Antibodies targeting disease associated antigen and γδ t cell receptors and uses thereof

Abstract

The present disclosure relates to antibodies such as bispecific antibodies targeting a disease associated antigen and δ1 and δ2 subtypes of γδ T cell receptors, to facilitate the elimination of disease cells by γδ T cells. The bispecific antibody optionally comprises an attenuated IL-15 and the sushi domain of IL-15Ra (SD-IL15) fused at the C-terminus of the heavy chains as immune modulator component that can stimulate the proliferation and activation of γδ T cells.

Description

[0001] Attorney Docket No.15271.0015-00304 Antibodies Targeting Disease Associated Antigen and γδ T Cell Receptors and Uses Thereof Cross Reference to Related Application [1] This application claims the priority to and benefits of U.S. Provisional Patent Application No. 63 / 604,970, filed on December 1, 2023, which is incorporated herein by reference in its entirety. Sequence Listing [2] This application contains a Sequence Listing electronically submitted as an XML file entitled “152710015-00304” having a size of 236,724 bytes and created on November 26, 2024. The information contained in the Sequence Listing is incorporated herein by reference. Field of Disclosure [3] The present disclosure provides antibodies such as bispecific antibodies targeting disease antigens (e.g., tumor associated antigen 5T4) and γδ T cell receptors with optional immune modulator component and the application of disclosed antibodies such as bispecific antibodies as therapeutics for diseases such as heme and solid tumors (e.g., cancers with 5T4 expression) and autoimmune diseases. Background of Disclosure [4] Cancer immunotherapy has made tremendous progress over the last decade with the central theme of reinvigorating anti-tumor efficacy of a patient’s own immune cells, mainly αβ T cells of the adaptive immune system (Mellman, Coukos et al. 2011, Waldman, Fritz et al. 2020). For instance, immune checkpoint inhibitors, functioning through de-repression of antigen- specific αβ T cells, now offer durable remissions and even cures for some patients with otherwise treatment-refractory solid tumors (Akinleye and Rasool 2019). Bispecific T cell engagers (BiTE) can redirect αβ T cells to kill tumor cells and showed good efficacy in clinical settings (Dempke, Fenchel et al. 2017). Adoptive cell therapy using chimeric antigen receptor (CAR) bearing αβ T cells and tumor infiltrating T cells (TILs) have also demonstrated promising efficacy, albeit predominantly in hematological malignancies (Maude, Frey et al. 2014, Han and Kwon 2018). However, these treatments only benefit a minority of patients and efforts to improve efficacy through combination therapies have proven to be difficult. [5] Besides αβ T cells, a growing number of studies have shown that γδ T cells play a pivotal role in the clearance of tumors and pathogen-infected cells with their potent cytotoxic, Attorney Docket No.15271.0015-00304 cytolytic, and unique immune-modulating functions (Nussbaumer and Koslowski 2019). So far most reported γδ T cell-based therapeutics have only shown suboptimal clinical efficacy so there is a clear need for the development of better therapeutic strategies that can stimulate γδ T cells such as δ1 and δ2 T cell subsets for the elimination of tumor cells. Molecules that can stimulate and maintain a large quantity of activated γδ T cells will allow for better therapeutic interventions. The present disclosure provides bispecific antibodies for a novel and effective immunotherapeutic approach harnessing γδ T cells. Summary of Disclosure [6] In one aspect, the present disclosure provides a bispecific antibody that is capable of simultaneously targeting a disease associated antigen (e.g., tumor associated antigen) and a γδ T cell receptor. By binding an antigen on tumor cells and a receptor on γδ T cells, the bispecific antibody is capable of activating and redirecting the γδ T cells in the reduction or elimination of nearby tumor cells. [7] In some embodiments, the bispecific antibody disclosed herein comprises a Fab arm with binding specificity to disease associated antigens, including but not limited to, 5T4, CD123, CD20, and CDH17. [8] As a non-limiting example, the disclosure provides for an anti-5T4 antibody or antigen-binding fragment thereof (e.g., a Fab arm) comprising a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 1, 2, and 3, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 4, 5, and 6, respectively. [9] As a non-limiting example, the disclosure provides for an anti-CD123 antibody or antigen-binding fragment thereof (e.g., a Fab arm) comprising a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 are chosen from SEQ ID NOs: 13 to 15, 19-21, and 25-27, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 are chosen from SEQ ID NOs: 16-18, 22-24, and 28-30, respectively. Attorney Docket No.15271.0015-00304

[0010] As a non-limiting example, the disclosure provides for an anti-CD20 antibody or antigen-binding fragment thereof (e.g., a Fab arm) comprising a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 31, 32, and 33, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 34, 35, and 36, respectively.

[0011] As a non-limiting example, the disclosure provides for an anti-CDH17 antibody or antigen-binding fragment thereof (e.g., a Fab arm) comprising a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 37, 38, and 39, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 40, 41, and 42, respectively.

[0012] In some embodiments, the bispecific antibody disclosed herein comprises a Fab arm or VHH with binding specificity to a γδ T cell receptor (γδTCR).

[0013] As a non-limiting example, the disclosure provides for antibody or antigen- binding fragment thereof (e.g., a Fab arm) targeting δ1 and / or δ2 subtypes of γδ T cell receptors (γδTCR) comprising a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 7, 8, and 9, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 10, 11, and 12, respectively.

[0014] As a non-limiting example, the disclosure provides for a VHH targeting δ1 and / or δ2 subtypes of γδ T cell receptors (γδTCR) comprising a variable region comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 are chosen from SEQ ID NOs: 43 to 72, respectively.

[0015] In some embodiments, the present disclosure provides a bispecific antibody that is in a human IgG1, IgG2, IgG3, and / or IgG4 framework with one or more optional IgG Fc Attorney Docket No.15271.0015-00304 mutations that can modulate antibody pharmacokinetic properties, effector functions, heterodimerization, and / or clustering activities.

[0016] In some embodiments, the present disclosure provides a 5T4 x γδTCR bispecific antibody comprising a 5T4 antibody heavy chain sequence as set forth in SEQ ID NO: 201, a 5T4 antibody light chain sequence as set forth in SEQ ID NO: 202, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210 to 214.

[0017] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody comprising a CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 215, 217, 218, 221, and 222, a CD123 antibody light chain sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210 to 214.

[0018] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody comprising a CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 233 to 237, a CD123 antibody light chain sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, and a γδTCR VHH sequence chosen from SEQ ID NOs: 229 to 232.

[0019] In some embodiments, the present disclosure provides a CD20 x γδTCR bispecific antibody comprising a CD20 antibody heavy chain sequence as set forth in SEQ ID NO: 225, a CD20 antibody light chain sequence as set forth in SEQ ID NO: 226, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210 to 214.

[0020] In some embodiments, the present disclosure provides a CDH17 x γδTCR bispecific antibody comprising a CDH17 antibody heavy chain sequence as set forth in SEQ ID NO: 227, a CDH17 antibody light chain sequence as set forth in SEQ ID NO: 228, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210 to 214.

[0021] In some embodiments, the bispecific antibody disclosed herein optionally comprises an immune modulator component that can stimulate the proliferation and / or activation Attorney Docket No.15271.0015-00304 of γδ T cells, which may facilitate more efficient and durable elimination of tumor cells by γδ T cells.

[0022] As a non-limiting example, the optional immune modulator component comprises an attenuated IL-15 and / or the sushi domain of IL-15Ra (SD-IL15) fused at the C- terminus of one or more of the heavy chains of the bispecific antibody disclosed herein.

[0023] As a non-limiting example, the attenuated IL-15 and the sushi domain of IL- 15Ra (SD-IL15) disclosed herein comprises an amino acid sequence as set forth in SEQ ID NO: 301, SEQ ID NO: 302, or SEQ ID NO: 303.

[0024] In some embodiments, the present disclosure provides a 5T4 x γδTCR bispecific antibody with SD-IL15 fused at the C-terminus of its heavy chain comprising a 5T4 antibody heavy chain sequence chosen from SEQ ID NOs: 201, 304, 305, and 306, a 5T4 antibody light chain sequence as set forth as SEQ ID NO: 202, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs. 307 to 315, 328, 329, and 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210 to 214.

[0025] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody with SD-IL15 fused at the C-terminus of its heavy chain comprising a CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 215, 217, 218, 221, 222, and 330 to 334, a CD123 antibody light chain sequence chosen from SEQ ID NOs. 216, 219, 220, 223, and 224, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 307 to 315, 328, 329, and 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210 to 214.

[0026] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody with SD-IL15 fused at the C-terminus of its heavy chain comprising a CD123 antibody heavy chain sequence chosen from SEQ ID NO: 337 to 341, a CD123 antibody light chain sequence chosen from SEQ ID NOs. 216, 219, 220, 223, and 224, and a γδTCR VHH sequence chosen from SEQ ID NOs: 229 to 232.

[0027] In some embodiments, the present disclosure provides a CD20 x γδTCR bispecific antibody with SD-IL15 fused at the C-terminus of its heavy chain comprising a CD20 antibody heavy chain sequence as set forth as SEQ ID NO: 335, a CD20 antibody light chain sequence as set forth as SEQ ID NO: 226, a γδTCR antibody heavy chain sequence chosen from Attorney Docket No.15271.0015-00304 SEQ ID NOs: 307 to 315, 328, 329, and 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210 to 214.

[0028] In some embodiments, the present disclosure provides a CDH17 x γδTCR bispecific antibody with SD-IL15 fused at the C-terminus of its heavy chain comprising a CDH17 antibody heavy chain sequence as set forth as SEQ ID NO: 336, a CDH17 antibody light chain sequence as set forth as SEQ ID NO: 228, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 307 to 315, 328, 329, and 203 to 209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs. 210 to 214.

[0029] In some embodiments, the present disclosure provides a nucleic acid encoding one or more amino acid chains of the antibodies, antigen-binding fragments, and bispecific antibodies as disclosed herein.

[0030] In some embodiments, the present disclosure provides a recombinant expression vector comprising the nucleic acid disclosed herein.

[0031] In some embodiments, the present disclosure provides a recombinant expression transformant comprising the recombinant expression vector disclosed herein.

[0032] In some embodiments, the present disclosure provides a method for producing the antibodies, antigen-binding fragments, and bispecific antibodies disclosed herein comprising steps of culturing the recombinant expression transformant disclosed herein and obtaining the antibodies, antigen-binding fragments, and bispecific antibodies from the culture.

[0033] In some embodiments, the present disclosure provides a bispecific antibody that can effectively stimulate γδ T cell proliferation and activation by crosslinking to a disease associated antigen (such as a tumor associated antigen) on cells such as tumor cells.

[0034] In some embodiments, the present disclosure provides a bispecific antibody that can effectively redirect γδ T cells to eliminate tumor cells expressing disease associated antigens such as 5T4, CD123, CD20, and CDH17.

[0035] The present disclosure provides a method for treating a patient, e.g., suffering from cancers or autoimmune diseases, comprising administering to the patient the antibodies, antigen-binding fragments, and bispecific antibodies targeting disease associated antigen and γδ T cell receptors disclosed herein. Attorney Docket No.15271.0015-00304

[0036] In some embodiments, the present disclosure provides an application of the antibodies, antigen-binding fragments, and bispecific antibodies disclosed herein in the manufacture of a medicament for the treatment or prevention of cancer or autoimmune diseases.

[0037] In some embodiments, the present disclosure provides dosage and route of administration of the medicament using the antibodies, antigen-binding fragments, and bispecific antibodies disclosed herein for treating cancers, preferably, pancreatic cancer, triple negative breast cancer, lung cancer or any cancer with disease associated antigen expression.

[0038] In some embodiments, the antibodies, antigen-binding fragments, and bispecific antibodies disclosed herein can be used in a combination regimen, e.g., with chemotherapy.

[0039] These and other embodiments of the present disclosure will be described in greater detail herein. Brief Description of the Drawings

[0040] Figure 1A-1B. Schematic drawings of a bispecific antibody targeting disease associated antigen (DAA) (e.g., tumor associated antigen TAA such as 5T4) and γδ T cell receptor (γδTCR). Fragments of the heavy and light chains of the antibody and VHH are depicted as rectangles.

[0041] Figure 2A-2D. Schematic drawings of a bispecific antibody targeting disease associated antigen (DAA) and γδ T cell receptor (γδTCR) with IL-15 and sushi domain of IL-15 receptor α (SD-IL15). One or two SD-IL15 fusion domains are respectively attached at the C- terminus of the antibody heavy chains. Fragments of the heavy and light chains of the antibody are depicted as rectangles. The SD-IL15 fusion domains are shown as ovals. In some context of the disclosure, a “heavy chain” may refer to a heavy chain of an antibody or VHH fused to an immune modulator component such as an SD-IL15 fusion domain.

[0042] Figure 3. Flow cytometry of exemplary δ1 T cell (Figure 3A) and δ2 T cell (Figure 3B) population expanded from human PBMC. The X axe is Fluorescence Intensity of CD3 and Y axe is Fluorescence Intensity of γδ T cell.

[0043] Figure 4. Gel images of protein bands of humanized γδTCR 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum3HChum1LC_IgG1_L234AL235A (hum3HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), Attorney Docket No.15271.0015-00304 γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), and γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC) subjected to SDS-PAGE analysis under reduced and non-reduced conditions, respectively.

[0044] Figure 5. Binding assays of humanized γδTCR 5A6E9 antibodies. Figures 5A to 5D show ELISA assay results showing humanized γδTCR 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum3HChum1LC_IgG1_L234AL235A (hum3HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC), control δ1TCR specific antibody (δ1 antibody), and control δ2TCR specific antibody (δ2 antibody), binding to human Vγ9Vδ1 (5A), human Vγ9Vδ2 (5B), human Vγ4Vδ1 (5C), and human Vγ4Vδ2 (5D) recombinant γδTCRs, respectively. The Y axes units are in Absorbance values at 450 nm. The x axes units are the concentration of the respective test articles in ng / mL units. Figure 5E shows flow cytometry-based binding assay results showing dose-dependent binding to Vγ9Vδ2 T cells by humanized γδTCR 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum3HChum1LC_IgG1_L234AL235A (hum3HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), and γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC). The Y axes units are folds of Mean Fluorescence Intensity over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units. Figure 5F shows flow cytometry-based binding assay results showing dose-dependent binding to Vδ1 T cells by humanized γδTCR 5A6E9 antibody γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC) and δ1TCR and δ2TCR antibodies. The Y axes units are folds of Mean Fluorescence Intensity over isotype control antibody. The X axes units are the concentration of the respective test articles in nM units.Figure 6. Jurkat cell NFAT reporter assays showing immobilized humanized γδTCR 5A6E9 antibodies in activating reporter gene expression. Humanized γδTCR 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum3HChum1LC_IgG1_L234AL235A (hum3HChum1LC), Attorney Docket No.15271.0015-00304 γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC), control δ1TCR specific antibody (δ1 antibody), and control δ2TCR specific antibody (δ2 antibody) were coated on a plate and their dose-dependent activation of luciferase reporter gene expression in Jurkat NFAT reporter cells transfected with human Vγ9Vδ1 TCR (6A) and human Vγ9Vδ2 TCR (6B) were quantitated. The Y axes units are luminescence Relative Light Units (RLU). The x axes units are the concentration of the respective test articles in ng / mL units.

[0045] Figure 7. Flow cytometry-based γδ T cell activation assays showing humanized γδTCR 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), and γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC), in inducing the activation and cytokine production of PBMC-derived Vγ9Vδ2 T cells. The data points represent relative expression of the activation markers CD25 (Figure 7A), CD69 (Figure 7B) and IFNγ (Figure 7C) in activated Vγ9Vδ2 T cells. The Y axes units are fold of Mean Fluorescence Intensity (MFI, Figure 7A and 7B) or the percent of positive cells (Figure 7C) over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units. Figure 7D shows γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC) antibody, δ1TCR antibody and δ2TCR antibody in inducing the activation of CD69 production from activated Vδ1 T cells. The Y axes units are fold of Mean Fluorescence Intensity over isotype control antibody. The X axes units are the concentration of the respective test articles in nM units.

[0046] Figure 8. Flow cytometry-based γδ T cell proliferation assays showing humanized γδTCR 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), and γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC), in inducing the proliferation of γδ T cells when PBMCs were cultured with antibodies coated on plates for 7 days. The data points represent relative quantity of activated γδ T cells. The Y axes units are fold Attorney Docket No.15271.0015-00304 of γδ T cell numbers over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units.

[0047] Figure 9. Characterizations of bispecific antibody 5T4 x γδTCR. Figure 9A shows gel images of protein bands of bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), subjected to SDS- PAGE analysis under reduced and non-reduced conditions. Figure 9B shows a cation exchange chromatography (CEX) analysis of bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC). The Y axes units are in Absorbance values at 280 nm. The x axes units are retention time in minutes.

[0048] Figure 10. ELISA assay of bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A hum4HChum1LC), binding to human 5T4. The Y axes units are in Absorbance values at 450 nm. The x axes units are the concentration of the respective test articles in ng / mL units.

[0049] Figure 11. Flow cytometry-based binding assay showing dose-dependent binding of bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A hum4HChum1LC), to 5T4 expressing NCI-H226 cells (Figure 11A) and MCF-7 cells (Figure 11B). The Y axes units are Mean Fluorescence Intensity (MFI). The X axes units are the concentration of the respective test articles in ng / mL units.

[0050] Figure 12. Flow cytometry-based binding assay showing dose-dependent binding of bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the Attorney Docket No.15271.0015-00304 corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A hum4HChum1LC), to Vγ9Vδ2 T cells. The Y axes units are folds of Mean Fluorescence Intensity over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units.

