ULBP2 antibodies (e.g., bispecific antibodies binding ULBP2 and CD3). In some embodiments, this disclosure provides a method of treating metastatic cancer in a subject, the method comprising administering to the subject an anti-ULBP2 antibody provided herein and a chemotherapy agent, radiotherapy, and / or an immune checkpoint inhibitor.

[0470] Pharmaceutical Composition

[0471] The antibodies disclosed herein (also referred to herein as “active compounds”) and their derivatives, fragments, analogs, and homologs may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Suitable carriers are described in the latest edition of the standard reference book in the art, Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers such as non-volatile oils may also be used. Such media and reagents for the use of pharmaceutically active substances are well known in the art. Their use in the compositions may be considered unless any conventional media or reagent is incompatible with the active compound. Additional active compounds may also be incorporated into the compositions.

[0472] The pharmaceutical compositions disclosed herein are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate; and a reagent for adjusting osmolality, such as sodium chloride or glucose. pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral formulation may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0473] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the water-soluble case) or dispersions, as well as sterile powders for immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, and Cremophor EL. TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be an easily injectable fluid. It must be stable under manufacturing and storage conditions and must be preserved to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, maintaining the desired particle size in the case of a dispersion, and using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride, are preferably included in the composition. The absorption of the injectable composition can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0474] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above into a suitable solvent as required, followed by sterile filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a base dispersion medium and the other desired ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0475] Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be combined with excipients and administered in tablet, lozenge, or capsule form. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally, rinsed, and spat out or swallowed. Pharmaceutically compatible binders and / or excipients may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0476] For inhalation administration, the compound is delivered as an aerosol spray from a pressure vessel or dispenser or sprayer containing a suitable propellant, such as a gas like carbon dioxide.

[0477] Systemic administration can also be performed via transmucosal or transdermal routes. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are well known in the art and, for example, include detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated as an ointment, cream, gel, or cream, as is known in the art.

[0478] The compound can also be prepared in the form of suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0479] In one embodiment, the active compound is prepared using a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are readily apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes containing monoclonal antibodies against viral antigens that target infected cells) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0480] For ease of administration and dosage uniformity, it is particularly advantageous to formulate oral or parenteral compositions in dosage units. As used herein, dosage units refer to physically discrete units suitable as unit doses for use on a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect when combined with a desired drug carrier. The specifications of the dosage unit forms disclosed herein are determined by and directly depend on the unique properties of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations in the techniques for formulating such active compounds for individual treatment.

[0481] The pharmaceutical composition may be included in a container, package, or applicator along with the instructions for use.

[0482] This disclosure will be further described in the following embodiments, which do not limit the scope of this disclosure as described in the claims.

[0483] definition

[0484] Unless otherwise defined, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terms and techniques described herein that are used in connection with cell and tissue culture, molecular biology, protein and oligonucleotide or polynucleotide chemistry and hybridization are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid staining). Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications or as commonly performed in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods known in the art, as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual* (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The terminology, laboratory procedures, and techniques used in connection with analytical chemistry, synthetic organic chemistry, pharmaceuticals, and medicinal chemistry described herein are well-known and commonly used in the field. Standard techniques are applied to chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0485] When used in accordance with this disclosure, unless otherwise stated, the following terms shall be understood to have the following meanings: The term "ULBP2-positive cancer" refers to cancer comprised of cells that express ULBP2 on their surface. In some implementations, the expression of ULBP2 on the cell surface is determined, for example, using methods such as immunohistochemistry or FACS, with antibodies against ULBP2. Alternatively, the expression of ULBP2 mRNA is considered to be associated with ULBP2 expression on the cell surface and can be determined by methods selected from in situ hybridization and RT-PCR (including quantitative RT-PCR). The term "ULBP2-positive cell" refers to a cell that expresses ULBP2 on its surface. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of this type of cancer include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer (including triple-negative (ER- / PR- / Her2-) breast cancer), colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancer.