[0051] Figure 13. Jurkat cell NFAT reporter assays showing 5T4 x γδTCR bispecific antibodies in mediating 5T4-expression tumor cells in activating reporter gene expression in Jurkat NFAT reporter cells transfected with human γδ TCR. 5T4 x γδTCR 5A6E9 bispecific antibodies, 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC), 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC), 5T4_PFA1 x γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (5T4 x hum5HChum4LC), 5T4_PFA1 x γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (5T4 x hum6HChum4LC), 5T4_PFA1 x control δ1TCR specific antibody (5T4 x δ1 antibody), and 5T4_PFA1 x control δ2TCR specific antibody (5T4 x δ2 antibody) were incubated with 5T4- expressing MCF-7 cells and Jurkat NFAT reporter cells transfected with human Vγ9Vδ1 TCR (13A), 5T4-expressing MCF-7 cells and Jurkat NFAT reporter cells transfected with human Vγ9Vδ2 TCR (13B), 5T4-expressing NCI-H226 cells and Jurkat NFAT reporter cells transfected with human Vγ9Vδ1 TCR (13C), 5T4-expressing NCI-H226 cells and Jurkat NFAT reporter cells transfected with human Vγ9Vδ2 TCR (13D) and their dose-dependent activation of luciferase reporter gene expression in transfected Jurkat NFAT reporter cells were quantitated. The Y axes units are luminescence Relative Light Units (RLU). The x axes units are the concentration of the respective test articles in ng / mL units.

[0052] Figure 14. Flow cytometry-based γδ T cell activation assays showing bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A hum4HChum1LC), in inducing the activation and cytokine production of PBMC derived Vγ9Vδ2 T cells. The data points represent relative expression of the activation markers CD69 (Figure 14A) and IFNγ (Figure 14B) in activated Vγ9Vδ2 T cells. The Y axes units are fold of Mean Fluorescence Intensity over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units. Attorney Docket No.15271.0015-00304

[0053] Figure 15. Flow cytometry-based γδ T cell cytotoxic degranulation assays showing bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), in inducing the expression of CD107a marker in activated PBMC derived Vγ9Vδ2 T cells. The Y axes units are percentage of CD107a-positive Vγ9Vδ2 T cells. The X axes units are the concentration of the respective test articles in ng / mL units.

[0054] Figure 16. Flow cytometry-based γδ T cell cytotoxicity assays showing bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and the corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), mediating Vγ9Vδ2 T cell cytotoxicity against 5T4 expressing MCF-7 cells in vitro. The Y axes units are percentage of MCF-7 cell lysis. The X axes units are the concentration of the respective test articles in ng / mL units.

[0055] Figure 17. Humanized xenograft BxPC-3 tumor model showing the tumor growth inhibition efficacy of bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC) in the treatment of BxPC-3 tumors along with inoculated γ9δ2 T cells expanded from human donor PBMC. Figure 17A shows tumor tumor volume upon treatment (n=5). The Y axes units are tumor volume. The X axes units are days post treatment. The arrows mark the timing of γ9δ2 T cell inoculations. Figure 17B shows mice body weight upon treatment. The Y axes units are body weight. The X axes units are days post treatment.

[0056] Figure 18 Characterizations of bispecific antibody 5T4 x γδTCR with attenuated IL-15. Figure 18A shows gel images of protein bands of bispecific antibodies 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K), and their corresponding parental antibodies, subjected to SDS-PAGE analysis under reduced and non-reduced conditions, respectively. Figure 18B shows Attorney Docket No.15271.0015-00304 a cation exchange chromatography (CEX) analysis of the 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and its corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A_SD-IL15_I68K (5T4_SD-IL15_I68K) and γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (hum2HChum1LC_SD- IL15_I68K). The Y axes units are in Absorbance values at 280 nm. The x axes units are retention time in minutes.

[0057] Figure 19. Characterizations of IL-15 functional activities of bispecific antibody 5T4 x γδTCR with attenuated IL-15. Figure 19A. IL-15 reporter assays showing bispecific antibodies 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K) and the 5T4_PFA1 antibody with native SD-IL15 (5T4_SD-IL15) in inducing the expression of reporter gene expression. The Y axes units are in Absorbance values at 650 nm. The X axes units are the concentration of the respective test articles in ng / mL units. Figure 19B. CTLL-2 cell proliferation assays showing bispecific antibodies 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K) and the 5T4_PFA1 antibody with native SD-IL15 (5T4_SD-IL15) in driving CTLL-2 cell proliferation. The Y axes units are luminescence Relative Light Units (RLU). The x axes units are the concentration of the respective test articles in ng / mL units.

[0058] Figure 20. Flow cytometry-based γδ T cell activation assays showing γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (hum2HChum1LC_SD- IL15_I68K) and γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (hum2HChum1LC_SD-IL15_S7TI68K) antibodies in inducing the activation and cytokine production of PBMC derived Vγ9Vδ2 T cells when coated on plates for overnight. The data points represent relative expression of the activation markers CD25 (Figure 20A), CD69 Attorney Docket No.15271.0015-00304 (Figure 20B) and IFNγ (Figure 20C) in activated Vγ9Vδ2 T cells. The Y axes units are fold of Mean Fluorescence Intensity (MFI, Figure 20A and 20B) or the percent of positive cells (Figure 20C) over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units.

[0059] Figure 21. Flow cytometry-based γδ T cell proliferation and activation assays showing γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (hum2HChum1LC_SD- IL15_I68K) and γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (hum2HChum1LC_SD-IL15_S7TI68K) antibodies in inducing the proliferation and activation γδ T cells when PBMCs were cultured with antibodies coated on plates for 7 days. The data points represent relative γδ T cell numbers (Figure 21A) and the expression of the activation marker CD25 (Figure 21B) in activated γδ T cells. The Y axes units are fold of γδ T cell numbers (Figure 21A) and Mean Fluorescence Intensity (Figure 21B) over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units.

[0060] Figure 22. Flow cytometry-based γδ T cell proliferation and activation assays showing bispecific antibodies 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC), 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K) in inducing the proliferation and activation of γδ T cells upon CD3+T cells enriched from PBMCs co-culturing with MCF-7 cells for 7 days. The data points represent relative γδ T cell numbers (Figure 22A) and the expression of the activation marker CD25 (Figure 22B) in activated γδ T cells. The Y axes units are fold of γδ T cell numbers (Figure 22A) and Mean Fluorescence Intensity (Figure 22B) over isotype control antibody. The X axes units are the concentration of the respective test articles in ng / mL units.

[0061] Figure 23. Flow cytometry-based γδ T cell cytotoxicity assays showing bispecific antibodies 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC), 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD- IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4 x Attorney Docket No.15271.0015-00304 γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K) in mediating Vγ9Vδ2 T cell cytotoxicity against 5T4 expressing NCI-H226 cells in vitro. The Y axes units are percentage of NCI-H226 cell lysis. The X axes units are the concentration of the respective test articles in ng / mL units.

[0062] Figure 24A. ELISA assay of anti-CD123 antibodies binding to human CD123. The Y axes units are in Absorbance values at 450 nm. The X axes units are the concentration of the respective test articles in ng / mL units. Figure 24B and Figure 24C. Flow cytometry-based binding assays showing dose-dependent binding of anti-CD123 antibodies binding to MOLM-13 cells. The Y axes units are Mean Fluorescence Intensity (MFI). The X axes units are the concentration of the respective test articles in ng / mL units.

[0063] Figure 25A and Figure 25B. Flow cytometry-based binding assays showing dose-dependent binding of anti-CD123 antibodies with SD-attenuated IL-15 binding to MOLM- 13 cells. The Y axes units are Mean Fluorescence Intensity (MFI). The X axes units are the concentration of the respective test articles in ng / mL units.

[0064] Figure 26. ELISA assays showing exemplar CD123 x γδTCR bispecific antibodies binding to human CD123 (Figure 26A) or γ9δ2 TCR (Figure 26B). The Y axes units are in Absorbance values at 450 nm. The X axes units are the concentration of the respective test articles in pM units.

[0065] Figure 27A-F. Jurkat cell NFAT reporter assays showing CD123 x γδTCR bispecific antibodies in mediating CD123-expression tumor cells in activating reporter gene expression in Jurkat NFAT reporter cells transfected with human γδ TCR. The Y axes units are luminescence Relative Light Units (RLU). The X axes units are the concentration of the respective test articles in pM units.

[0066] Figure 28A, C, E, and G. Flow cytometry-based γδ T cell degranulation assays showing CD123-flotetuzumab x 5A6E9 bispecific antibody along with null control antibodies in inducing the expression of CD107a marker in activated PBMC derived δ1 T cells or δ2 T cells. The Y axes units are percentage of CD107a-positive γδ T cells. The X axes units are the concentration of the respective test articles in pM units. Figure 28B, D, F, and H. Flow cytometry-based γδ T cell cytotoxicity assays showing CD123-flotetuzumab x 5A6E9 bispecific antibody along with null control antibodies in mediating δ1 T cells or δ2 T cells cytotoxicity Attorney Docket No.15271.0015-00304 against CD123 expressing cells in vitro. The Y axes units are percentage of tumor cell lysis. The X axes units are the concentration of the respective test articles in pM units.

[0067] Figure 29. Characterizations of IL-15 functional activities of bispecific antibody CD123 x γδTCR with attenuated IL-15. Figure 29A. IL-15 reporter assays showing CD123- flotetuzumab x 5A6E9 bispecific antibodies with different numbers of SD-IL15_S7TI68K in inducing the expression of reporter gene expression. The Y axes units are in Absorbance values at 650 nm. The X axes units are the concentration of the respective test articles in pM units. Figure 29B. CTLL-2 cell proliferation assays showing CD123-flotetuzumab x 5A6E9 with different numbers of SD-IL15_S7TI68K in driving CTLL-2 cell proliferation. The Y axes units are luminescence Relative Light Units (RLU). The X axes units are the concentration of the respective test articles in pM units.

[0068] Figure 30. Flow cytometry-based γδ T cell activation assays showing null x 5A6E9 bispecific antibodies with different numbers of SD-IL15_S7TI68K in inducing the activation and cytokine production of PBMC derived Vγ9Vδ2 T cells when coated on plates for overnight. The data points represent relative expression of the activation markers CD69 (Figure 30A), CD25 (Figure 30B) and IFNγ (Figure 30C) in activated Vγ9Vδ2 T cells. The Y axes units are fold of Mean Fluorescence Intensity (MFI, A and B) or the percent of positive cells (C) over isotype control antibody. The X axes units are the concentration of the respective test articles in nM units.

[0069] Figure 31. Flow cytometry-based T cell proliferation and activation assays showing CD123-flotetuzumab x 5A6E9 bispecific antibody and related null control antibodies with one, two, and no SD-IL15_S7TI68K engineered on the molecules in inducing the proliferation and activation of T cells upon PBMCs co-culturing with MV-4-11 cells for 7 days. The data points represent the percent γδ T cell proliferation (Figure 31A, B, and C), the expression of the activation marker CD69 on activated γδ T cells (Figure 31D, E, and F), the expression of the activation marker CD25 on activated γδ T cells (Figure 31 G, H, and I), and the proliferation and activation of αβ T cells (Figure 31 J, K, and L). The Y axes units are percentage of T cell proliferation (Figure 31 A, B, C, and J) and Mean Fluorescence Intensity (Figure 31D, E, F, G, H, I, K, and L) over isotype control antibody. The X axes units are the concentration of the respective test articles in pM units. Attorney Docket No.15271.0015-00304

[0070] Figure 32. Flow cytometry-based cytotoxicity assays showing CD123- flotetuzumab x 5A6E9 bispecific antibody and related null control antibodies with no (Figure 32A), one (Figure 32B), and two (Figure 32C) SD-IL15_S7TI68K engineered on the molecules in mediating cytotoxicity of γδ T cells from PBMC against CD123 expressing MV-4-11 cells in vitro. The Y axes units are quantity of remaining live tumor cells. The X axes units are the concentration of the respective test articles in pM units.

[0071] Figure 33. Humanized xenograft MOLM-13 tumor model showing the tumor growth inhibition efficacy of CD123 x γδTCR bispecific antibodies in the treatment of MOLM- 13 tumors along with inoculated γ9δ2 T cells expanded from human donor PBMC. Figure 33A shows the study design and dosing scheme. Figure 33B shows total flux luc signal (p / s) from ex vivo live imaging of tumor burden upon treatment. Figure 33C shows average mice body weights upon treatment. Figure 33D shows representative images of ex vivo live imaging of tumor burden upon treatment.

[0072] Figure 34. Humanized xenograft MOLM-13 tumor model showing the tumor growth inhibition efficacy of CD123 x γδTCR bispecific antibodies in the treatment of MOLM- 13 tumors along with human donor PBMC. Figure 34A shows the study design and dosing scheme. Figure 34B shows total flux luc signal (p / s) from ex vivo live imaging of tumor burden upon treatment. Figure 34C shows average mice body weights upon treatment. Figure 34D shows representative images of ex vivo live imaging of tumor burden upon treatment.

[0073] Figure 35A-D. Jurkat cell NFAT reporter assays showing CD20 x γδTCR bispecific antibodies in mediating CD20-expression tumor cells in activating reporter gene expression in Jurkat NFAT reporter cells transfected with human γδ TCR. The Y axes units are luminescence Relative Light Units (RLU). The X axes units are the concentration of the respective test articles in pM units. Figure 35E. Flow cytometry-based γδ T cell cytotoxicity assays showing CD20-rituximab x 5A6E9 bispecific antibody along with null control antibodies in mediating δ2 T cell cytotoxicity against CD20 expressing cells in vitro. The Y axes units are percentage of tumor cell lysis. The X axes units are the concentration of the respective test articles in pM units.

[0074] Figure 36. Jurkat cell NFAT reporter assays showing CDH17 x γδTCR bispecific antibodies in mediating CDH17-expression tumor cells in activating reporter gene expression in Jurkat NFAT reporter cells transfected with human γδ TCR. The Y axes units are Attorney Docket No.15271.0015-00304 luminescence Relative Light Units (RLU). The X axes units are the concentration of the respective test articles in pM units.

[0075] Figure 37. ELISA-binding assays showing various γδTCR VHH binding to human Vγ9Vδ1 (Figure 37A and B), human Vγ9Vδ2 (Figure 37C and D), human Vγ4Vδ1 (Figure 37E and F), and human Vγ4Vδ2 (Figure 37G and H) recombinant γδTCRs, respectively. The Y axes units are in Absorbance values at 450 nm. The X axes units are the concentration of the respective test articles in pM units.

[0076] Figure 38. Jurkat cell NFAT reporter assays showing CD123 x γδTCR VHH bispecific antibodies in mediating CD123-expression tumor cells in activating reporter gene expression in Jurkat NFAT reporter cells transfected with human γδ TCR. The Y axes units are luminescence Relative Light Units (RLU). The X axes units are the concentration of the respective test articles in pM units.

[0077] Figure 39. Characterization of CD123 x γδTCR VHH bispecific antibodies with different CD123 binding arms paired with Pan-371VHH. ELISA-binding assays showing tested CD123 x γδTCR VHH bispecific antibodies binding to human CD123 (Figure 39A) and human Vγ9Vδ2 (Figure 39B). Jurkat cell NFAT reporter assays showing tested CD123 x γδTCR VHH bispecific antibodies in mediating CD123-expression tumor cells in activating reporter gene expression in Jurkat NFAT reporter cells transfected with human γ9δ1 TCR (Figure 39C) and γ9δ1 TCR (Figure 39D). The Y axes units are luminescence Relative Light Units (RLU). The X axes units are the concentration of the respective test articles in pM units. Detailed description Definitions

[0078] All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth. If certain content of a reference cited herein contradicts or is inconsistent with the present disclosure, the present disclosure controls.

[0079] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Attorney Docket No.15271.0015-00304

[0080] Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, exemplary materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

[0081] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0082] “Antibodies” is meant in a broad sense and includes immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, trispecific or multi-specific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity.

[0083] “Full length antibody molecules” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g., IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CH1, hinge, CH2and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions may be further subdivided into regions of hyper variability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VHand VLis composed of three CDRs and four FR segments, arranged from amino-to-carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0084] “Complementarity determining regions (CDR)” are “antigen binding sites” in an antibody. CDRs may be defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) “Hypervariable regions,” “HVR,” or “HV,” three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3) refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk 1987). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides a Attorney Docket No.15271.0015-00304 standardized numbering and definition of antigen-binding sites. The correspondence between CDRs, HVs and IMGT delineations are described (Lefranc, Pommie et al. 2003). The term “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2” and “LCDR3” as used herein includes CDRs defined by any of the methods described supra, Kabat, Chothia or IMGT, unless otherwise explicitly stated in the specification.

[0085] Immunoglobulins may be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant region amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species may be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant regions.

[0086] “Antibody fragments” refers to a portion of an immunoglobulin molecule that retains the heavy chain and / or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include well known Fab, F(ab’)2, Fd and Fv fragments as well as domain antibodies (dAb) consisting of one VH domain. VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains may pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Disclosure Publ. Nos. WO1998 / 44001, WO1988 / 01649, WO1994 / 13804 and WO1992 / 01047.

[0087] “Monoclonal antibody” refers to an antibody population with single amino acid composition in each heavy and each light chain, except for possible well-known alterations such as removal of C-terminal lysine from the antibody heavy chain. Monoclonal antibodies typically bind one antigenic epitope, except that multispecific monoclonal antibodies bind multiple distinct antigenic epitopes. Monoclonal antibodies may have heterogeneous glycosylation within the antibody population. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent.

[0088] “Isolated antibody” refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities. “Isolated antibody” Attorney Docket No.15271.0015-00304 encompasses antibodies that are isolated to a higher purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0089] “Humanized antibody” refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework so that the framework may not be an exact copy of expressed human immunoglobulin or human immunoglobulin germline gene sequences.

[0090] “Human antibody” refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding site are derived from sequences of human origin. If the antibody contains a constant region or a portion of the constant region, the constant region also is derived from sequences of human origin.

[0091] “Anti-target” refers to an antibody or antibody domain that can bind to the specified target molecule such as 5T4 or γδ T cell receptor (γδTCR) (i.e., anti-5T4 is an antibody or antibody domain that can bind to . The style “5T4” refers to a 5T4 protein or 5T4 gene product.

[0092] The numbering of amino acid residues in the antibody constant region throughout the specification is according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991), unless otherwise explicitly stated.

[0093] Conventional one and three-letter amino acid codes are used herein as shown in Table 1. Table 1 Three-letter Amino acid One-letter code Attorney Docket No.15271.0015-00304 Glutamine Glu Q Glycine Gly G

[0094] The polypeptides, nucleic acids, fusion proteins, and other compositions provided herein may encompass polypeptides, nucleic acids, fusion proteins, and the like that have a recited percent identity to an amino acid sequence or DNA sequence provided herein. The term “identity” refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. “Percent identity,” “percent homology,” “sequence identity,” or “sequence homology” and the like mean the percent of identical residues between the amino acids or nucleotides in the compared molecules and is calculated based on the size of the smallest of the molecules being compared. For these calculations, gaps in alignments (if any) are preferably addressed by a particular mathematical model or computer program (an algorithm). Methods that can be used to calculate the identity of the aligned nucleic acids or polypeptides include those described in Computational Molecular Biology (Lesk, A. M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. Attorney Docket No.15271.0015-00304 and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. In calculating percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences.

[0095] The constant region sequences of the mammalian IgG heavy chain are designated in sequence as CH1-hinge-CH2-CH3. The “hinge,” “hinge region” or “hinge domain” of an IgG is generally defined as including Glu216 and terminating at Pro230 of human IgG1 according to the EU Index but functionally, the flexible portion of the chain may be considered to include additional residues termed the upper hinge region referred to as residues Glu216 to Gly237 and the lower hinge referred to as residues Glu233 to Ser239 of the Fc region where FcγR binding was generally attributed. Hinge regions of other IgG isotypes may be aligned with the IgG1sequence by placing the first and last cysteine residues forming inter-heavy chain S-S bonds. Although boundaries may vary slightly, as numbered according to the EU Index, the CH1 domain is adjacent to the VH domain and amino terminal to the hinge region of an immunoglobulin heavy chain molecule and includes the first (most amino terminal) constant region of an immunoglobulin heavy chain, e.g., from about EU positions 118-215. The Fc domain extends from amino acid 231 to amino acid 447; the CH2 domain is from about Ala231 to Lys340 or Gly341; and the CH3from about Gly341 or Gln342 to Lys447. The residues of the IgG heavy chain constant region of the CH1 region typically terminate at Lys. An Fc domain containing molecule may comprise at least the CH2 and the CH3 domains of an antibody constant region, e.g., comprising at least a region from about Ala231 to Lys447 of IgG heavy chain constant region. An Fcdomain containing molecule may optionally comprise at least a portion of the hinge region.

[0096] “Epitope” refers to a portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be composed of contiguous and / or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acids from differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule. Antibody “epitope” typically depends on the methodology used to identify the epitope. Attorney Docket No.15271.0015-00304

[0097] A “leader sequence” as used herein includes any signal peptide that can be processed by a mammalian cell, including the human B2M leader. Such sequences are well- known in the art.

[0098] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms also include polypeptides that have co-translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and the like.

[0099] Furthermore, as used herein, a “polypeptide” refers to a protein that includes modifications, such as deletions, additions, and substitutions (generally conservative in nature as would be known to a person in the art) to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods.

[0100] The term “recombinant,” as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term “recombinant,” as used with respect to a protein or polypeptide, refers to a polypeptide produced by expression from a recombinant polynucleotide. The term “recombinant,” as used with respect to a host cell or a virus, refers to a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to a material (e.g., a cell, a nucleic acid, a protein, or a vector) that has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0101] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to include a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Attorney Docket No.15271.0015-00304

[0102] “Vector” refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system, such as a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The vector polynucleotide may be DNA or RNA molecules, cDNA, or a hybrid of these, single stranded or double stranded.

[0103] “Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0104] As used herein, the term “heterologous” used in reference to nucleic acid sequences, proteins or polypeptides, means that these molecules are not naturally occurring in the cell from which the heterologous nucleic acid sequence, protein or polypeptide was derived. For example, the nucleic acid sequence coding for a human polypeptide that is inserted into a cell that is not a human cell is a heterologous nucleic acid sequence in that particular context. Whereas heterologous nucleic acids may be derived from different organism or animal species, such nucleic acid need not be derived from separate organism species to be heterologous. For example, in some instances, a synthetic nucleic acid sequence or a polypeptide encoded therefrom may be heterologous to a cell into which it is introduced in that the cell did not previously contain the synthetic nucleic acid. As such, a synthetic nucleic acid sequence or a polypeptide encoded therefrom may be considered heterologous to a human cell, e.g., even if one or more components of the synthetic nucleic acid sequence or a polypeptide encoded therefrom was originally derived from a human cell.

[0105] A “host cell,” as used herein, denotes an in vivo or in vitro eukaryotic or prokaryotic cell or a cell from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, wherein the cell can be, or have been, used as recipients for a nucleic acid (e.g., an expression vector that comprises a nucleotide sequence encoding a multimeric polypeptide of the present disclosure), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A “recombinant host cell” Attorney Docket No.15271.0015-00304 (also referred to as a “genetically modified host cell”) is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector. For example, a genetically modified eukaryotic host cell is genetically modified by virtue of introduction into a suitable eukaryotic host cell a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell, or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.

[0106] “Specific binding” or “specifically binds” or “binds” or “targets” or “targeting” refer to an antibody binding to a specific antigen with greater affinity than for other antigens. Typically, the antibody “specifically binds” when the equilibrium dissociation constant (KD) for binding is about 1×10-8M or less, for example about 1×10-9M or less, about 1×10-10M or less, about 1×10-11M or less, or about 1×10-12M or less, typically with the KDthat is at least one hundred-fold less than its KD for binding to a non-specific antigen (e.g., BSA, casein). The KD may be measured using standard procedures.

[0107] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0108] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.

[0109] A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent, or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated. Attorney Docket No.15271.0015-00304

[0110] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0111] The following examples described the invention in further detail which are not intended to limit the scope of protection for the invention. γδ T cell-based Therapies

[0112] Unlike αβ T cells, γδ T cells can detect cancers through innate natural killer receptors without the obligate requirement for cognate tumor associated antigen presentation on major histocompatibility complex (MHC) molecules. γδ T cells can produce large quantities of cytokines and chemokines rapidly while residing in the blood circulation or in the non-lymphoid tissues (e.g., skin, intestines, and lungs); thereby providing a first line of immunosurveillance against aberrant cell growth and infectious diseases to bridge the innate and adaptive immune responses (Silva-Santos, Serre et al. 2015). γδ T-cells were identified as the prognostically most favorable immune cell subset in tumor infiltrates from 18,000 tumors across 39 malignancies (Gentles, Newman et al. 2015). A more recent study confirmed the relative abundance of Vγ9Vδ2 T-cells in TILs and their association with improved patient outcome (Tosolini, Pont et al. 2017). Thus, higher γδ T-cell frequency in tumor from cancer patients correlates with better clinical outcomes in different malignancies (Donia, Ellebaek et al. 2012, Meraviglia, Lo Presti et al. 2017, Wang, Lin et al. 2017, Lu, Dai et al. 2020).

[0113] In humans, γδ T-cells represent 1 to 10% of total CD3+ T-cells and express a combination of 7 different Vγ TCR (T cell receptor) chains (Vγ2, 3, 4, 5, 8, 9, and 11), paired with either of 4 Vδ (Vδ1, 2, 3, and 5) chains (Adams, Gu et al. 2015, Zhao, Niu et al. 2018). The Vγ9Vδ2 T-cell subset, with Vγ9 paired with Vδ2, is the predominant pro-inflammatory effector γδ T-cell subset in peripheral blood to combat tumor malignancy and infection (Saura-Esteller, de Jong et al. 2022). Vγ9Vδ2 T-cells participate in the defense against malignant cells by sensing small phosphorylated metabolites (phosphoantigen (pAg) molecules) produced by cholesterol biosynthesis or by pathogens (Tanaka, Morita et al. 1995). Increased levels of intracellular pAg levels arising from stress conditions like infection or malignant transformation can activate Attorney Docket No.15271.0015-00304 Vγ9Vδ2 T-cells to initiate target cell killing. The Vγ9Vδ2 T-cells can also be activated by ligands that include butyrophilin (BTN) 3A1 and BTN2A1 (Cano, Pasero et al. 2021).

[0114] Vδ1 T-cells, which are less abundant in circulation than the Vγ9Vδ2 T-cells, are enriched in tissues including the skin, large intestine, spleen, liver, and the tumor infiltrating lymphocytes (TILs). The Vδ1 T-cell subset, with Vδ1 paired with different Vγ TCR chains, contains both effector T-cells as well as regulatory T-cells that can promote tumor growth (Zhao, Niu et al. 2018). Several studies have shown that Vδ1 T-cells have TCR recognizing different ligands, including pathogenic and self-lipids presented by CD1d (Luoma, Castro et al. 2013) (Mangan, Dunne et al. 2013).

[0115] Considering the promising role of γδ T-cells in the immunosurveillance against tumor cells, novel γδ T-cell-based immunotherapies are being developed in recent years (Saura- Esteller, de Jong et al. 2022). Since Vδ2 T cells have historically been easier to isolate and expand compared to Vδ1 T cells, Vγ9Vδ2 T cells were employed for tumor killing in earlier clinical studies. Such Vγ9Vδ2 T cells were either stimulated and expanded in vivo by systemic administration of pAgs or expanded ex vivo followed by adoptive transfer of autologous Vγ9Vδ2 T cells. Another promising approach is to use a bispecific molecule capable of redirecting the γδ T cells to kill tumor cells in a potent and specific manner. Both δ2 T cells and δ1 T cells can be used as a cancer immunotherapy since δ1 T cells constitute the dominant subset of γδ T cells that can infiltrate into tumor tissues.

[0116] So far most reported γδ T cell-based therapeutics have only shown suboptimal clinical efficacy so there is a clear need for the development of better therapeutic strategies that can stimulate both δ1 and δ2 T cell subsets for the elimination of tumor cells. Molecules that can stimulate and maintain a large quantity of activated γδ T cells will allow for better therapeutic interventions. The bispecific antibody disclosed herein offer a novel and effective immunotherapeutic approach harnessing γδ T cells. Antibodies such as bispecific antibodies targeting disease associated antigens and γδ T cell receptors

[0117] In some embodiments, the present disclosure provides molecular entities, such as antibodies or antigen-binding fragments, comprising one or more sequences set forth in Table Attorney Docket No.15271.0015-00304 8. In some embodiments, the antibodies or antigen-binding fragments disclosed herein are multispecific (such as bispecific).

[0118] In some embodiments, the present disclosure provides a bispecific antibody that has dual binding specificities to both disease associated antigen (DAA) and γδ T cell receptor (γδTCR). The bispecific antibody disclosed herein can mediate the engagement of γδ T cells to tumor cells with the expression of the specific disease associated antigen and facilitate the activated γδ T cells to eliminate the engaged tumor cells.

[0119] In some embodiments, the disease associated antigen (DAA) disclosed herein includes, but not limited to, 5T4, CD123, HER2, EGFR, cMET, Nectin-4, ROR1, CD19, CD20, CD22, CD38, CD79b, CD318, LRRC15, mesothelin, MUC1, MUC16, MUC17, FAP, and CDH17. In some embodiments, the bispecific antibody disclosed herein binds to more than a single DAA comprising some of the aforementioned antigens.

[0120] In some embodiments, the bispecific antibody disclosed herein comprises one set of heavy chain and light chain with binding specificity to a disease associated antigen as the DAA-binding arm and another set of heavy chain and light chain or VHH with binding specificity to either of or both δ1 and δ2 subtypes of γδ T cell receptors (γδTCR), as the γδTCR binding arm. As a non-limiting example, the disclosure provides illustrations of configurations of such bispecific antibody disclosed herein in Figure 1A to 1B.

[0121] The present disclosure provides a bispecific antibody that can be prepared using well established point mutations in the CH1, CH2, and CH3 domains via bispecific antibody technology such as controlled Fab arm exchange or via co-expression. In some embodiments, all controlled Fab arm exchange or co-expression constructs are symmetric so that there is no preference for the selection of point mutations of the respective parental antibodies.

[0122] In some embodiments, the therapeutic antibodies and fragments applicable for the bispecific antibody design of the present disclosure encompass full length antibody comprising two heavy chains and two light chains. The antibodies can be human or humanized antibodies. Humanized antibodies include chimeric antibodies and CDR-grafted antibodies. Chimeric antibodies are antibodies that include a non-human antibody variable region linked to a human constant region. CDR-grafted antibodies are antibodies that include the CDRs from a non-human “donor” antibody linked to the framework region from a human “recipient” antibody. Exemplary human or humanized antibodies include IgG, IgM, IgE, IgA, and IgD antibodies. The Attorney Docket No.15271.0015-00304 present antibodies can be of any class (IgG, IgM, IgE, IgA, IgD, etc.) or isotype. For example, a human antibody can comprise an IgG Fc domain, such as at least one of isotypes, IgG1, IgG2, IgG3 or IgG4.

[0123] In some embodiments, the present disclosure provides a humanized antibody targeting 5T4, wherein the humanized antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 1, 2, and 3 respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 4, 5, and 6, respectively. In some embodiment, the humanized antibody targeting 5Τ4 comprises a heavy chain variable region having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 101, and a light chain variable region SEQ ID NO: 102 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 102.

[0124] In some embodiments, the present disclosure provides a humanized antibody targeting δ1 and / or δ2 subtypes of γδ T cell receptors, wherein the humanized antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 is selected from: SEQ ID NOs: 7, 43, 46, 49, 52, 55, 58, 61, 64, 67, and 70; the HCDR2 is selected from: SEQ ID NOs: 8, 44, 47, 50, 53, 56, 59, 62, 65, 68, and 71; and the HCDR3 is selected from: SEQ ID NOs: 9, 45, 48, 51, 54, 57, 60, 63, 66, 69, and 72. In some embodiments, the HCDR1, HCDR2, and HCDR3 comprise: SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 46, 47, and 48; SEQ ID NOs: 49, 50, and 51; SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 55, 56, and 57; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 61, 62, and 63; Attorney Docket No.15271.0015-00304 SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 67, 68, and 69; or SEQ ID NOs: 70, 71, and 72; respectively;

[0125] In some embodiments, the present disclosure provides a humanized antibody targeting δ1 and / or δ2 subtypes of γδ T cell receptors, wherein the humanized antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 7, 8, and 9 respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 10, 11, and 12. In some embodiment, the humanized antibody targeting δ1 and / or δ2 subtypes of γδ T cell receptors comprises a heavy chain variable region selected from SEQ ID NOs: 103-109 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 103-109, and / or a light chain variable region selected from SEQ ID NOs: 110-114 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 110- 114.

[0126] In some embodiments, the present disclosure provides a humanized antibody targeting CD123, wherein the humanized antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 13, 14, and 15, respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 16, 17, and 18, respectively. In some embodiment, the humanized antibody targeting CD123 comprises a heavy chain variable region SEQ ID NO: 115 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 115, and / or a light chain variable region SEQ ID NO: 116 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 116.

[0127] In some embodiments, the present disclosure provides a humanized antibody targeting CD123, wherein the humanized antibody comprises Attorney Docket No.15271.0015-00304 a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 is selected from: SEQ ID NOs: 19 and 25; the HCDR2 is selected from: SEQ ID NOs: 20 and 26; and the HCDR3 is selected from: SEQ ID NOs: 21 and 27; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1 is selected from: SEQ ID NOs: 22 and 28; the LCDR2 is selected from: SEQ ID NOs: 23 and 29; and the LCDR3 is selected from: SEQ ID NOs: 24 and 30.

[0128] In some embodiments, the present disclosure provides a humanized antibody targeting CD123, wherein the humanized antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 19, 20, and 21 respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 22, 23, and 24, respectively. In some embodiment, the humanized antibody targeting CD123 comprises a heavy chain variable region selected from SEQ ID NOs: 117 and 118, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 117 and 118, and a light chain variable region selected from SEQ ID NOs: 119 and 120, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 119 and 120.

[0129] In some embodiments, the present disclosure provides a humanized antibody targeting CD123, wherein the humanized antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 25, 26, and 27 respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 28, 29, and 30, respectively. In some embodiment, the Attorney Docket No.15271.0015-00304 humanized antibody targeting CD123 comprises a heavy chain variable region selected from SEQ ID NOs: 121 and 122, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 121 and 122, and a light chain variable region selected from SEQ ID NOs: 123 and 124, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 123 and 124.

[0130] In some embodiments, the present disclosure provides an antibody targeting CD20, wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 31, 32, and 33 respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 34, 35, and 36, respectively. In some embodiment, the antibody targeting CD20 comprises a heavy chain variable region as set forth as SEQ ID NO: 125, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NOs: 125, and a light chain variable region as set forth as SEQ ID NO: 126, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 126.

[0131] In some embodiments, the present disclosure provides an antibody targeting CDH17, wherein the antibody comprises a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 37, 38, and 39 respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 40, 41, and 42, respectively. In some embodiment, the antibody targeting CDH17 comprises a heavy chain variable region as set forth as SEQ ID NO: 127, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NOs: 127, and a light chain variable region as set forth as SEQ ID NO: 128, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 128. Attorney Docket No.15271.0015-00304

[0132] In some embodiments, the present disclosure provides a VHH targeting δ1 and / or δ2 subtypes of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 43, 44, and 45, respectively. In some embodiment, the VHH targeting δ1 and / or δ2 subtypes of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 129 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 129.

[0133] In some embodiments, the present disclosure provides a VHH targeting δ1 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 46, 47, and 48 respectively. In some embodiment, the VHH targeting δ1 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 130 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 130.

[0134] In some embodiments, the present disclosure provides a VHH targeting δ1 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 49, 50, and 51 respectively. In some embodiment, the VHH targeting δ1 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 131 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 131.

[0135] In some embodiments, the present disclosure provides a VHH targeting δ1 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 52, 53, and 54 respectively. In some embodiment, the VHH targeting δ1 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 132 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 132.

[0136] In some embodiments, the present disclosure provides a VHH targeting δ1 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region Attorney Docket No.15271.0015-00304 (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 55, 56, and 57 respectively. In some embodiment, the VHH targeting δ1 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 133 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 133.

[0137] In some embodiments, the present disclosure provides a VHH targeting δ2 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 58, 59, and 60 respectively. In some embodiment, the VHH targeting δ2 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 134 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 134.

[0138] In some embodiments, the present disclosure provides a VHH targeting δ2 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 61, 62, and 63 respectively. In some embodiment, the VHH targeting δ2 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 135 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 135.