[0486] The phrases “reference sample,” “reference cell,” or “reference tissue” refer to a sample having at least one known characteristic that can be used for comparison with a sample having at least one unknown characteristic. In some embodiments, the reference sample can be used as a positive or negative indicator. In contrast to the levels of proteins and / or mRNA present in a sample with unknown characteristics, the reference sample can be used to establish the levels of proteins and / or mRNA present, for example, in healthy tissue. In some embodiments, the reference sample is from the same subject being tested, but from a different site within the subject. In some embodiments, the reference sample is from a tissue region surrounding or adjacent to cancer. In some embodiments, the reference sample is not from the subject being tested, but from a sample from a subject known to have or not have the discussed barrier (e.g., a specific cancer or ULBP2-related barrier). In some embodiments, the reference sample is from the same subject, but from a time point prior to the development of cancer in the subject. In some embodiments, the reference sample is from a benign cancer sample (e.g., a benign breast cancer sample) from the same or different subjects. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample will indicate a level that a person skilled in the art would understand, given this disclosure, that the molecule is absent and / or present at a low level. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample will indicate a level that a person skilled in the art would understand, given this disclosure, that the molecule is present at a certain level.

[0487] As used in this article, the terms "disease" or "disorder" refer to a condition that requires and / or is expected to be treated.

[0488] Unless otherwise specified, the terms “tumor cell,” “cancer cell,” “cancer,” “tumor,” and / or “proliferation” are used interchangeably herein and refer to cells (or cells) exhibiting uncontrolled growth and / or abnormally increased cell survival and / or suppressed apoptosis that interfere with the normal function of organs and systems of the body. This definition includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases. The terms “cancer” and “tumor” cover solid cancers and hematologic / lymphomas, and also encompass malignant, pre-malignant, and benign growths, such as dysplasia. Furthermore, this definition also includes cells with abnormal proliferation that is not hindered by the immune system (e.g., immune evasion and immune escape mechanisms) (e.g., virus-infected cells). Exemplary tumor cells include, but are not limited to: basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancers, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer or renal cell carcinoma; laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung), melanoma, myeloma, neuroblastoma, oral cancer (lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancers, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, Cancers of the urinary system, vulvar cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas, including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleavage cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloid leukemia; other cancers and sarcomas; post-transplant lymphoproliferative disorder (PTLD), and abnormal angiogenesis associated with nevus hamartomatosis, edema (e.g., edema associated with brain tumors), and Meigs syndrome.

[0489] In some implementations, the term cancer may encompass lung cancer, breast cancer, head and neck cancer, ovarian cancer, and / or endometrial cancer. Where necessary, the distinction between "cancer" and "cancer cells" can be made by explicitly using the phrase "cancer cells"; however, the term "cancer" will encompass concepts such as a subject with cancer, multicellular tumors, and single cancer cells.

[0490] "Increase or decrease" refers to a statistically significant increase or decrease. Those skilled in the art will appreciate that "modulation" can also involve achieving changes (which may be increases or decreases) in the affinity, specificity, and / or selectivity of the target or antigen for one or more ligands, binding couplers, couplers bound to homomeric or heteromeric forms, or substrates; achieving changes (which may be increases or decreases) in the sensitivity of the target or antigen under one or more conditions in the presence of the target or antigen (e.g., pH, ionic strength, presence of cofactors, etc.) compared to the same conditions in the absence of antibodies, bispecific or multispecific peptide reagents. This can be determined, depending on the target involved, in any suitable manner and / or using any suitable assay known per se or described herein.

[0491] When used in this article, “immune response” is intended to encompass cellular and / or humoral immune responses sufficient to suppress or prevent the onset of a disease (such as cancer or cancer metastasis) or to improve the symptoms of a disease. “Immune response” can encompass aspects of both the innate and adaptive immune systems.

[0492] When used herein, “treatment” is a method of achieving a beneficial or desired clinical outcome. As used herein, “treatment” encompasses any administration or application of a therapeutic agent for a disease in mammals, including humans. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, any or more of the following: relief of one or more symptoms, reduction of the severity of the disease, prevention or delay of disease spread (e.g., metastasis, such as to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slow disease progression, improvement of the disease state, inhibition of the disease or disease progression, inhibition or slowing of the disease or its progression, prevention of its development, and remission (whether partial or complete). “Treatment” also encompasses reducing the pathological consequences of proliferative diseases. The methods provided herein take into account any or more of these aspects of treatment. Consistent with the foregoing, the term treatment does not require the complete elimination of all aspects of the obstruction.

[0493] "Improvement" refers to the reduction or improvement of one or more symptoms compared to not using anti-ULBP2 antibodies. "Improvement" also includes shortening or reducing the duration of symptoms.