[0139] In some embodiments, the present disclosure provides a VHH targeting δ2 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 64, 65, and 66 respectively. In some embodiment, the VHH targeting δ2 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 136 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 136.

[0140] In some embodiments, the present disclosure provides a VHH targeting δ2 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 67, 68, and 69 respectively. In some embodiment, the VHH targeting δ2 subtype Attorney Docket No.15271.0015-00304 of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 137 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 137.

[0141] In some embodiments, the present disclosure provides a VHH targeting δ2 subtype of γδ T cell receptors, wherein the VHH comprises complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 70, 71, and 72 respectively. In some embodiment, the VHH targeting δ2 subtype of γδ T cell receptors comprises a variable region sequence as set forth as SEQ ID NO: 138 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 138.

[0142] In some embodiments, the bispecific antibody disclosed herein comprises an anti-5T4 antibody PFA1 heavy chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 1, 2, and 3 respectively; and / or a light chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 4, 5, and 6, respectively.

[0143] In some embodiments, the bispecific antibody disclosed herein comprises an anti-5T4 antibody PFA1 heavy chain variable region amino acid sequence as set forth as SEQ ID NO: 101 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 101 and a light chain variable region amino acid sequence as set forth as SEQ ID NO: 102 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 102.

[0144] In some embodiments, the bispecific antibody disclosed herein comprises an anti-5T4 antibody PFA1 heavy chain with variable region amino acid sequence and a IgG1 Fc with L234A / L235A mutations as set forth as SEQ ID NO: 201 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 201, and anti-5T4 antibody light chain with variable region amino acid sequence as set forth as SEQ ID NO: 202 or an amino acid sequence having at least 85% (e.g., at Attorney Docket No.15271.0015-00304 least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 202.

[0145] In some embodiments, the bispecific antibody disclosed herein comprises antibody 5A6E9 targeting either of or both δ1 and δ2 subtypes of γδ T cell receptors (γδTCR) comprising a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 7, 8, and 9, respectively; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the bispecific antibody disclosed herein comprises antibody 5A6E9 targeting both δ1 and δ2 subtypes of γδ T cell receptors (γδTCR).

[0146] In some embodiments, the bispecific antibody disclosed herein comprises humanized anti-γδTCR antibody 5A6E9 heavy chain variable region amino acid sequence chosen from SEQ ID NOs: 103 to 109 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 103-109, and light chain variable region amino acid sequence chosen from SEQ ID NOs: 110 to 114 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 110- 114.

[0147] In some embodiments, the bispecific antibody disclosed herein comprises humanized anti-γδTCR antibody 5A6E9 heavy chain with variable region amino acid sequence and a IgG1 Fc with L234A / L235A mutations chosen from SEQ ID NOs: 203 to 209 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 203 to 209, and anti-γδTCR antibody 5A6E9 light chain with variable region amino acid sequence chosen from SEQ ID NOs: 210 to 214 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NOs: 210 to 214.

[0148] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody flotetuzumab heavy chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 13, 14, and 15 respectively; and / or a Attorney Docket No.15271.0015-00304 light chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 16, 17, and 18, respectively.

[0149] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody flotetuzumab heavy chain variable region amino acid sequence as set forth as SEQ ID NO: 115 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 115 and light chain variable region amino acid sequence as set forth as SEQ ID NO: 116 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 116.

[0150] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody flotetuzumab heavy chain with variable region amino acid sequence and a IgG1 Fc with L234A / L235A mutations as set forth as SEQ ID NO: 215 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 215, and anti-CD123 antibody light chain with variable region amino acid sequence as set forth as SEQ ID NO: 216 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 216.

[0151] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 10D1 heavy chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 19, 20, and 21, respectively; and / or a light chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 22, 23, and 24, respectively.

[0152] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 10D1 heavy chain variable region amino acid sequence chosen from SEQ ID NOs: 117 and 118 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 117 and 118, and light chain variable region amino acid sequence chosen from SEQ ID NOs: 119 and Attorney Docket No.15271.0015-00304 120, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 119 and 120.

[0153] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 10D1 heavy chain with variable region amino acid sequence and a IgG1 Fc with L234A / L235A mutations chosen from SEQ ID NOs: 217 and 218, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 217 and 218, and anti-CD123 antibody light chain with amino acid sequence chosen from SEQ ID NOs: 219 and 220, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 219 and 220.

[0154] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 1E12 heavy chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 25, 26, and 27, respectively; and / or a light chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 28, 29, and 30, respectively.

[0155] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 1E12 heavy chain variable region amino acid sequence chosen from SEQ ID NOs: 121 and 122 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 121 and 122, and light chain variable region amino acid sequence chosen from SEQ ID NOs: 123 and 124, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 123 and 124.

[0156] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 1E12 heavy chain with variable region amino acid sequence and a IgG1 Fc with L234A / L235A mutations chosen from SEQ ID NOs: 221 and 222, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 221 and 222, and anti-CD123 antibody light chain with amino acid sequence chosen from SEQ ID NOs: 223 and 224, or an amino acid sequence Attorney Docket No.15271.0015-00304 having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 223 and 224.

[0157] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD20 antibody rituximab heavy chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 31, 32, and 33 respectively; and / or a light chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 34, 35, and 36, respectively.

[0158] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD20 antibody rituximab heavy chain variable region amino acid sequence as set forth as SEQ ID NO: 125 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 125 and light chain variable region amino acid sequence as set forth as SEQ ID NO: 126 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 126.

[0159] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD20 antibody rituximab heavy chain with variable region amino acid sequence and a IgG1 Fc with L234A / L235A mutations as set forth as SEQ ID NO: 225 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 225, and anti-CD20 antibody light chain with variable region amino acid sequence as set forth as SEQ ID NO: 226 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 226.

[0160] In some embodiments, the bispecific antibody disclosed herein comprises anti- CDH17 antibody v1 heavy chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 37, 38, and 39 respectively; and / or a light chain variable region amino acid sequence comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprises SEQ ID NOs: 40, 41, and 42, respectively. Attorney Docket No.15271.0015-00304

[0161] In some embodiments, the bispecific antibody disclosed herein comprises anti- CDH17 antibody v1 heavy chain variable region amino acid sequence as set forth as SEQ ID NO: 127 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 127 and light chain variable region amino acid sequence as set forth as SEQ ID NO: 128 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 128.

[0162] In some embodiments, the bispecific antibody disclosed herein comprises anti- CDH17 antibody v1 heavy chain with variable region amino acid sequence and a IgG1 Fc with L234A / L235A mutations as set forth as SEQ ID NO: 227 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 227, and anti-CDH17 antibody light chain with variable region amino acid sequence as set forth as SEQ ID NO: 228 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 228.

[0163] In some embodiments, the bispecific antibody disclosed herein comprises VHH Pan-371 targeting either or both δ1 and δ2 subtypes of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 43, 44, and 45, respectively.

[0164] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR Pan-371 VHH variable region amino acid sequence as set forth as SEQ ID No. 129 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 129.

[0165] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR Pan-371 VHH amino acid sequence and a IgG1 Fc with L234A / L235A / Y349C / T366S / L368A / Y407V mutations as set forth as SEQ ID NO: 229 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 229.

[0166] In some embodiments, the bispecific antibody disclosed herein comprises VHH D1-275 targeting δ1 subtype of γδ T cell receptors (γδTCR) comprising complementarity Attorney Docket No.15271.0015-00304 determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 46, 47, and 48, respectively.

[0167] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D1-275 VHH variable region amino acid sequence as set forth as SEQ ID No. 130 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 130.

[0168] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D1-275 VHH amino acid sequence and a IgG1 Fc with L234A / L235A / Y349C / T366S / L368A / Y407V mutations as set forth as SEQ ID NO: 230 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 230.

[0169] In some embodiments, the bispecific antibody disclosed herein comprises VHH D1-130 targeting δ1 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 49, 50, and 51, respectively.

[0170] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D1-130 VHH variable region amino acid sequence as set forth as SEQ ID No. 131 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 131.

[0171] In some embodiments, the bispecific antibody disclosed herein comprises VHH D1-154 targeting δ1 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 52, 53, and 54, respectively.

[0172] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D1-154 VHH variable region amino acid sequence as set forth as SEQ ID No. 132 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 132.

[0173] In some embodiments, the bispecific antibody disclosed herein comprises VHH D1-124 targeting δ1 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 55, 56, and 57, respectively. Attorney Docket No.15271.0015-00304

[0174] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D1-124 VHH variable region amino acid sequence as set forth as SEQ ID No. 133 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 133.

[0175] In some embodiments, the bispecific antibody disclosed herein comprises VHH D2-263 targeting δ2 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 58, 59, and 60, respectively.

[0176] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D2-263 VHH variable region amino acid sequence as set forth as SEQ ID No. 134 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 134.

[0177] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D2-263 VHH amino acid sequence and a IgG1 Fc with L234A / L235A / Y349C / T366S / L368A / Y407V mutations as set forth as SEQ ID NO: 231 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 231.

[0178] In some embodiments, the bispecific antibody disclosed herein comprises VHH D2-368 targeting δ2 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 61, 62, and 63, respectively.

[0179] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D2-368 VHH variable region amino acid sequence as set forth as SEQ ID No. 135 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 135.

[0180] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D2-368 VHH amino acid sequence and a IgG1 Fc with L234A / L235A / Y349C / T366S / L368A / Y407V mutations as set forth as SEQ ID NO: 232 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 232. Attorney Docket No.15271.0015-00304

[0181] In some embodiments, the bispecific antibody disclosed herein comprises VHH D2-129 targeting δ2 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 64, 65, and 66, respectively.

[0182] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D2-129 VHH variable region amino acid sequence as set forth as SEQ ID No. 136 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 136.

[0183] In some embodiments, the bispecific antibody disclosed herein comprises VHH D2-142 targeting δ2 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 67, 68, and 69, respectively.

[0184] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D2-142 VHH variable region amino acid sequence as set forth as SEQ ID No. 137 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 137.

[0185] In some embodiments, the bispecific antibody disclosed herein comprises VHH D2-355 targeting δ2 subtype of γδ T cell receptors (γδTCR) comprising complementarity determining region (CDR) sequences CDR1, CDR2, and CDR3, wherein the CDR1, CDR2, and CDR3 comprises SEQ ID NOs: 70, 71, and 72, respectively.

[0186] In some embodiments, the bispecific antibody disclosed herein comprises anti- γδTCR D2-355 VHH variable region amino acid sequence as set forth as SEQ ID No. 138 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 138.

[0187] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody flotetuzumab heavy chain with variable region amino acid sequence and a IgG1 Fc with S354C / T366W mutations as set forth as SEQ ID NO: 233 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 233, and anti-CD123 antibody light chain with variable region amino acid sequence as set forth as SEQ ID NO: 216 or an amino acid sequence having at least Attorney Docket No.15271.0015-00304 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 216.

[0188] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 10D1 heavy chain with variable region amino acid sequence and a IgG1 Fc with S354C / T366W mutations chosen from SEQ ID NOs: 234 and 235, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 234 and 235, and anti-CD123 antibody light chain with amino acid sequence chosen from SEQ ID NOs: 219 and 220, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 219 and 220.

[0189] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD123 antibody 1E12 heavy chain with variable region amino acid sequence and a IgG1 Fc with S354C / T366W mutations chosen from SEQ ID NOs: 236 and 237, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 236 and 237, and anti-CD123 antibody light chain with amino acid sequence chosen from SEQ ID NOs: 223 and 224, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 223 and 224.

[0190] In some embodiments, the bispecific antibody disclosed herein comprises anti- 5T4 antibody PFA1 heavy chain with sequence as set forth as SEQ ID NO: 201, anti-5T4 antibody PFA1 light chain with sequence as set forth as SEQ ID NO: 202, anti-γδTCR antibody 5A6E9 heavy chain with sequence chosen from SEQ ID NO: 203 to 209, and anti-γδTCR antibody 5A6E9 light chain with sequence chosen from SEQ ID NO: 210 to 214 (e.g., as listed in Table 2). Table 2. 5T4 x γδTCR bispecific antibody 5T4 5T4 γδTCR γδTCR 0 0 Attorney Docket No.15271.0015-00304 HChum1LC_IgG1_L234AL235A 5T4 PFA1 x γδTCR 5A6E9hum4 0 3 3 , CD123 antibody heavy chain with sequence chosen from SEQ ID NOs: 215, 217, 218, 221, and 222, anti-CD123 antibody light chain with sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, anti-γδTCR antibody 5A6E9 heavy chain with sequence chosen from SEQ ID NOs: 203 to 209, and anti-γδTCR antibody 5A6E9 light chain with sequence chosen from SEQ ID NO: 210 to 214 (e.g., as listed in Table 3). Table 3. CD123 x γδTCR bispecific antibody Abbreviation CD12 CD12 γδTC γδTC 3 3 t n : : : : Attorney Docket No.15271.0015-00304 CD123_10D1hum2HChum2LC CD123- x γδTCR_5A6E9hum6 10D1h 2HCh 2L SEQ: SEQ: SEQ: SEQ: : : : :

[0192] In some embodiments, the bispecific antibody disclosed herein comprises anti- CD20 antibody heavy chain with sequence ae set forth as SEQ ID NO: 225, anti-CD20 antibody light chain with sequence as set forth as SEQ ID NO: 226, anti-γδTCR antibody 5A6E9 heavy chain with sequence chosen from SEQ ID NOs: 203 to 209, and anti-γδTCR antibody 5A6E9 light chain with sequence chosen from SEQ ID NOs: 210 to 214 (e.g., as listed in Table 6).

[0193] In some embodiments, the bispecific antibody disclosed herein comprises anti- CDH17 antibody heavy chain with sequence ae set forth as SEQ ID NO: 227, anti-CDH17 antibody light chain with sequence as set forth as SEQ ID NO: 228, anti-γδTCR antibody 5A6E9 heavy chain with sequence chosen from SEQ ID NOs: 203 to 209, and anti-γδTCR antibody 5A6E9 light chain with sequence chosen from SEQ ID NOs: 210 to 214 (e.g., as listed in Table 6).

[0194] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody with CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 233- Attorney Docket No.15271.0015-00304 237, a CD123 antibody light chain sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, and a γδTCR VHH sequence chosen from SEQ ID NOs: 229-232 (e.g., as listed in Table 7). IL-15 and sushi domain of IL-15Rα fusion protein to stimulate the proliferation and activation of γδ T cells

[0195] In some embodiments, the bispecific antibody targeting disease associated antigen and γδ T cell receptors disclosed herein optionally comprises an immune modulator component that can stimulate the proliferation and activation of γδ T cells. As a result, the number of γδ T cells increases significantly and the activated γδ T cells may eliminate tumor cells more efficiently.

[0196] In some embodiments, the optional immune modulator component comprises proinflammatory cytokine IL-15 which plays an important role in the stimulation of proliferation and activation of T cells, including γδ T cells, and NK cells. Human IL-15 comprises amino acid sequence as set forth as SEQ ID NO: 316.

[0197] In some embodiments, the optional immune modulator component comprises a fusion protein comprising the sushi domain (SD) of IL-15 receptor α and cytokine IL-15 (SD- IL15). In comparison to IL-15 alone, SD-IL15 is a much more potent agonist in stimulating the proliferation and activation of T cells, including γδ T cells, and NK cells. The sushi domain (SD) of IL-15 receptor α comprises amino acid sequence as set forth as SEQ ID NO: 317. The SD- IL15 fusion protein comprises amino acid sequence as set forth as SEQ ID NO: 318.

[0198] In some embodiments, the optional immune modulator component comprises a fusion protein comprising the sushi domain (SD) and an attenuated form of cytokine IL-15. Although the fusion protein with attenuated IL-15 has reduced potency in stimulating T cells and NK cells, it nonetheless can enhance in vivo half-life, sustained exposure, and / or tolerability.

[0199] As a non-limiting example, the attenuated IL-15 has an amino acid sequence comprising one or more mutations selected from S7N, S7Q, S7A, S7D, S7E, S7F, S7G, S7H, S7I, S7K, S7L, S7M, S7P, S7R, S7T, S7V, S7W, S7Y, D8E, D8S, D8T, D8Q, K10E, K10Q, D30E, D30N, D30P, D30Q, H32N, H32P, S54V, S54L, S54P, D61Q, E64Q, N65D, N65Q, I68A, I68V, I68F, I68G, I68K, I68R, I68L, I68M, I68Q, I68D, I68E, I68H, I68N, I68P, I68S, I68T, I68W, I68Y, L69A, L69V, L69D, L69E, L69F, L69G, L69H, L69I, L69K, L69M, L69N, Attorney Docket No.15271.0015-00304 L69P, L69Q, L69R, L69S, L69T, L69W, L69Y, Q108N, I111L, I111P, and N112Q substitutions, compared to SEQ ID NO: 316.

[0200] In some embodiments, the optional immune modulator component comprises a fusion protein comprising the sushi domain (SD) and IL-15 with I68K mutation (SD-IL15_I68K) with amino acid sequence as set forth as SEQ ID NO: 301.

[0201] In some embodiments, the optional immune modulator component comprises a fusion protein comprising the sushi domain (SD) and IL-15 with S7T and I68K mutations (SD- IL15_S7TI68K) with amino acid sequence as set forth as SEQ ID NO: 302.

[0202] In some embodiments, the optional immune modulator component comprises a fusion protein comprising the sushi domain (SD) and IL-15 with I68K and L69S mutations (SD- IL15_I68KL69S) with amino acid sequence as set forth as SEQ ID NO: 303.

[0203] In some embodiments, the bispecific antibody disclosed herein comprises one set of heavy chain and light chain with binding specificity to disease associated antigen (e.g., tumor associated antigen) as the DAA-binding arm (e.g., TAA-binding arm) and another set of heavy chain and light chain or VHH with binding specificity to either of or both δ1 and δ2 subtypes of γδ T cell receptors (γδTCR), as the γδTCR binding arm. In some embodiments, the bispecific antibody disclosed herein comprises an attenuated IL-15 and the sushi domain of IL- 15Rα (SD-IL15) fused at the C-terminus of one or both of the two heavy chains of the bispecific antibody. As a non-limiting example, the disclosure provides illustrations of configurations of such a bispecific antibody disclosed herein in Figure 2A to 2D.

[0204] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-5T4 antibody PFA1 heavy chain variable region, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_I68K, SD-IL15_S7TI68K, or SD-IL15_I68KL69S) as set forth as SEQ ID NO: 304, 305, or 306, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 304-306, and an anti-5T4 antibody PFA1 light chain with variable region amino acid sequence PFA1VL as set forth as SEQ ID NO: 202 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 202.

[0205] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-γδTCR antibody heavy chain with variable Attorney Docket No.15271.0015-00304 region amino acid sequence 5A6E9hum2HC, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_I68K, SD-IL15_S7TI68K, or SD-IL15_I68KL69S) as set forth as SEQ ID NO: 307, 308, or 309 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 307-309, and anti-γδTCR antibody light chain with variable region amino acid sequence 5A6E9hum1LC as set forth as SEQ ID NO: 210 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 210.

[0206] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-γδTCR antibody heavy chain with variable region amino acid sequence 5A6E9hum3HC, a IgG1 Fc with L234A / L235A mutations, and SD- IL15_I68K, SD-IL15_S7TI68K, or SD-IL15_I68KL69S) as set forth as SEQ ID NO: 310, 311, or 312 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 310-312, and anti- γδTCR antibody light chain with variable region amino acid sequence 5A6E9hum1LC as set forth as SEQ ID NO: 210 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 210.

[0207] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-γδTCR antibody heavy chain with variable region amino acid sequence 5A6E9hum4HC, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_I68K, SD-IL15_S7TI68K, or SD-IL15_I68KL69S) as set forth as SEQ ID NO: 313, 314, or 315 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 313-315, and anti-γδTCR antibody light chain with variable region amino acid sequence 5A6E9hum1LC as set forth as SEQ ID NO: 210 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 210.

[0208] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-γδTCR antibody heavy chain with variable region amino acid sequence 5A6E9hum5HC, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_S7TI68K) as set forth as SEQ ID NO: 328 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 328, and anti-γδTCR antibody light chain with variable region amino acid sequence Attorney Docket No.15271.0015-00304 5A6E9hum4LC as set forth as SEQ ID NO: 213 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 213.

[0209] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-γδTCR antibody heavy chain with variable region amino acid sequence 5A6E9hum6HC, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_S7TI68K) as set forth as SEQ ID NO: 329 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 329, and anti-γδTCR antibody light chain with variable region amino acid sequence 5A6E9hum4LC as set forth as SEQ ID NO: 213 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 213.

[0210] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-CD123 antibody flotetuzumab heavy chain variable region, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_S7TI68K) as set forth as SEQ ID NO: 330, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 330, and an anti- CD123 antibody flotetuzumab light chain with variable region amino acid sequence as set forth as SEQ ID NO: 216 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 216.

[0211] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-CD123 antibody 10D1 heavy chain variable region, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_S7TI68K) chosen from SEQ ID NOs: 331 and 332, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NO: 331 and 332, and an anti-CD123 antibody 10D1 light chain with variable region amino acid sequence chosen from SEQ ID NOs: 219 and 220, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 219 and 220.

[0212] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-CD123 antibody 1E12 heavy chain variable Attorney Docket No.15271.0015-00304 region, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_S7TI68K) chosen from SEQ ID NOs: 333 and 334, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NO: 333 and 334, and an anti-CD123 antibody 1E12 light chain with variable region amino acid sequence chosen from SEQ ID NOs: 223 and 224, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 223 and 224.

[0213] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-CD123 antibody flotetuzumab heavy chain variable region, an IgG1 Fc with S354C / T366W mutations, and SD-IL15_S7TI68K) as set forth as SEQ ID NO: 337, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 337, and an anti- CD123 antibody flotetuzumab light chain with variable region amino acid sequence as set forth as SEQ ID NO: 216 or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to SEQ ID NO: 216.

[0214] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-CD123 antibody 10D1 heavy chain variable region, an IgG1 Fc with S354C / T366W mutations, and SD-IL15_S7TI68K) chosen from SEQ ID NOs: 338 and 339, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NO: 338 and 339, and an anti-CD123 antibody 10D1 light chain with variable region amino acid sequence chosen from SEQ ID NOs: 219 and 220, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 219 and 220.

[0215] In some embodiments, the bispecific antibody disclosed herein comprises a heavy chain amino acid sequence (including an anti-CD123 antibody 1E12 heavy chain variable region, an IgG1 Fc with S354C / T366W mutations, and SD-IL15_S7TI68K) chosen from SEQ ID NOs: 340 and 341, or an amino acid sequence having at least 85% (e.g., at least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NO: 340 and 341, and an anti-CD123 antibody 1E12 light chain with variable region amino acid sequence chosen from SEQ ID NOs: 223 and 224, or an amino acid sequence having at least 85% (e.g., at Attorney Docket No.15271.0015-00304 least 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identity to any one of SEQ ID NOs: 223 and 224.

[0216] In some embodiments, the present disclosure provides a 5T4 x γδTCR bispecific antibody with SD-IL15_I68K fused at the C-terminus of its heavy chains comprising a 5T4 antibody heavy chain sequence as set forth as SEQ ID NO: 304, a 5T4 antibody light chain sequence as set forth as SEQ ID NO: 202, a γδTCR antibody heavy chain sequence chosen from SEQ ID NO: 307, 310, and 313, and a γδTCR antibody light chain sequence as set forth as SEQ ID NO: 210 (e.g., as listed in Table 4).

[0217] In some embodiments, the present disclosure provides a 5T4 x γδTCR bispecific antibody with SD-IL15_S7TI68K fused at the C-terminus of its heavy chains comprising a 5T4 antibody heavy chain sequence as set forth as SEQ ID NO: 305, a 5T4 antibody light chain sequence as set forth as SEQ ID NO: 202, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 308, 311, 314, 328, and 329, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs. 210-214 (e.g., as listed in Table 4).

[0218] In some embodiments, the present disclosure provides a 5T4 x γδTCR bispecific antibody with SD-IL15_I68KL69S fused at the C-terminus of its heavy chains comprising a 5T4 antibody heavy chain sequence as set forth as SEQ ID NO. 306, a 5T4 antibody light chain sequence as set forth as SEQ ID NO: 202, a γδTCR antibody heavy chain sequence chosen from SEQ ID NO: 309, 312, and 315, and a γδTCR antibody light chain sequence as set forth as SEQ ID NO: 210 (e.g., as listed in Table 4). Table 4. 5T4 x γδTCR bispecific antibody with SD and attenuated IL-15 5T4 5T4 γδTCR γδTCR 5T4 x δTCR bis ecific antibod with 0 0 0 Attorney Docket No.15271.0015-00304 HChum1LC_IgG1_L234AL235A_SD- IL15_I68KL69S 0 0 0 0 0 0

[0219] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody with SD-IL15_S7TI68K fused at the C-terminus of both heavy chains comprising a CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 330-334, a CD123 antibody light chain sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 308, 311, 314, 328, and 329, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210-214 (e.g., as listed in Table 5).

[0220] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody with SD-IL15_S7TI68K fused at the C-terminus of one of the two heavy chains comprising a CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 330-334, a CD123 antibody light chain sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 203-209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210-214 (e.g., as listed in Table 5). Attorney Docket No.15271.0015-00304

[0221] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody with SD-IL15_S7TI68K fused at the C-terminus of one of the two heavy chains comprising a CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 215, 217, 218, 221, and 222, a CD123 antibody light chain sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 308, 311, 314, 328, and 329, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210- 214 (e.g., as listed in Table 5). Table 5. CD123 x γδTCR bispecific antibody with SD and attenuated IL-15 γδTC CD1 γδTC CD123 x γδTCR bispecific CD123 23 R ht n : : : : : Attorney Docket No.15271.0015-00304 CD123_10D1hum2HChum1LC_Ig CD123- G1_L234AL235A_SD- 1 D1h 2H h E E E EQ: : : : : :

[0222] In some embodiments, the present disclosure provides a CD20 x γδTCR bispecific antibody with SD-IL15_S7TI68K fused at the C-terminus of a CD20 antibody heavy chain sequence as set forth as SEQ ID NO: 335, a CD20 antibody light chain sequence as set forth as SEQ ID NO: 226, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs. 203-209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210-214 (e.g., as listed in Table 6). Attorney Docket No.15271.0015-00304

[0223] In some embodiments, the present disclosure provides a CDH17 x γδTCR bispecific antibody with SD-IL15_S7TI68K fused at the C-terminus of a CDH17 antibody heavy chain sequence as set forth as SEQ ID NO: 336, a CDH17 antibody light chain sequence as set forth as SEQ ID NO: 228, a γδTCR antibody heavy chain sequence chosen from SEQ ID NOs: 203-209, and a γδTCR antibody light chain sequence chosen from SEQ ID NOs: 210-214 (e.g., as listed in Table 6). Table 6. Bispecific antibodies anti-CD20 and anti-CDH17 Abbreviation DAA DAA γδTCR γδTCR DAA x γδTCR bispecific antibody Heav Li ht Heavy Light n : : : :

[0224] In some embodiments, the present disclosure provides a CD123 x γδTCR bispecific antibody with SD-IL15_S7TI68K fused at the C-terminus of a CD123 antibody heavy chain sequence chosen from SEQ ID NOs: 337-341, a CD123 antibody light chain sequence chosen from SEQ ID NOs: 216, 219, 220, 223, and 224, a γδTCR VHH sequence chosen from SEQ ID NOs: 229-232 (e.g., as listed in Table 7). Table 7. Bispecific antibodies with anti-CD123 and γδTCR VHH Abbreviation CD123 x γδTCR bispecific γδTCR Attorney Docket No.15271.0015-00304 Heavy Light chain chain : : : : : : : : : : : Attorney Docket No.15271.0015-00304 IL15_S7TI68K x γδTCR_Pan- 371VHH_IgG1_L234AL235A

[0225] In certain embodiments, a leader peptide is chosen to drive the secretion of the antibody described in this disclosure into the cell culture supernatant as a secreted respective parental antibody protein. Any leader peptide for any known secreted proteins / peptides can be used.

[0226] As used herein, a “leader peptide” or “signal peptide” or “leader sequence” includes a short peptide, usually 16-30 amino acids in length, that is present at the N-terminus of most of newly synthesized proteins that are destined towards the secretory pathway. Although lead peptides are extremely heterogeneous in sequence, and many prokaryotic and eukaryotic lead peptides are functionally interchangeable even between different species, the efficiency of protein secretion may be strongly determined by the sequence of the lead / signal peptide.

[0227] In certain embodiments, the leader peptide is from a protein residing either inside certain organelles (such as the endoplasmic reticulum, Golgi, or endosomes), secreted from the cell, or inserted into most cellular membranes may be used.

[0228] In certain embodiments, the leader peptide is from a eukaryotic protein.

[0229] In certain embodiments, the leader peptide is from a secreted protein, e.g., a protein secreted outside a cell.

[0230] In certain embodiments, the leader peptide is from a transmembrane protein.

[0231] In certain embodiments, the leader peptide contains a stretch of amino acids that is recognized and cleaved by a signal peptidase.

[0232] In certain embodiments, the leader peptide does not contain a cleavage recognition sequence of a signal peptidase.

[0233] In certain embodiments, the leader peptide is a signal peptide for tissue plasminogen activator (tPA), herpes simplex virus glycoprotein D (HSVgD), a growth hormone, a cytokine, a lipoprotein export signal, CD2, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR, or S. cerevisiae mating factor α-1 signal peptide.

[0234] In some embodiments, a leader sequence as described herein may be a mammalian CD4 or CD8 leader sequence, including but not limited to, e.g., a human CD4 or CD8 leader sequence, a non-human primate CD4 or CD8 leader sequence, a rodent CD4 or CD8 Attorney Docket No.15271.0015-00304 leader sequence, and the like. In some embodiments, a CD4 or CD8 leader comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity with the human CD4 or CD8 leader sequences. IgG Fc of bispecific antibody targeting disease associated antigen and γδ T cell receptors

[0235] The present bispecific antibody targeting disease associated antigen (e.g., tumor associated antigen) and γδ T cell receptors may comprise a modified IgG Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a native Fc region. In some embodiments, the bispecific antibodies disclosed herein are provided with a modified Fcregion where a naturally-occurring Fcregion is modified to extend the half-life of the antibody when compared to the parental native antibody in a biological environment, for example, the serum half-life or a half-life measured by an in vitro assay. Exemplary mutations that may be made singularly or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0236] In certain embodiments, the extension of half-life can be realized by engineering the M252Y / S254T / T256E mutations, collectively known as YTE mutations, in IgG1 Fc residue (Dall'Acqua, Kiener et al. 2006).

[0237] In certain embodiments, the extension of half-life can also be realized by engineering the M428L / N434S mutations, collectively known as LS mutations, in IgG1Fcas SEQ ID NO: 319 (Zalevsky, Chamberlain et al. 2010).

[0238] In certain embodiments, the extension of half-life can also be realized by engineering the T250Q / M428L mutations in IgG1Fc(Hinton, Xiong et al. 2006).

[0239] In certain embodiments, the extension of half-life can also be realized by engineering the N434A mutations in IgG1 Fc (Shields, Namenuk et al. 2001).

[0240] In certain embodiments, the extension of half-life can also be realized by engineering the T307A / E380A / N434A mutations in IgG1 Fc (Petkova, Akilesh et al. 2006).

[0241] The effect of Fc engineering on the extension of antibody half-life can be evaluated in PK studies in mice relative to antibodies with native IgG Fc.

[0242] In some embodiments, the bispecific antibodies disclosed herein are provided with a modified Fc region where a naturally-occurring Fc region is modified to enhance the Attorney Docket No.15271.0015-00304 antibody resistance to proteolytic degradation by a protease that cleaves the wild-type antibody between or at residues 222-237 (EU numbering).

[0243] In certain embodiments, the resistance to proteolytic degradation can be realized by engineering E233P / L234A / L235A mutations in the hinge region with G236 deleted when compared to a parental native antibody, such as in SEQ ID NO: 320, residue numbering according to the EU Index (Kinder, Greenplate et al. 2013).

[0244] In instances where effector functionality is to be enhanced, the antibodies of the disclosure may be engineered to introduce at least one mutation in the antibody Fc that increases the binding of the antibody to an activating Fcγ receptor (FcγR) and / or increases Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and / or phagocytosis (ADCP). Such modifications can comprise low or null Fc fucosylation, and / or engineering of Fc mutations such as S239E / I332E / A330L, S239D / I332E / A330L, S239D / I332E, S239D, I332E, S298A / E333A / K334A.

[0245] In certain embodiments, the enhancement of effector functions can be realized by engineering F243L / R292P / Y300L / V305I / P396L mutations, collectively known as LPLIL mutations, when compared to a parental native antibody, residue numbering according to the EU Index (Stavenhagen, Gorlatov et al. 2008).

[0246] In certain embodiments, the enhancement of the effector functions can be realized by engineering E345R or other Fc clustering mutations on the bispecific antibody using residue numbering according to the EU Index.

[0247] In certain embodiments, the enhancement of effector functions can be realized by engineering F243L / R292P / Y300L / V305I / P396L mutations and E345R mutation, collectively known as E345R / LPLIL mutations, when compared to a parental native antibody, residue numbering according to the EU Index.

[0248] In instances where effector functionality is not desired, the antibodies of the disclosure may be engineered to introduce at least one mutation in the antibody Fc that reduces binding of the antibody to an activating Fcγ receptor (FcγR) and / or reduces Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell- mediated cytotoxicity (ADCC), or phagocytosis (ADCP).

[0249] Fcpositions that may be mutated to reduce binding of the antibody to the activating FcγR and subsequently to reduce effector functions are those described for example in Attorney Docket No.15271.0015-00304 (Xu, Alegre et al. 2000) (Vafa, Gilliland et al. 2014) (Bolt, Routledge et al. 1993) (Chu, Vostiar et al. 2008) (Shields, Namenuk et al. 2001).

[0250] Exemplary mutations that may be made singularly or in combination are K214T, E233P, L234V, L234A, deletion of G236, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S mutations on IgG1, IgG2, IgG3 or IgG4.

[0251] Exemplary combination mutations that may be made to reduce ADCC are L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236- deleted / A327G / P331A / D365E / L358M on IgG1, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgG1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236-deleted / G237A / P238S on IgG4. Hybrid IgG2 / 4Fcdomains may also be used, such as Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4.

[0252] In certain embodiments, the reduction of effector functions can be realized by engineering L234A / L235A mutations, collectively known as AA mutations, compared to a parental native IgG1 antibody Fc, such as in SEQ ID NO: 321, residue numbering according to the EU Index.

[0253] In certain embodiments, the reduction of effector functions can be realized by engineering L234F / L235E / D265A mutations, collectively known as FEA mutations, compared to a parental native IgG1 antibody Fc, residue numbering according to the EU Index.

[0254] In certain embodiments, the reduction of effector functions can be realized by using IgG2 or IgG4 Fc instead of IgG1 on the bispecific antibodies disclosed herein.

[0255] In some embodiments, the bispecific antibodies disclosed herein are provided with a modified Fc region where a naturally-occurring Fc region is modified to facilitate the generation of bispecific antibody by Fc heterodimerization.

[0256] In certain embodiments, the Fc heterodimerization can be realized by engineering F405L (as SEQ ID NO: 322) and K409R (as SEQ ID NO: 323) mutations on two parental antibodies and the generation of multispecific antibody in a process known as Fab arm exchange (Labrijn, Meesters et al. 2014). Attorney Docket No.15271.0015-00304

[0257] In certain embodiments, the Fc heterodimerization can also be realized by Fc mutations to facilitate Knob-in-Hole strategy (see, e.g., Intl. Publ. No. WO 2006 / 028936). An amino acid with a small side chain (hole) is introduced into one Fc domain and an amino acid with a large side chain (knob) is introduced into the other Fc domain. After co-expression of the two heavy chains, a heterodimer is formed as a result of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob” (Ridgway, Presta et al. 1996). Exemplary Fc mutation pairs forming a knob and a hole are: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S / L368A / Y407V.

[0258] In certain embodiments, the Fc heterodimerization can be realized by engineering T366W and T366S / L368A / Y407V mutations on two parental antibodies and the generation of multispecific antibody by co-expression or by a process known as Fab arm exchange.