[0494] The term "biological sample" refers to a quantity of material derived from or formerly from a living organism. Such material includes, but is not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0495] The term "control" refers to a composition known to contain no analyte ("negative control") or a composition containing analyte ("positive control"). A positive control may contain a known concentration of analyte. "Control," "positive control," and "calibrator" are used interchangeably herein and refer to a composition containing a known concentration of analyte. A "positive control" can be used to establish the performance characteristics of an assay and is a useful indicator of the integrity of reagents (e.g., analytes).

[0496] "Predetermined cutoff value" and "predetermined level" generally refer to assay cutoff values ​​used to assess diagnostic / prognostic / treatment efficacy outcomes by comparing assay results to predetermined cutoff values / levels, where the predetermined cutoff values / levels have been associated with or correlated with various clinical parameters, such as disease severity, progression / non-progression / improvement, etc. While this disclosure may provide exemplary predetermined levels, it is well known that cutoff values ​​can vary depending on the nature of the immunoassay (e.g., the antibodies used). Those skilled in the art are also fully capable of adapting the disclosure herein to other immunoassays to obtain assay-specific cutoff values ​​for those other immunoassays based on this disclosure. Although the precise values ​​of predetermined cutoff values / levels may vary from assay to assay, the relevance described herein, if any, is likely to apply generally.

[0497] The term "inhibition" refers to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the incidence, extent, or likelihood of that trait. "Reduction" or "inhibition" means a decrease, reduction, or cessation of activity, function, and / or amount compared to a reference. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 20% or more. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 50% or more. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 75%, 85%, 90%, 95%, or more. In some embodiments, the aforementioned amounts are inhibited or reduced over the same time period relative to a control (e.g., placebo) over a period of time.

[0498] When used herein, “delay disease progression” means to postpone, hinder, slow, delay, stabilize, inhibit, and / or slow the development of a disease (such as cancer). Such delay can have varying durations, depending on the individual’s medical history and / or the individual being treated. As will be apparent to those skilled in the art, adequate or significant delay can effectively encompass prevention, as the individual does not develop the disease. For example, the development of advanced cancer, such as metastasis, can be delayed.

[0499] When used herein, “prevention” includes providing prevention of the onset or recurrence of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it. Unless otherwise specified, the terms “reduction,” “suppression,” or “prevention” do not imply or require complete prevention at all times.

[0500] When used herein, "inhibitory" function or activity means a reduction in said function or activity compared to the same conditions other than the condition or parameter of interest, or compared to another condition. For example, an antibody that inhibits tumor growth reduces the growth rate of said tumor (compared to the tumor growth rate in the absence of said antibody).

[0501] The term "antibody-binding region" refers to a region of an antigen containing the epitope to which the antibody binds. The antibody-binding region can be determined using epitope binning with biolayer interferometry, alanine scanning, or domain mixing assays (using antigen constructs in which a region of the antigen is exchanged with an antigenic region of another species, and determining whether the antibody still binds to the antigen). The amino acids within the antibody-binding region that interact with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and / or by crystallography of the antibody bound to the antigen.

[0502] As used herein, the term "antibody" refers to both immunoglobulin molecules (i.e., molecules containing antigen-binding sites that specifically bind to antigens (responding to an immune response to an antigen)) and the immunologically active portion of immunoglobulin (Ig) molecules. "Specific binding," "immune response to," or "immunospecific binding" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other peptides or with much lower affinity (K+). d > 10 -6 ) binding. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, F ab F ab’ and F (ab')2 Fragments, scFv and F ab Expression Library.

[0503] The basic structural unit of an antibody is known to be a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, primarily responsible for effector function. Generally, antibody molecules derived from humans involve any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another due to the nature of the heavy chain present in the molecule. Some classes also have subclasses, such as IgG1, IgG2, IgG4, etc. Furthermore, in humans, the light chain may be either a κ chain or a λ chain.

[0504] As used herein, the term "monoclonal antibody" (MAb) or "monoclonal antibody composition" refers to a population of antibody molecules containing only one type of antibody molecule, said antibody molecule being composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining region (CDR) of a monoclonal antibody is identical in all molecules of said population. MAbs contain antigen-binding sites capable of inducing an immune response to a specific epitope of an antigen, characterized by a unique binding affinity to said epitope.