[0259] In certain embodiments, the Fc heterodimerization can also be realized by Fc mutations to facilitate the electrostatically-matched interactions strategy (Gunasekaran, Pentony et al. 2010). Mutations can be engineered to generate positively charged residues at one Fc domain and negatively charged residues at the other Fc domain as described in US Patent Publ. No. US2010 / 0015133; US Patent Publ. No. US2009 / 0182127; US Patent Publ. No. US2010 / 028637 or US Patent Publ. No. US2011 / 0123532. Heavy chain heterodimerization can be formed by electrostatically-matched interactions between two mutated Fc.

[0260] In some embodiments, the bispecific antibodies disclosed herein are provided with a modified Fc region where a naturally-occurring Fc region is modified to facilitate the multimerization of the antibody upon interaction with cell surface receptors, although such engineered antibody exists as a monomer in solution. The Fcmutations that facilitate antibody multimerization include, but not limited to, E345R mutation, E430G mutation, E345R / E430G mutations, E345R / E430G / Y440R mutations as described in (Diebolder, Beurskens et al. 2014). Such mutations may also include, but not limited to, T437R mutation, T437R / K248E mutations, T437R / K338A mutations as described in (Zhang, Armstrong et al. 2017).

[0261] Antibodies of the disclosure further comprising conservative modifications are within the scope of the disclosure. “Conservative modifications” refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody Attorney Docket No.15271.0015-00304 containing the amino acid sequences. Conservative modifications include amino acid substitutions, additions and deletions. Conservative substitutions are those in which the amino acid is replaced with an amino acid residue having a similar side chain. The families of amino acid residues having similar side chains are well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amide (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for alanine scanning mutagenesis. Amino acid substitutions to the antibodies of the disclosure may be made by known methods for example by PCR mutagenesis (US Disclosure No. 4,683,195). Alternatively, libraries of variants may be generated for example using random (NNK) or non-random codons, for example DVK codons, which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants may be evaluated for their characteristics using assays described herein.

[0262] The antibodies of the disclosure may be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation or non-naturally occurring covalent modification such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modifications may occur in vivo or in vitro. For example, the antibodies of the disclosure may be conjugated to polyethylene glycol (PEGylated) to improve their pharmacokinetic profiles. Conjugation may be conducted by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics while not interfering with function.

[0263] Antibodies of the disclosure may be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desirable biological or biophysical property are within the scope of the disclosure. Stability of an antibody is influenced by a number of factors, including (1) core packing of individual domains that affects their intrinsic stability, (2) protein / protein interface interactions that have impact upon the HC and LC pairing, (3) burial of Attorney Docket No.15271.0015-00304 polar and charged residues, (4) H-bonding network for polar and charged residues; and / or (5) surface charge and polar residue distribution among other intra- and inter-molecular forces (Worn and Pluckthun 2001). Potential structure destabilizing residues may be identified based upon the crystal structure of the antibody or by molecular modelling in certain cases, and the effect of the residues on antibody stability may be assessed by generating and evaluating variants harboring mutations in the identified residues. One of the ways to increase antibody stability is to raise the thermal transition midpoint (Tm) as measured by differential scanning calorimetry (DSC). In general, the protein Tm is correlated with its stability and inversely correlated with its susceptibility to unfolding and denaturation in solution and the degradation processes that depend on the tendency of the protein to unfold. A number of studies have found correlation between the ranking of the physical stability of formulations measured as thermal stability by DSC and physical stability measured by other methods. Formulation studies suggest that a Fab Tm has implication for long-term physical stability of a corresponding mAb.

[0264] Antibodies of the disclosure may have amino acid substitutions in the Fcregion that improve manufacturing and drug stability. An example for IgG1 is H224S (or H224Q) in the hinge 221-DKTHTC-226 (EU numbering) which blocks radically induced cleavage; and for IgG4, the S228P mutation blocks half-antibody exchange. Expression and purification of antibodies

[0265] The antibodies disclosed herein can be prepared as follows. In some embodiments, a bispecific antibody targeting disease associated antigen (e.g., tumor associated antigen) and γδ T cell receptors of the present disclosure can be encoded by a single nucleic acid (e.g., a single nucleic acid comprising nucleotide sequences that encode the light and heavy chain polypeptides of the antibody), or by two or more separate nucleic acids, each of which encodes a different part of the parental antibody.

[0266] As a non-limiting example, the present disclosure provides a nucleic acid sequence encoding an anti-5T4 antibody heavy chain with a variable region PFA1VH, an IgG1 Fc with L234A / L235A mutations, and SD-IL15_S7TI68K fused at the C-terminus, wherein the nucleic acid sequence comprises SEQ ID NO: 324.

[0267] As a non-limiting example, the present disclosure provides a nucleic acid sequence encoding an anti-5T4 antibody light chain with a variable region PFA1LC, wherein the nucleic acid sequence comprises SEQ ID NO: 325. Attorney Docket No.15271.0015-00304

[0268] As a non-limiting example, the present disclosure provides a nucleic acid sequence encoding an anti-γδTCR antibody heavy chain with a variable region 5A6E9hum4HC, a IgG1 Fc with L234A / L235A mutations, and SD-IL15_S7TI68K fused at the C-terminus, wherein the nucleic acid sequence comprises SEQ ID NO: 326.

[0269] As a non-limiting example, the present disclosure provides a nucleic acid sequence encoding an anti-γδTCR antibody light chain with a variable region 5A6E9hum1LC, wherein the nucleic acid sequence comprises SEQ ID NO: 327.

[0270] The nucleic acids described herein can be inserted into vectors, e.g., nucleic acid expression vectors and / or targeting vectors. Such vectors can be used in various ways, e.g., for the expression of an antibody described herein in a cell or transgenic animal. Vectors are typically selected to be functional in the host cell in which the vector will be used. A nucleic acid molecule encoding an antibody described herein may be amplified / expressed in prokaryotic, yeast, insect (baculovirus systems) and / or eukaryotic host cells. Selection of the host cell will depend in part on whether the bispecific antibodies described herein are to be post-translationally modified (e.g., glycosylated and / or phosphorylated). If so, yeast, insect, or mammalian host cells are preferable. Expression vectors typically contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.

[0271] In most cases, a leader or signal sequence is engineered at the N-terminus of the bispecific antibody described herein to guide its secretion. The secretion of the bispecific antibody described herein from a host cell will result in the removal of the signal peptide from the antibody. Thus, the bispecific antibody described herein will lack any leader or signal sequence. In some cases, such as where glycosylation is desired in a eukaryotic host cell expression system, one may manipulate the various pre-sequences to improve glycosylation or yield. For example, one may alter the peptidase cleavage site of a signal peptide, or add pre- sequences, which also may affect glycosylation.

[0272] The disclosure further provides a cell (e.g., an isolated or purified cell) comprising a nucleic acid or vector of the disclosure. The cell can be any type of cell capable of Attorney Docket No.15271.0015-00304 being transformed with the nucleic acid or vector of the disclosure so as to produce a polypeptide encoded thereby. To express the bispecific antibody described herein, DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.

[0273] Methods of introducing nucleic acids and vectors into isolated cells and the culture and selection of transformed host cells in vitro are known in the art and include the use of calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, and electroporation.

[0274] After introducing the nucleic acid or vector of the disclosure into the cell, the cell is cultured under conditions suitable for expression of the encoded sequence. The antibody, antigen binding fragment, or portion of the antibody then can be isolated from the cell.

[0275] In certain embodiments, two or more vectors that together encode the bispecific antibodies described herein, can be introduced into the cell.

[0276] Purification of the bispecific antibody described herein which has been secreted into the cell media can be accomplished using a variety of techniques including affinity, immunoaffinity or ion exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing, chromatofocusing, and high-pressure liquid chromatography. For example, antibodies comprising an Fcregion may be purified by affinity chromatography with Protein A, which selectively binds the Fc region.

[0277] Modified forms of the bispecific antibody described herein may be prepared with affinity tags, such as hexahistidine (SEQ ID NO: 342) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen) at either its carboxyl or amino terminus and purified by a one-step affinity column. For example, polyhistidine binds with great affinity and specificity to nickel, thus an affinity column of nickel can be used for purification of polyhistidine-tagged selective binding agents. In some instances, more than one purification step may be employed.

[0278] The heterodimerization of the bispecific antibody described herein can be generated by a process known as controlled Fabarm exchange from two parental antibodies with Attorney Docket No.15271.0015-00304 F405L and K409R (EU numbering) mutation in IgG Fc respectively (Labrijn, Meesters et al. 2014). The controlled Fab arm exchange reaction is the result of a disulfide-bond isomerization reaction and dissociation-association of CH3 domains. First, two parental antibodies are generated, one bearing the F405L Fcmutation, and one bearing the K409R Fcmutation. The heavy chain disulfide bonds in the hinge regions of the parental antibodies are reduced and the heavy chains of the parental antibodies are separated. The F405L and K409R mutations favor heterodimerization over homodimerization of the heavy chains. Therefore, the resulting free cysteines of one of the parental antibodies form an inter heavy-chain disulfide bond with cysteine residues of a second parental antibody. The resulting product is a heterodimerized antibody with one half coming from one parental antibody and the other half coming from another parental antibody.

[0279] The bispecific antibodies described herein are generated from one parental antibody targeting DAA (e.g., 5T4) with F405L Fc mutation and another parental antibody targeting γδ T cell receptor with K409R Fc mutation by controlled Fab arm exchange.

[0280] The bispecific antibodies described herein may be generated by other Fcmutations and engineering processes that facilitate Fcheterodimerization, including, but not limited to, Knob-in-Hole and the electrostatically-matched interactions.

[0281] In the Knob-in-Hole strategy (see, e.g., Intl. Publ. No. WO 2006 / 028936, incorporated by reference), selected amino acids forming the interface of the CH3domains in human IgG can be mutated at positions affecting CH3 domain interactions to promote heterodimer formation. An amino acid with a small side chain (hole) is introduced into one Fcdomain and an amino acid with a large side chain (knob) is introduced into the other Fcdomain of the parental antibodies. After co-expression of the two heavy chains, a heterodimer is formed because of the preferential interaction of the heavy chain with a “hole” with the heavy chain with a “knob.” Exemplary CH3substitution pairs forming a knob and a hole include: T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S_L368A / Y407V.

[0282] In the electrostatically-matched interactions strategy, mutations can be engineered to generate positively charged residues at one CH3surface and negatively charged residues at a second CH3 surface as described in US 2010 / 0015133 A1; US 2009 / 0182127 A1; Attorney Docket No.15271.0015-00304 US 2010 / 028637 A1, or US 2011 / 0123532 A1. Heterodimerization of heavy chains can be formed by electrostatically-matched interactions between two mutated Fc.

[0283] The formation of bispecific antibody can be assessed by analytical HPLC if there is a detectable difference in the biophysical properties of the two parental antibodies. A difference in pI may lead to two separate peaks for the two parental antibodies on Cation Exchange chromatography and the bispecific antibody may migrate as a peak in between. A difference in hydrophobicity may lead to two separate peaks for the two parental antibodies on hydrophobic interaction chromatography and the bispecific antibody may migrate as a peak in between. The analytical HPLC not only demonstrates the formation of bispecific antibody, but also allows the quantitation of percentage of bispecific antibody formed. Target binding and functional activity of bispecific antibody targeting disease associated antigen (DAA) and γδ T cell receptors

[0284] In vitro binding and cell-based assays are well described in the art for use in determining the bispecific antibody targeting DAA and γδ T cell receptors of the present disclosure in binding to its antigens. For example, the binding of antibody disclosed herein may be determined by ELISA by immobilizing recombinant or purified antigen, sequestering the antibody with the immobilized antigen and determining the amount of bound antibody. This can also be performed using a Biacore® instrument for kinetic analysis of binding interactions. For cell-based binding assay, the binding of antibody may be determined by flow cytometry by incubating the antibody with cells expressing antigens on cell surface and determining the amount of antibody bound to cell surface antigen.

[0285] In vitro and cell-based functional assays are well described in the art for use in determining the functional activity of the bispecific antibody targeting DAA and γδ T cell receptors of the present disclosure. The effects of bispecific antibodies described herein on the stimulation of the activation and proliferation of γδ T cells can be assessed by flow-cytometry based assays to check the expression of activation markers, such as CD107a, CD69, CD25, or proliferation markers, such as Ki67, on purified γδ T cells. The effects of bispecific antibodies described herein on directing γδ T cells in the killing of cells with DAA expression can be assessed by flow cytometry based cell cytotoxicity assay by mixing purified γδ T cells with target cells in a certain effector cell: target cell ratio. Besides, the effects of bispecific antibodies described herein on the expansion and activation of γδ T cell population in the whole PBMC can Attorney Docket No.15271.0015-00304 be assessed by flow cytometry based assay to check the increase in percentage of γδ T cells in PBMC and the activation markers of the γδ T cell population.

[0286] For IL-15, the functional activity of IL-15 component of the bispecific antibody described herein can be assessed by reporter-based cell assay and CTLL-2 cell proliferation assay. The enhancement by IL-15 of the bispecific antibody described herein on the activation and proliferation of γδ T cells can be assessed by flow-cytometry based assays to check the expression of activation markers, such as CD107a, CD69, CD25, or proliferation markers, such as Ki67, on purified γδ T cells. The enhancement by IL-15 of the bispecific antibodies described herein on directing γδ T cells in the killing of tumor cells with TAA expression can be assessed by flow cytometry based cell cytotoxicity assay by mixing purified γδ T cells with tumor target cells in a certain effector cell: target cell ratio. Besides, the effects of IL-15 component of the bispecific antibodies described herein on the expansion and activation of γδ T cell population in the whole PBMC can be assessed by flow cytometry-based assay to check the increase in percentage of γδ T cells in PBMC and the activation markers of the γδ T cell population.

[0287] Besides in vitro functional activity, animal models and ex vivo functional assays are well described in the art for use in determining the in vivo efficacy of the bispecific antibody targeting DAA and γδ T cell receptor of the present disclosure. The anti-tumor efficacy of the bispecific antibodies described herein can be assessed by humanized xenograft mouse models with human tumor cell lines and patient derived primary tumor cells, along with purified and amplified human γδ T cells or PBMC from donors. Pharmaceutical Compositions

[0288] The antibodies disclosed herein including the bispecific antibody targeting DAA and γδ T cell receptors for use according to the present disclosure can be formulated in compositions, especially pharmaceutical compositions, for use in the methods herein. Such compositions comprise a therapeutically or prophylactically effective amount of the multispecific antibody described in this disclosure in mixture with a suitable carrier, e.g., a pharmaceutically acceptable agent. Typically, the bispecific antibody described in this disclosure is sufficiently purified for administration to an animal before formulation in a pharmaceutical composition.

[0289] Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, coloring, flavoring and diluting agents, emulsifying agents, Attorney Docket No.15271.0015-00304 suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffering agents, antimicrobials, and surfactants.

[0290] The composition can be in liquid form or in a lyophilized or freeze-dried form and may include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents.

[0291] Compositions can be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to a skilled worker, such as intraarticular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral, or inhaled routes.

[0292] Pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides local concentration of the product (e.g., bolus, depot effect) for a sustained release and / or increased stability or half-life in a particular local environment. Methods of Use

[0293] The antibodies disclosed herein including the bispecific antibodies targeting DAA (e.g., 5T4) and γδ T cell receptors described herein are useful for the treatment or prevention of cancers, such as breast, ovarian, gastric, lung, pancreatic and other cancers. In some embodiments, the cancer is selected from pancreatic cancer, triple negative breast cancer, lung cancer, and any cancer with TAA (e.g., 5T4) expression. In some embodiments, the cancer is any cancer with DAA expression. In particular, the bispecific antibody described herein is useful for the treatment of tumor types with high percentage of residential γδ T cells in the tumor mass.

[0294] The antibodies disclosed herein including the bispecific antibodies targeting DAA and γδ T cell receptors described herein are useful for the treatment or prevention of autoimmune diseases. In some embodiments, the autoimmune disease is any autoimmune disease in which the disease causing cells express DAA. In some embodiments, the autoimmune disease is selected from multiple sclerosis, N-methyl-d-aspartate receptor (NMDAR) encephalitis, myasthenia gravis, systemic lupus erythematosus (SLE), rheumatoid arthritis, myelin– oligodendrocyte glycoprotein (MOG) spectrum disorder (MOGSD) and neuromyelitis optica spectrum disorder (NMOSD) Attorney Docket No.15271.0015-00304

[0295] The disclosure also provides use of bispecific antibody targeting DAA and γδ T cell receptors described herein or a composition comprising bispecific antibody targeting DAA and γδ T cell receptors described herein in the manufacture of a medicament for treating or preventing a disease or condition, e.g., cancer or an autoimmune disease, comprising administering to the subject a therapeutically effective amount of the bispecific antibody or the composition.

[0296] The disclosure also provides bispecific antibody targeting DAA and γδ T cell receptors described herein or a composition comprising bispecific antibody targeting DAA and γδ T cell receptors described herein for use in a method of treating or preventing a disease or condition, e.g., cancer or an autoimmune disease, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bispecific antibody or the composition.

[0297] The disclosure also provides bispecific antibody targeting DAA and γδ T cell receptors described herein that can be used in a combination regimen, e.g., with chemotherapy, radiotherapy, and / or cell therapy.

[0298] All combinations of the various elements described herein are within the scope of the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

[0299] This disclosure will be better understood from the following Experimental Details. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the disclosure. Example 1: Expansion of γδ T cells from PBMC

[0300] For Vγ9Vδ2 T cell expansion, donor PBMCs (peripheral blood mononuclear cells) were seeded in a 6-well flat-bottomed plate at a density of 1×106cells / mL in complete RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 1x Pen / Strep and 50 IU / mL IL-2. On the first day, zoledronic acid (ZOL, 5 µM, Sigma) was added into the culture medium to stimulate the expansion of Vγ9Vδ2 T cells. Two days later, the culture media was supplemented with IL-2 at 100 IU / mL and IL-15 at 10 ng / mL and this was repeated every second day for 14 days.

[0301] The purity of the Vγ9Vδ2 T cell population was assessed starting on day 14 by gating CD3 and δ2 by flow cytometry. If higher than 90 % purity of Vγ9Vδ2 T-cell is required, αβ T cells can be depleted from the cultured cells by an αβ T cell selection kit. Figure 3A Attorney Docket No.15271.0015-00304 demonstrated an exemplary cell expansion experiment resulting in 96.2% purity of Vγ9Vδ2 T cells.

[0302] For Vδ1 T cell expansion, donor PBMC in which the Vδ1 T cells were the dominant γδ T cell population were seeded at a density of 1×106cells / mL in a 24-well flat- bottomed plate pre-coated with 2 µg / mL of 5A6E9-IL-15 antibody. The culture media was supplemented with IL-2 at 100 IU / mL every other day for 8 days. Then the cells were harvested and resuspended in a new 5A6E9-IL-15 coated 24-well plate for a re-stimulation for another 7 days. The purity of the Vδ1 T cell population was assessed by gating CD3 and δ1 by flow cytometry. Figure 3B demonstrated an exemplary cell expansion experiment resulting in 85.9% purity of Vδ1 T cells. Example 2: Antibody binding to both δ1 and δ2 subtypes of γδ T cell receptors

[0303] A monoclonal mouse anti-human γδ T cell receptor antibody with clone number 5A6E9 was humanized with the inclusion of CDRs into human germline scaffolds while preserving a few key residues to achieve higher stability and better expression and minimizing immunogenicity. Seven humanized VH variants (hum1HC, hum2HC, hum3HC, hum4HC, hum5HC, hum6HC, hum7HC) and five humanized VL variants (hum1LC, hum2LC, hum3LC, hum4LC, hum5LC) were designed. By combination of the seven humanized VH variants paring with the five humanized VL variants, thirty five humanized 5A6E9 antibodies with IgG1 Fc with L234A / L235A / K409R mutations were expressed by co-transfection of plasmids encoding the heavy and light chains into Expi293F cells following the transfection kit instructions (Thermo Scientific). Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 µm filter. The purifications of expressed antibodies were conducted by affinity chromatography over mAbSelectSure columns (Cytiva Life Sciences). The purified antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm.

[0304] Not all combinations of humanized heavy chain and light chain led to expression of humanized 5A6E9 antibodies and their expression levels varied. Several exemplary humanized 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2Hchum1LC), γδTCR_5A6E9hum3Hchum1LC_IgG1_L234AL235A (hum3Hchum1LC), γδTCR_5A6E9hum4Hchum1LC_IgG1_L234AL235A (hum4Hchum1LC), γδTCR_5A6E9hum5Hchum4LC_IgG1_L234AL235A (hum5Hchum4LC), and Attorney Docket No.15271.0015-00304 γδTCR_5A6E9hum6Hchum4LC_IgG1_L234AL235A (hum6Hchum4LC), were subjected to SDS-PAGE analysis (Figure 4). Under the reduced condition, all the tested humanized antibodies had heavy chains and light chains of the expected molecular weight. Under the non-reduced condition, all the humanized antibodies migrated as a major protein band with a molecular weight around 150 kDa.

[0305] ELISA-based binding assays were employed to evaluate the binding to γδ T cell receptors by the five exemplary humanized 5A6E9 antibodies. In this assay, 2 µg / mL Vγ9Vδ1 TCR, Vγ9Vδ2 TCR, Vγ4Vδ1 TCR, or Vγ4Vδ2 TCR as fusion proteins with human IgG Fc were coated on ELISA plate. Increasing concentrations of γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum3HChum1LC_IgG1_L234AL235A (hum3HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), and γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC), were applied on the coated ELISA plate and their binding to the recombinant Vγ9Vδ1, Vγ9Vδ2, Vγ4Vδ1, or Vγ4Vδ2 TCR were detected by HRP-conjugated anti-human IgG F(ab)’ secondary antibody. All five humanized 5A6E9 antibodies dose-dependently bound to recombinant Vγ9Vδ1 (Figure 5A), Vγ9Vδ2 (Figure 5B), Vγ4Vδ1 (Figure 5C), and Vγ4Vδ2 (Figure 5D) with similar potencies but hum3HChum1LC antibody had a lower efficacy in all cases. As controls, the binding of reference antibodies specific to either δ1 or δ2 subtype of γδ T cell receptors (δ1 antibody and δ2 antibody) were also assessed in these ELISA-based binding assays. These control antibodies showed binding specificities in these assays (Figure 5A-5D).

[0306] Besides ELISA assay detecting binding to recombinant γδTCR, flow cytometry- based binding assay was employed to evaluate the binding to γ9δ2 T cell receptors expressed on surface of purified γ9δ2 T cells by the five exemplary humanized 5A6E9 antibodies. In this assay, increasing concentrations of humanized 5A6E9 antibodies were applied to γ9δ2 T cells and their bindings to cell surface γδ T cell receptors were detected by PE-conjugated anti-human secondary antibody by MacsQuant flow cytometer. All five humanized 5A6E9 antibodies dose- dependently bound to cell surface γ9δ2 T cell receptors with varied potencies (Figure 5E). Among them, hum6HChum4LC showed most potent and efficacious binding while Attorney Docket No.15271.0015-00304 hum3HChum1LC showed relatively weakest binding. The binding to δ1 T cells by humanized 5A6E9 antibody demonstrated that γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC) antibody concentration-dependently bound to δ1 T cells (Figure 5F). Example 3: Activation of γδ T cell receptors by humanized 5A6E9 antibodies

[0307] The functional activities of exemplar humanized 5A6E9 antibodies in activating γδ T cell receptors were assessed by Jurkat cell NFAT reporter assays. In this assay, plasmids expressing combination of γ9 and δ1 subunits or combination of γ9 and δ2 subunits were transiently transfected into Jurkat NFAT reporter cell line (BPS Bioscience) to express γ9δ1TCR or γ9δ2TCR in transfected cells. Humanized γδTCR 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum3HChum1LC_IgG1_L234AL235A (hum3HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC), γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC), control δ1TCR specific antibody (δ1 antibody), and control δ2TCR specific antibody (δ2 antibody) were coated on plate and incubated with reporter cells overnight. The dose-dependent activation of luciferase reporter gene expression in Jurkat NFAT reporter cells transfected with human γ9δ1 TCR and human γ9δ2 TCR by the immobilized antibodies were quantitated.

[0308] It was observed that γ9δ1-expressing reporter cells were activated by δ1 antibody but not by δ2 antibody, while γ9δ2-expressing reporter cells were activated by δ2 antibody but not by δ1 antibody (Figure 6A and 6B), indicating the TCR subtype specific activation in these reporter assays. The four humanized 5A6E9 antibodies showed different potencies in activating γ9δ1 TCR and γ9δ2 TCR in these reporter assays. In the γ9δ1 reporter cell assay, hum6HChum4LC was the most potent while hum5HChum4LC was the weakest among these humanized 5A6E9 antibodies in activating γ9δ1 TCR-dependent reporter gene expression (Figure 6A). In the γ9δ2 reporter cell assay, both hum5HChum4LC and hum6HChum4LC were relatively more potent than hum2HChum1LC and hum4HChum1LC in activating γ9δ2 TCR-dependent reporter gene expression (Figure 6B).

[0309] The activations of γ9δ2 T cells by humanized 5A6E9 antibodies were also evaluated in a γδ T cell activation assay. In this assay, U-bottom 96-well cell culture plates were Attorney Docket No.15271.0015-00304 coated with serial dilutions of antibodies overnight at 4° C. Next day, wells were washed by PBS twice and 1×105PBMC derived γ9δ2 T cells were added into each well and incubated overnight at 37° C. In some wells, Golgistop (BD Biosciences) was added for intracellular cytokine testing. Flow cytometry was then used to determine CD25, CD69 and IFN-γ expression on γδ T cells.

[0310] CD25 and CD69 are the activation markers for activated γδ T cells. It was observed that γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), and γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC) dose-dependently activated both CD25 and CD69 expression on γ9δ2 T cells with similar potencies while γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (hum5HChum4LC) showed weaker activation (Figure 7A and 7B). Besides the CD25 and CD69 activation markers, the secretion of IFNγ is also a hallmark for activated γδ T cells. All four tested humanized 5A6E9 antibodies showed similar potencies but slightly different efficacies in stimulating the γ9δ2 T cells in the secretion of IFNγ (Figure 7C). The experiment of activation of δ1 T cells by humanized 5A6E9 antibody demonstrated that γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC) dose-dependently activated CD69 expression on δ1 T cells (Figure 7D).

[0311] The functional activity of the humanized 5A6E9 antibodies in the stimulation of the proliferation of γδ T cells was also evaluated by a PBMC-based assay. In this assay, U- bottom 96-well cell culture plates were coated with serial dilutions of antibodies overnight at 4°C. Next day, wells were washed by PBS twice and PBMC were added into each well and incubated for 7 days at 37° C. The change of the γδ T cell population in PBMC was quantified by flow cytometric counting beads (CountBright Absolute Counting Beads, ThermoFisher).

[0312] It was observed that all four tested humanized 5A6E9 antibodies, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC), γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum5HChum4LC), and γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (hum6HChum4LC), dose-dependently stimulated the proliferation of γδ T cells with similar potencies (Figure 8). Example 4: Generation of bispecific antibodies targeting 5T4 and γδ T cell receptors Attorney Docket No.15271.0015-00304

[0313] Bispecific antibodies targeting 5T4 and γδ T cell receptors were generated with combinations of heavy chain and light chain constructs listed in Table 2. The 5T4 and γδ T cell receptor antibodies comprise a humanized anti-5T4 antibody PFA1 Fab arm as the 5T4-binding arm and one of the humanized 5A6E9 antibody Fab arms as the γδ T cell receptor binding arm, and an IgG1 Fc with L234A / L235A mutations for the reduction of Fc effector functions.

[0314] The 5T4 x γδTCR bispecific antibodies listed in Table 2 were generated by the expression of two parental antibodies for each binding arm followed by the generation of bispecific antibodies in a process known as controlled Fab-arm exchange. One parental antibody is the anti-5T4 antibody PFA1 with an IgG1 Fc with L234A / L235A / F405L mutations. Another parental antibody is one of the humanized anti-γδTCR 5A6E9 antibodies with an IgG1 Fc with L234A / L235A / K409R mutations. Plasmids encoding heavy chains and light chains of parental antibodies were co-transfected into Expi293F cells following the transfection kit instructions (Thermo Scientific). Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 µm filter. The purifications of expressed parental antibodies were conducted by affinity chromatography over mAbSelectSure columns (Cytiva Life Sciences). The purified parental antibodies were buffer exchanged into DPBS, pH7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm.

[0315] For controlled Fab-arm exchange, equal molar amounts of both parental antibodies were mixed together and reduced for 5 hours in the presence of 75 mM 2- mercaptoethylamine (2-MEA). The reaction mixture was dialyzed against DPBS to allow the 5T4 x γδTCR bispecific antibody formation.

[0316] The bispecific antibodies targeting 5T4 and γδ T cell receptors were subjected to SDS-PAGE analysis and one example was shown as Figure 9A. Under reduced condition, the bispecific antibodies targeting 5T4 and γδ T cell receptors evaluated showed heavy chains and light chains with expected sizes. Under the non-reduced condition, the bispecific antibodies migrated as a major protein band with a molecular weight around 150 kDa.

[0317] The formation of 5T4 x γδTCR bispecific antibodies was assessed by Cation Exchange (CEX) chromatography. As an example, the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and its corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and Attorney Docket No.15271.0015-00304 γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), were loaded onto a Bio SCX NP5 ion exchange column (Agilent). The profiles of peak migration for these antibodies were shown in Figure 6B. The 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) bispecific antibody migrated as a major protein peak with the retention time in between the migrated major peaks of the two parental antibodies 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), indicating the formation of bispecific antibodies. Further calculation of the area under the curve (AUC) indicated that over 95% of the parental antibodies formed the bispecific antibodies by the Fab- arm exchange (Figure 9B). Example 5: Binding specificity of bispecific antibodies targeting 5T4 and γδ T cell receptors

[0318] ELISA-based binding assay was employed to evaluate the binding to 5T4 antigen by an exemplary bispecific antibody targeting 5T4 and γδ T cell receptors. In this assay, 1 µg / mL recombinant human 5T4 protein (Acrobiosystems) was coated on ELISA plate. Increasing concentrations of the bispecific antibody and its parental antibodies were applied on the coated ELISA plate and their binding to the recombinant human 5T4 were detected by HRP- conjugated anti-human IgG secondary antibody. Both the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and its parental antibody 5T4_PFA1_IgG1_L234AL235A (5T4) showed dose-dependent binding to 5T4 while the γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC) parental antibody did not (Figure 10). The parental antibody 5T4_PFA1_IgG1_L234AL235A (5T4) with two 5T4 binding arms showed better potency in binding to 5T4 than the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC).

[0319] Flow cytometry-based binding assays were employed to evaluate the binding to 5T4-expressing cells by an exemplary bispecific antibody targeting 5T4 and γδ T cell receptors. In this assay, increasing concentrations of the exemplary bispecific antibody and its parental antibodies were applied to 5T4-expressing NCI-H226 and MCF-7 cells and their bindings to cell surface 5T4 receptors were detected by PE-conjugated anti-human secondary antibody by MacsQuant flow cytometer. Both the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x Attorney Docket No.15271.0015-00304 γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and its parental antibody 5T4_PFA1_IgG1_L234AL235A (5T4) showed dose-dependent binding to 5T4 receptors expressed on NCI-H226 cells (Figure 11A) and MCF-7 cells (Figure 11B) while the γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC) parental antibody did not. The parental antibody 5T4_PFA1_IgG1_L234AL235A (5T4) with two 5T4 binding arms showed better potency in binding to 5T4 than the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC).

[0320] Flow cytometry-based binding assays were also employed to evaluate the binding to γ9δ2 T cells by an exemplary bispecific antibody targeting 5T4 and γδ T cell receptors. In this assay, increasing concentrations of the exemplary bispecific antibody and its parental antibodies were applied to expanded γ9δ2 T cells and their bindings to cell surface γδ T cell receptors were detected by PE-conjugated anti-human secondary antibody by MacsQuant flow cytometer. Both the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and its parental antibody γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC) showed dose-dependent binding to Vγ9Vδ2 T cells, while the 5T4_PFA1_IgG1_L234AL235A (5T4) parental antibody did not (Figure 12). The parental antibody γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC) with two γδ T cell receptor binding arms showed better potency in binding to γδ T cells than the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC). Example 6: Activation of γδ T cell receptor by 5T4 x γδ TCR bispecific antibodies

[0321] The functional activities of exemplar 5T4 x γδ TCR bispecific antibodies in mediating the activating γδ T cell receptors by 5T4-expressing tumor cells were assessed by Jurkat cell NFAT reporter assays. In this assay, plasmids expressing combination of γ9 and δ1 subunits or combination of γ9 and δ2 subunits were transiently transfected into Jurkat NFAT reporter cell line (BPS Bioscience) to express γ9δ1TCR or γ9δ2TCR in transfected cells. 5T4 x γδ TCR bispecific antibodies, 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC), 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC), 5T4_PFA1 x Attorney Docket No.15271.0015-00304 γδTCR_5A6E9hum5HChum4LC_IgG1_L234AL235A (5T4 x hum5HChum4LC), 5T4_PFA1 x γδTCR_5A6E9hum6HChum4LC_IgG1_L234AL235A (5T4 x hum6HChum4LC), control 5T4_PFA1 x δ1TCR specific antibody (5T4 x δ1 antibody), and control 5T4_PFA1 x δ2TCR specific antibody (5T4 x δ2 antibody) were incubated with reporter cells and 5T4-expressing tumor cell MCF-7 or NCI-H226 overnight. The dose-dependent activation of luciferase reporter gene expression in Jurkat NFAT reporter cells transfected with human γ9δ1 TCR and human γ9δ2 TCR by the bispecific antibody mediated ligation with 5T4-expressing cells were quantitated.

[0322] It was observed that γ9δ1-expressing reporter cells were activated by 5T4 x δ1 antibody but not by 5T4 x δ2 antibody in the presence of MCF-7 cells (Figure 13A) or NCI- H226 cells (Figure 13C), while γ9δ2-expressing reporter cells were activated by 5T4 x δ2 antibody but not by 5T4 x δ1 antibody in the presence of MCF-7 cells (Figure 13B) or NCI- H226 cells (Figure 13D), indicating the TCR subtype specific activation in these reporter assays. The bispecific antibodies with 5T4 pairing with the four humanized 5A6E9 antibodies showed different potencies in activating γ9δ1 TCR and γ9δ2 TCR in the presence of 5T4 expressing tumor cells in these reporter assays (Figure 13A-D). In all cases, 5T4 x hum6HChum4LC was the most potent one followed by 5T4 x hum5HChum4LC while 5T4 x hum2HChum1LC and 5T4 x hum4HChum1LC were relatively weaker in activating reporter gene expression.

[0323] The activation of γ9δ2 T cells by 5T4 x γδ TCR bispecific antibody and its parental antibodies was evaluated in a γδ T cell activation assay. In this assay, U-bottom 96-well cell culture plates were coated with serial dilutions of antibodies overnight at 4° C. Next day, wells were washed by PBS twice and 1×105PBMC derived γ9δ2 T cells were added into each well and incubated overnight at 37° C. In some wells, Golgistop (BD Biosciences) was added for intracellular cytokine testing. Flow cytometry was then used to determine CD69 and IFN-γ expression on γδ T cells.

[0324] CD69 is the activation marker for activated γδ T cells. It was observed that both the coated 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and its parental antibody γδTCR_5A6E9hum4Hchum1LC_IgG1_L234AL235A (hum4HChum1LC) dose-dependently activated CD69 expression on γ9δ2 T cells while the parental antibody Attorney Docket No.15271.0015-00304 5T4_PFA1_IgG1_L234AL235A (5T4) did not (Figure 14A). The parental antibody γδTCR_5A6E9hum4Hchum1LC_IgG1_L234AL235A (hum4HChum1LC) with two γδTCR binding arms showed better potency in γδT cell activation than the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4Hchum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC).