[0505] The terms "antigen-binding region," "antigen-binding site," or "binding portion" refer to the portion of an immunoglobulin molecule involved in antigen binding. An antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") region of the heavy ("H") and light ("L") chains. Three highly divergent segments (referred to as "hypervariates") within the V regions of the heavy and light chains are interspersed between more conserved flanking segments referred to as "framework regions" or "FRs." Therefore, the term "FR" refers to the naturally occurring amino acid sequence between and near the hypervariates in an immunoglobulin. In an antibody molecule, the three hypervariates of the light chain and the three hypervariates of the heavy chain are arranged opposite each other in three-dimensional space to form an antigen-binding surface. This antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariates of each heavy and light chain are referred to as "complementarity-determining regions" or "CDRs." Various methods for numbering the amino acid sequence of an antibody and identifying the complementarity-determining regions are known in the art. For example, the Kabat numbering system (see Kabat, EA et al., Sequences of Protein of Immunological Interest, 5th ed., US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (see IMGT ®, the internationalImMunoGeneTics information system ® The IMGT exon numbering system is available online at http: / / www.imgt.org / . It is routinely used and recognized in the art as a reliable and accurate system for determining amino acid positions in coding sequences, allele alignment, and facilitating comparison of sequences in immunoglobulins (IG) and T-cell receptors (TR) from all vertebrate species. The accuracy and consistency of IMGT data are based on IMGT-ONTOLOGY, the first and, to date, only ontology for immunogenetics and immunoinformatics (see Lefranc. MP et al., Biomolecules, 2014 Dec; 4(4), 1102-1139). IMGT tools and databases operate against the IMGT reference catalog built from large sequence libraries. In the IMGT system, IG V-DOMAIN and IG C-DOMAIN are delimited with exon delimitation taken into account where appropriate. Therefore, with the greater availability of sequences in the IMGT database, those skilled in the art can and "use" the IMGT exon numbering system to reliably determine amino acid positions in coding sequences and perform allele alignment. In addition, the correspondence between the IMGT unique number and other numbers (i.e., Kabat) can be obtained from the IMGT scientific chart (see Lefranc. MP et al., Biomolecules, 2014 Dec; 4(4), 1102-1139).

[0506] The terms "hypervariant region" or "variable region" refer to the amino acid residues of an antibody that are typically responsible for antigen binding. The hypervariant region usually contains amino acid residues from the "complementarity-determining region" or "CDR" (e.g., when numbered according to the Kabat numbering system, V...). L Residues approximately at positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the V... H Residues approximately 31–35 (H1), 50–65 (H2), and 95–102 (H3) in the sequence; Kabat et al., Sequences of Protein of Immunological Interest, 5th ed., Public Health Service, Nation...

Claims

1. A method of reducing a population of cancer cells in a subject having a cancer, the method comprising administering to the subject: a) an anti-ULBP2 antibody or antigen-binding fragment thereof, and b) a chemotherapeutic agent, radiation therapy, and / or an immune checkpoint inhibitor, wherein a plurality of cancer cells express UL16 binding protein 2 (ULBP2).

2. The method of claim 1, wherein the method results in a reduction of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% in the number of cancer cells expressing ULBP2 in the subject relative to the number of cancer cells expressing ULBP2 in the subject prior to administration of the anti-ULBP2 antibody or antigen-binding fragment thereof and the chemotherapeutic agent and / or radiation therapy.

3. The method of any one of claims 1-2, wherein the anti-ULBP2 antibody or antigen-binding fragment thereof and the chemotherapeutic agent, radiation therapy, and / or immune checkpoint inhibitor are administered sequentially.

4. The method of any one of claims 1-3, wherein the anti-ULBP2 antibody or antigen-binding fragment thereof and the chemotherapeutic agent, radiation therapy, and / or immune checkpoint inhibitor are administered simultaneously.

5. The method of any one of claims 1-4, wherein the cancer is a squamous carcinoma.

6. The method of claim 5, wherein the squamous carcinoma is a basal squamous carcinoma.

7. The method of any one of claims 1-6, wherein the subject has a bladder cancer, a lung cancer, a head and neck cancer, a cervical cancer, an esophageal cancer, an anal cancer, a vulvar cancer, a penile cancer, a thymus cancer, a skin cancer cell, or an adrenal cortex cancer.

8. The method of any one of claims 1-3, wherein the cancer is an adenocarcinoma.

9. The method of claim 8, wherein the adenocarcinoma is a lung adenocarcinoma, a pancreatic adenocarcinoma, a bladder adenocarcinoma, an esophageal adenocarcinoma, or a colon adenocarcinoma.

10. The method of any one of claims 1-9, wherein the subject has an EGFR activating mutation or an EGFR copy number alteration.

11. The method of claim 10, wherein the subject was previously treated with a tyrosine kinase inhibitor (TKI).

12. The method of claim 11, wherein the TKI is osimertinib.

13. The method of claim 11 or 12, wherein the TKI does not decrease the level of ULBP2 expression in the cancer.

14. The method of any one of claims 11-13, wherein the subject previously receiving a TKI has a recurrence of cancer following TKI treatment.

15. The method of any one of claims 1-3, wherein the cancer is a metastatic cancer.

16. The method of claim 15, wherein the subject having a metastatic cancer has a non- primary lesion in the liver, lung, bone, brain, or lymph node that is derived from a primary cancer.

17. The method of claim 15 or 16, wherein the primary cancer is a squamous carcinoma or an adenocarcinoma.

18. The method of claim 17, wherein the primary cancer is squamous cell carcinoma, including bladder cancer, lung cancer, head and neck cancer, cervical cancer, esophageal cancer, anal cancer, vulvar cancer, penile cancer, thymic cancer, skin cancer cells, or adrenocortical cancer.

19. The method of claim 17, wherein the primary cancer is an adenocarcinoma including lung adenocarcinoma, pancreatic adenocarcinoma, bladder adenocarcinoma, esophageal adenocarcinoma, or colon adenocarcinoma.

20. The method according to any one of claims 1-19, wherein the subject has previously been administered an immune checkpoint inhibitor.

21. The method according to any one of claims 1-20, wherein the immune checkpoint inhibitor is a PD1 / PDL1 inhibitor or a CTLA4 inhibitor.

22. The method according to any one of claims 1-21, wherein the immune checkpoint inhibitor is averumarub, durvalumab, atezolizumab, pembrolizumab, nivolumab, atezolizumab, ipilimumab, or trimemumab.

23. The method according to any one of claims 20-22, wherein the immune checkpoint inhibitor does not reduce the ULBP2 expression level in the cancer.

24. The method according to any one of claims 1-23, wherein the anti-ULBP2 antibody or a fragment thereof comprises: a) Complementarity-determining region 1 (VH2 CDR1 ) comprising the amino acid sequence of SEQ ID NO: 428; complementarity-determining region 2 (VH2 CDR2 ) comprising the amino acid sequence of SEQ ID NO: 430; complementarity-determining region 3 (VH2 CDR3 ) comprising the amino acid sequence of SEQ ID NO: 432; complementarity-determining region 1 (VL2 CDR1 ) comprising the amino acid sequence of SEQ ID NO: 433; complementarity-determining region 2 (VL2 CDR2 ) comprising the amino acid sequence of SEQ ID NO: 434; and complementarity-determining region 3 (VL2 CDR3 ) comprising the amino acid sequence of SEQ ID NO: 435; or b) a complementarity determining region 1 (VH2 ) comprising the amino acid sequence of SEQ ID NO: 5; a complementarity determining region 2 (VH2 ) comprising the amino acid sequence of SEQ ID NO: 7; a complementarity determining region 3 (VH2 ) comprising the amino acid sequence of SEQ ID NO: 9; a complementarity determining region 1 (VL2 ) comprising the amino acid sequence of SEQ ID NO: 10; a complementarity determining region 2 (VL2 ) comprising the amino acid sequence of SEQ ID NO: 11 ; and a complementarity determining region 3 (VL2 ) comprising the amino acid sequence of SEQ ID NO:

12. CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 ​​​​​​ 25. The method according to any one of claims 1-24, wherein the anti-ULBP2 antibody or a fragment thereof is a bispecific antibody that specifically binds to ULBP2 and CD3, or wherein the anti-ULBP2 antibody or a fragment thereof is a bispecific antibody that specifically binds to ULBP2 and PD1.

26. The method of claim 24 or 25, wherein the CD58 polypeptide or a fragment thereof is fused to the C-terminus of the heavy chain polypeptide of the bispecific antibody.

27. The method of claim 26, wherein the CD58 comprises the amino acid sequence of SEQ ID NO:

49.

28. The method according to any one of claims 1-27, wherein the chemotherapeutic agent is selected from topoisomerase inhibitors, anthracycline drugs doxorubicin, DNA-inducing antibiotics, pyrimidine antagonists, and platinum alkylating agents.

29. The method according to any one of claims 1-28, wherein the radiotherapy is selected from 3D conformal radiotherapy (3DCRT), image-guided radiotherapy (IGRT), intensity modulated radiotherapy (IMRT), volume modulated arc therapy (VMAT), brachytherapy, intraoperative radiotherapy (IORT), stereotactic radiosurgery (SRS), proton therapy, MRI linear accelerator and / or stereotactic body radiotherapy (SBRT).

30. A method for identifying human subjects suffering from cancer suitable for treatment with an anti-UL16 binding protein 2 (ULBP2) antibody or an antigen-binding fragment thereof, the method comprising the steps of: a) Obtaining biological samples from human subjects with cancer; b) Detect the expression level of ULBP2; c) Compare the detected ULBP2 expression levels with the control levels of ULBP2 expression; and d) When the detected ULBP2 expression level is greater than the control level of ULBP2 expression, the subject is identified as a responder.

31. The method according to claim 30, further comprising: e) When the subject is identified as a responder, administer an anti-ULBP2 antibody or a fragment thereof in an amount sufficient to alleviate the symptoms of the cancer.

32. A method for treating a human subject suffering from cancer, the method comprising the steps of: a) Obtaining biological samples from human subjects with cancer; b) Detect the expression level of ULBP2; c) Compare the detected ULBP2 expression levels with the control levels of ULBP2 expression; d) When the detected ULBP2 expression level is greater than the control level of ULBP2 expression, the subject is identified as a responder; and e) When the subject is identified as a responder, administer an anti-ULBP2 antibody or a fragment thereof in an amount sufficient to alleviate the symptoms of the cancer.

33. The method according to any one of claims 30-32, wherein the anti-ULBP2 antibody or a fragment thereof comprises: a) Complementarity-determining region 1 (VH2) of the amino acid sequence containing SEQ ID NO: 428 CDR1 ); containing complementarity-determining region 2 (VH2) of the amino acid sequence of SEQ ID NO: 430 CDR2 ); containing complementarity-determining region 3 (VH2) of the amino acid sequence of SEQ ID NO: 432 CDR3 ); containing complementarity-determining region 1 (VL2) of the amino acid sequence of SEQ ID NO: 433 CDR1 ); Complementarity-determining region 2 (VL2) of the amino acid sequence containing SEQ ID NO: 434 CDR2 ); and complementarity-determining region 3 (VL2) containing the amino acid sequence of SEQ ID NO:

435. CDR3 );or b) a complementarity determining region 1 (VH2) comprising the amino acid sequence of SEQ ID NO: 5; a complementarity determining region 2 (VH2) comprising the amino acid sequence of SEQ ID NO: 7; a complementarity determining region 3 (VH2) comprising the amino acid sequence of SEQ ID NO: 9; a complementarity determining region 1 (VL2) comprising the amino acid sequence of SEQ ID NO: 10; a complementarity determining region 2 (VL2) comprising the amino acid sequence of SEQ ID NO: 11; and a complementarity determining region 3 (VL2) comprising the amino acid sequence of SEQ ID NO:

12. CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 ; a complementarity determining region 2 (VL2) comprising the amino acid sequence of SEQ ID NO: 11; and a complementarity determining region 3 (VL2) comprising the amino acid sequence of SEQ ID NO: 12.​​​​​ 34. The method according to any one of claims 30-33, wherein the anti-ULBP2 antibody or a fragment thereof is a bispecific antibody that specifically binds to ULBP2 and CD3.

35. The method of claim 34, wherein the CD58 polypeptide or a fragment thereof is fused to the C-terminus of the heavy chain polypeptide of the bispecific antibody.

36. The method of claim 35, wherein the CD58 comprises the amino acid sequence of SEQ ID NO: 49 or 50.

37. The method according to any one of claims 30-36, wherein the cancer is squamous cell carcinoma.

38. The method of claim 37, wherein the squamous cell carcinoma is basal squamous cell carcinoma.

39. The method according to claim 37 or 38, wherein the subject has bladder cancer, lung cancer, head and neck cancer, cervical cancer, esophageal cancer, anal cancer, vulvar cancer, penile cancer, thymic cancer, skin cancer cells, or adrenocortical cancer.

40. The method according to any one of claims 30-36, wherein the cancer is adenocarcinoma.

41. The method of claim 40, wherein the adenocarcinoma is lung adenocarcinoma, pancreatic adenocarcinoma, bladder adenocarcinoma, esophageal adenocarcinoma, or colon adenocarcinoma.

42. The method according to any one of claims 30-41, wherein the subject has an EGFR activating mutation or an EGFR copy number alteration.

43. The method according to any one of claims 30-36, wherein the cancer is a metastatic cancer.

44. The method of claim 43, wherein the subject with metastatic cancer has non-primary lesions in the liver, lungs, bones, brain, or lymph nodes originating from the primary cancer.

45. The method according to claim 43 or 44, wherein the primary cancer is squamous cell carcinoma or adenocarcinoma.

46. ​​The method of claim 45, wherein the primary cancer is squamous cell carcinoma, including bladder cancer, lung cancer, head and neck cancer, cervical cancer, esophageal cancer, anal cancer, vulvar cancer, penile cancer, thymic cancer, skin cancer cells, or adrenocortical cancer.

47. The method of claim 46, wherein the primary cancer is an adenocarcinoma including lung adenocarcinoma, pancreatic adenocarcinoma, bladder adenocarcinoma, esophageal adenocarcinoma, or colon adenocarcinoma.

48. The method according to any one of claims 30-47, wherein the subject is simultaneously or pre-administered a therapeutically effective amount of the therapeutic agent.

49. The method of claim 48, wherein the therapeutic agent is a chemotherapeutic agent selected from topoisomerase inhibitors, anthracycline drugs doxorubicin, DNA-fracture-inducing antibiotics, pyrimidine antagonists, and platinum alkylating agents.

50. The method of claim 48, wherein the therapeutic agent is a radiotherapy selected from 3D conformal radiotherapy (3DCRT), image-guided radiotherapy (IGRT), intensity-modulated radiotherapy (IMRT), volume-modulated arc therapy (VMAT), brachytherapy, intraoperative radiotherapy (IORT), stereotactic radiosurgery (SRS), proton therapy, MRI linear accelerator and / or stereotactic body radiotherapy (SBRT).

51. The method of claim 48, wherein the therapeutic agent is a CAR-T cell therapy, an immune checkpoint inhibitor, a co-stimulatory ligand, or a cytokine.

52. The method according to any one of claims 25-29 or 34-51, wherein the bispecific antibody comprises an anti-ULBP2 antibody or an antigen-binding fragment thereof, the anti-ULBP2 antibody or the antigen-binding fragment thereof comprising a light chain variable region (VL2) containing the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region (VH2) containing the amino acid sequence of SEQ ID NO:

629.

53. The method according to any one of claims 25-29 or 34-52, wherein the anti-ULBP2 antibody or its antigen-binding fragment comprises a light chain (L2) containing the amino acid sequence of SEQ ID NO: 67 or a heavy chain (H2) containing the amino acid sequence of SEQ ID NO:

621.

54. The method according to any one of claims 25-29 or 34-53, wherein the bispecific antibody comprises an anti-CD3 antibody or an antigen-binding fragment thereof, the anti-CD3 antibody or the antigen-binding fragment thereof comprising a light chain variable region (VL1) containing the amino acid sequence of SEQ ID NO: 22 and a heavy chain variable region (VH1) containing the amino acid sequence of SEQ ID NO:

17.

55. The method according to any one of claims 25-29 or 24-54, wherein the CD58 polypeptide or a fragment thereof is fused to the C-terminus of the heavy chain of the anti-CD3 antibody of the bispecific antibody or an antigen-binding fragment thereof.

56. The method according to any one of claims 25-29 or 24-55, wherein the anti-CD3 antibody or its antigen-binding fragment comprises a light chain (L1) containing the amino acid sequence of SEQ ID NO: 69 and a heavy chain (H1) fused to the CD58 polypeptide containing the amino acid sequence of SEQ ID NO: 622.

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