[0325] Besides the CD69 activation marker, the secretion of IFNγ is also a hallmark for activated γδ T cells. It was observed that both the coated 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) and its parental antibody γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC) dose-dependently activated IFNγ production on activated γ9δ2 T cells while the parental antibody 5T4_PFA1_IgG1_L234AL235A (5T4) did not (Figure 14B). The parental antibody γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC) with two γδTCR binding arms showed better potency in the promotion of IFNγ secretion than the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC). Example 7: Cytotoxic degranulation of γ9δ2 T cells by 5T4 x γδ TCR bispecific antibody

[0326] The cytotoxic degranulation of γ9δ2 T cells by 5T4 x γδ TCR bispecific antibody upon co-culturing with 5T4-expressing cells was evaluated in a γδ T cell degranulation assay. In this assay, 5T4 expressing MCF-7 cells were preincubated with serial dilutions of antibodies. Thirty minutes later, 1×105PBMC derived γ9δ2 T cells were co-cultured with 1×105MCF-7 cells (E:T ratio is 1:1) in a master mix including anti-CD107a antibody and Golgistop (BD Biosciences). After four-hour incubation, the expression of CD107a on γ9δ2 T cells was checked by flow cytometry.

[0327] It was observed that 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) dose- dependent activated CD107a expression on γ9δ2 T cells when co-cultured with 5T4-expressing MCF-7 cells (Figure 15). The parental antibody 5T4_PFA1_IgG1_L234AL235A (5T4) did not induce degranulation of Vγ9Vδ2 T cells at all and the parental antibody γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC) showed much less potency in driving CD107a expression on γ9δ2 T cells compared to the 5T4 x γδTCR bispecific Attorney Docket No.15271.0015-00304 antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC). Example 8: Cytotoxicity of target cells by γ9δ2 T cell mediated by 5T4 x γδ TCR bispecific antibody

[0328] The antibody mediated cytotoxicity of 5T4-expressing target cells by γ9δ2 T cells were evaluated in a γδ T cell cytotoxicity assay. In this assay, expanded γ9δ2 T cells were co-cultured with 5T4 expressing MCF-7 cells at 1:1 E:T ratio in the presence of serial dilutions of antibodies for 24 hours. At the end of the incubation time, the ability of γδ T cells to lyse target cells was determined by flow cytometry.

[0329] It was observed that the 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (5T4 x hum4HChum1LC) dose- dependently facilitated the killing of 5T4-expressing MCF-7 cells by Vγ9Vδ2 T cells (Figure 16), while neither of the two parental antibodies, 5T4_PFA1_IgG1_L234AL235A (5T4) and γδTCR_5A6E9hum4HChum1LC_IgG1_L234AL235A (hum4HChum1LC), mediated significant target cell killing by the lack of the ability of crosslinking MCF-7 cells with γ9δ2 T cells. Example 9: Efficacy of bispecific antibody targeting 5T4 and γδ T cell receptors in humanized tumor xenograft model

[0330] The efficacy of bispecific antibody targeting 5T4 and γδ T cell receptors in tumor cell killing was evaluated in a humanized tumor xenograft model. In this model, 5T4- expressing BxPC-3 cells were subcutaneously inoculated into NCG mice with 5 mice per study group. Human γ9δ2 T cells expanded from donor PBMC were intravenously injected into the mice at a level of 3 million cells per mouse at day 0, 7, and 14 after tumor cell inoculation. Bispecific antibody targeting 5T4 and γδ T cell receptor 5T4_PFA1 x γδTCR_5ª6E9hum2Hchum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC) was dosed intraperitoneally at 5 mg / kg twice per week starting at day 0 post tumor cell inoculation for five weeks. The volume of the tumor and the weight of mice were measured.

[0331] It was observed that 5T4 x hum2HChum1LC antibody treatment led to 28% BxPC-3 tumor cell growth inhibition compared to the PBS treatment group (Figure 17A). No difference in body weight between antibody treatment group and vehicle control group (Figure 17B). This data indicated tumor killing efficacy of bispecific antibody targeting 5T4 and γδ T cell receptors. Attorney Docket No.15271.0015-00304 Example 10: Generation of bispecific antibodies targeting 5T4 and γδ T cell receptors with Sushi domain and attenuated IL-15 (SD-IL15)

[0332] Bispecific antibodies targeting 5T4 and γδ T cell receptors with the sushi domain and attenuated IL-15 (SD-IL15) were generated with combinations of heavy chain and light chain constructs listed in Table 3. The 5T4 and γδ T cell receptor bispecific antibodies with SD-attenuated IL15 comprise a humanized anti-5T4 antibody PFA1 Fab arm as the 5T4-binding arm and humanized 5A6E9 antibody Fab arms as the γδ T cell receptor binding arm, an IgG1 Fc with L234A / L235A mutations for the reduction of Fc effector functions, and a SD-attenuated IL15 component fused at the C-terminus of both heavy chains (see, e.g., Figure 2).

[0333] The 5T4 x γδTCR bispecific antibodies with SD-attenuated IL15 listed in Table 4 were generated by the expression of two parental antibodies for each binding arm followed by the generation of bispecific antibodies in a process known as controlled Fab-arm exchange. One parental antibody is an anti-5T4 antibody PFA1 with an IgG1 Fc with L234A / L235A / F405L mutations and C-terminus SD-attenuated IL15. Another parental antibody is a humanized anti- γδTCR 5A6E9 antibody with an IgG1 Fc with L234A / L235A / K409R mutations and C-terminus SD-IL15 with attenuation mutations. Plasmids encoding heavy chains and light chains of the parental antibodies were co-transfected into Expi293F cells following the transfection kit instructions (Thermo Scientific). Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 µm filter. The purifications of expressed parental antibodies were conducted by affinity chromatography over mAbSelectSure columns (Cytiva Life Sciences). The purified parental antibodies were buffer-exchanged into DPBS, pH7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm.

[0334] For controlled Fab-arm exchange, equal molar amounts of both parental antibodies were mixed together and reduced for 5 hours in the presence of 75 mM 2- mercaptoethylamine (2-MEA). The reaction mixture was dialyzed against DPBS to allow the 5T4 x γδTCR bispecific antibody formation.

[0335] The bispecific antibodies targeting 5T4 and γδ T cell receptors with SD- attenuated IL-15 were subjected to SDS-PAGE analysis and examples were shown in Figure 18A. Under reduced condition, the bispecific antibodies 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x Attorney Docket No.15271.0015-00304 hum2HChum1LC_SD-IL15_I68K) and 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K), and their corresponding parental antibodies, showed heavy chains and light chains with expected sizes. Under the non-reduced condition, the bispecific antibodies migrated as a major protein band with a molecular weight around 200 kDa.

[0336] The formation of 5T4 x γδTCR bispecific antibody with SD-attenuated IL-15 was assessed by Cation Exchange (CEX) chromatography. As an example, the 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and its corresponding parental antibodies, 5T4_PFA1_IgG1_L234AL235A_SD-IL15_I68K (5T4_SD-IL15_I68K) and γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (hum2HChum1LC_SD- IL15_I68K), were loaded onto Bio SCX NP5 ion exchange column (Agilent). The profiles of peak migration for these antibodies were shown in Figure 18B. The 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) bispecific antibody migrated as a major protein peak with the retention time in between the migrated major peaks of the two parental antibodies 5T4_PFA1_IgG1_L234AL235A_SD-IL15_I68K (5T4_SD-IL15_I68K) and γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (hum2HChum1LC_SD- IL15_I68K), indicating the formation of bispecific antibodies (Figure 18B). Example 11: IL-15 activities of bispecific antibodies targeting 5T4 and γδ T cell receptors with SD-IL15 with attenuation mutations

[0337] The functional IL-15 activities of the SD-attenuated IL-15 component of the bispecific antibodies targeting 5T4 and γδ T cell receptors were evaluated in a HEK-Blue IL-15 reporter assay (Invivogen). Increasing concentrations of exemplary bispecific antibodies, 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K), were applied to HEK-Blue cells expressing IL-15 heterodimeric receptor complexes composed of CD122 and CD132 and secreted embryonic alkaline phosphatase (SEAP) reporter construct activated by JAK / STAT5 (Invivogen). The IL- 15 driven SEAP reporter activation was quantitated by Quanti-Blue kit (Invivogen). The Attorney Docket No.15271.0015-00304 5T4_PFA1 antibody with native SD-IL15 (5T4_SD-IL15) and the two bispecific antibodies with mutated IL-15 showed dose-dependent activities in driving reporter gene expression (Figure 19A). However, relative to native SD-IL15, the I68K and S7TI68K mutations weakened the IL- 15 potency so the bispecific antibodies with mutated IL-15 showed reduced potencies in driving the reporter gene activation.

[0338] The functional IL-15 activities of the SD-IL15 component of the bispecific antibodies targeting 5T4 and γδ T cell receptors were also evaluated in a CTLL-2 cell proliferation assay. CTLL-2 is a mouse T lymphocyte cell line whose proliferation depends on IL-15. In this assay, increasing concentrations of exemplary bispecific antibodies targeting 5T4 and γδ T cell receptors with SD-IL15 were applied to CTLL-2 cells and the proliferation of CTLL-2 cells was quantitated by CellTiter-Glo kit (Promega). The 5T4_PFA1 antibody with native SD-IL15 (5T4_SD-IL15) and the two bispecific antibodies with mutated IL-15, 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K), showed dose-dependent activities in driving the proliferation of CTLL-2 cells (Figure 19B). However, relative to native SD-IL15, the I68K and S7TI68K mutations weakened the IL-15 potency so the bispecific antibodies with mutated IL-15 showed reduced potencies in driving CTLL-2 cell proliferation. Example 12: Stimulation of the proliferation and activation of γ9δ2 T cells by antibody targeting γδ T cell receptors with SD-IL15 with attenuation mutations

[0339] The functional activity of the antibody targeting γδ T cell receptors with SD- IL15 with attenuation mutations in the stimulation of the activation of γ9δ2 T cells was evaluated by a T cell-based assay. In this assay, U-bottom 96-well cell culture plates were coated with serial dilutions of antibodies overnight at 4° C. Next day, wells were washed by PBS twice and 1×105PBMC-derived γ9δ2 T cells were added into each well and incubated overnight at 37 °C. In some wells, Golgistop (BD Biosciences) was added for intracellular cytokine testing. Flow cytometry was then used to determine CD25, CD69 and IFN-γ expression on γδ T cells.

[0340] CD25 and CD69 are the activation markers for activated γδ T cells. It was observed that while coated γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC) antibody showed poor potency in activating γ9δ2 T cells, the two γδTCR Attorney Docket No.15271.0015-00304 antibodies with SD-attenuated IL-15, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (hum2HChum1LC_SD- IL15_I68K) and γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (hum2HChum1LC_SD-IL15_S7TI68K), dose-dependently activated both CD25 and CD69 expression on γ9δ2 T cells with much improved potency (Figure 20A and 20B). Besides, the SD- attenuated IL-15 component also significantly facilitated γδTCR antibody with much increased potency in stimulating the γ9δ2 T cells in the secretion of IFNγ (Figure 20C).

[0341] The functional activity of the antibody targeting γδ T cell receptors with SD- IL15 with attenuation mutations in the stimulation of the proliferation and activation of γδ T cells was also evaluated by a PBMC-based assay. In this assay, U-bottom 96-well cell culture plates were coated with serial dilutions of antibodies overnight at 4° C. Next day, wells were washed by PBS twice and PBMC were added into each well and incubated for 7 days at 37 °C. The change of the γδ T cell population in PBMC was quantified by flow cytometric counting beads (CountBright Absolute Counting Beads, ThermoFisher). Flow cytometry was also used to determine CD25 expression on γδ T cells.

[0342] It was observed that while the coated γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (hum2HChum1LC) antibody barely stimulated the proliferation of γδ T cells, the two γδTCR antibodies with SD-attenuated IL-15, γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (hum2HChum1LC_SD- IL15_I68K) and γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (hum2HChum1LC_SD-IL15_S7TI68K), dose-dependently stimulated the proliferation of γδ T cells with significant potency (Figure 21A). Besides, the SD-attenuated IL-15 component also significantly facilitated γδTCR antibody with higher potency in stimulating CD25 expression on the activated γδ T cells (Figure 21B). Example 13: Stimulation of the proliferation and activation of γδ T cells by bispecific antibodies targeting 5T4 and γδ T cell receptors with Sushi domain and attenuated IL-15

[0343] The activation of γδ T cells by bispecific antibodies targeting 5T4 and γδ T cell receptors with Sushi domain and attenuated IL-15 upon co-culturing with 5T4-expressing cells was evaluated in a γδ T cell activation assay. In this assay, CD3+T cells, including both αβ and γδ T cells, were enriched from PBMC by negative selection. Purified T cells were co-cultured Attorney Docket No.15271.0015-00304 with 5T4 expressing MCF-7 cells (E:T ratio is 10:1) in the presence of tested bispecific antibodies for 7 days. The change of the γδ T cell population was quantified by flow cytometric counting beads (CountBright Absolute Counting Beads, ThermoFisher). Flow cytometry was also used to determine CD25 expression on γδ T cells.

[0344] It was observed that 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC) only weakly stimulated the proliferation of γδ T cells when co-cultured with 5T4-expressing MCF-7 cells (Figure 22A). The two bispecific antibodies with SD-attenuated IL-15, 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K), dose-dependently stimulated the proliferation of γδ T cells with much higher potency relative to 5T4 x γδTCR bispecific antibody without the SD- IL15 component (Figure 22A), indicating the activity of IL-15 in enhancing γδ T cell proliferation.

[0345] Besides, it was also observed that the two bispecific antibodies with SD- attenuated IL-15, 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K), dose-dependently stimulated CD25 expression on γδ T cells with higher potencies relative to 5T4 x γδTCR bispecific antibody without the SD-IL15 component, 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC) (Figure 22B). Example 14: Cytotoxicity of target cells by γ9δ2 T cells mediated by 5T4 x γδ TCR bispecific antibody with SD-attenuated IL-15

[0346] The antibody mediated cytotoxicity of 5T4-expressing target cells by γ9δ2 T cells were evaluated in a γδ T cell cytotoxicity assay. In this assay, expanded γ9δ2 T cells were incubated with serial dilutions of 5T4 x γδTCR bispecific antibodies with SD-attenuated IL-15 for 48 hours. Then 5T4-expressing NCI-H226 cells were added. Four hours later, the cytotoxicity of the target cells by γδ T cells was determined by flow cytometry. Attorney Docket No.15271.0015-00304 It was observed that 5T4 x γδTCR bispecific antibody 5T4_PFA1 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A (5T4 x hum2HChum1LC) dose- dependently facilitated the killing of 5T4-expressing NCI-H226 cells by γ9δ2 T cells (Figure 23). The two bispecific antibodies with SD-attenuated IL-15, 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_I68K (5T4 x hum2HChum1LC_SD-IL15_I68K) and 5T4 x γδTCR_5A6E9hum2HChum1LC_IgG1_L234AL235A_SD-IL15_S7TI68K (5T4 x hum2HChum1LC_SD-IL15_S7TI68K), dose-dependently mediated the cytotoxicity of NCI- H226 cells with higher efficacies relative to 5T4 x γδTCR bispecific antibody without the SD- IL15 component (Figure 23), indicating the activity of IL-15 in enhancing γδ T cell ability in cytotoxicity. Example 15: Generation of bispecific antibodies targeting CD123 and γδ T cell receptors

[0347] Bispecific antibodies targeting CD123 and γδ T cell receptors were generated with combinations of heavy chain and light chain constructs listed in Tables 3 and 5. The CD123 and γδ T cell receptor antibodies comprise a humanized anti-CD123 antibody Fab arm as the CD123-binding arm and a humanized 5A6E9 antibody Fab arm as the γδ T cell receptor binding arm, and an IgG1 Fc with L234A / L235A mutations for the reduction of Fc effector functions.

[0348] The CD123 x γδTCR bispecific antibodies listed ins Table 3 and 5 were generated by the expression of two parental antibodies for each binding arm followed by the generation of bispecific antibodies in a process known as controlled Fab-arm exchange. Plasmids encoding heavy chains and light chains of parental antibodies were co-transfected into Expi293F cells following the transfection kit instructions (Thermo Scientific). Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 µm filter. The purifications of expressed parental antibodies were conducted by affinity chromatography over mAbSelectSure columns (Cytiva Life Sciences). The purified parental antibodies were buffer exchanged into DPBS, pH7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm. For controlled Fab-arm exchange, equal molar amounts of both parental antibodies were mixed together and reduced for 5 hours in the presence of 75 mM 2- mercaptoethylamine (2-MEA). The reaction mixture was dialyzed against DPBS to allow for the oxidation and formation of CD123 x γδTCR bispecific antibody. Attorney Docket No.15271.0015-00304 Example 16: Binding specificity of bispecific antibodies targeting CD123 and γδ T cell receptors

[0349] ELISA-based binding assay was employed to evaluate the binding to CD123 antigen by exemplary antibodies targeting CD123. In this assay, 1 µg / mL recombinant human CD123 protein (Acrobiosystems) was coated on an ELISA plate. Increasing concentrations of the CD123 antibodies were applied on the coated ELISA plate and their binding to the recombinant human CD123 were detected by HRP-conjugated anti-human IgG secondary antibody. The CD123 antibodies used to generate the CD123 x γδTCR bispecific antibodies listed in Table 3 showed dose-dependent binding to CD123 with comparable good potencies (Figure 24A).

[0350] Flow cytometry-based binding assays were employed to evaluate the binding to CD123-expressing cells by exemplary CD123 antibodies. In this assay, increasing concentrations of the exemplary CD123 antibodies were applied to CD123-expressing MOLM-13 cells and their bindings to cell surface CD123 receptors were detected by PE-conjugated anti-human secondary antibody by MacsQuant flow cytometer. The CD123 antibodies used to generate the CD123 x γδTCR bispecific antibodies listed in Table 3 showed dose-dependent binding to MOLM-13 cells with comparable good potencies (Figures 24B and 24C).

[0351] Flow cytometry-based binding assays were employed to evaluate the binding to CD123-expressing cells by exemplary CD123 antibodies with SD-attenuated IL-15. In this assay, increasing concentrations of the exemplary CD123 antibodies with SD-attenuated IL-15 were applied to CD123-expressing MOLM-13 cells and their bindings to cell surface CD123 receptors were detected by PE-conjugated anti-human secondary antibody by MacsQuant flow cytometer. The CD123 antibodies with SD-IL15_S7TI68K used to generate the CD123 x γδTCR bispecific antibodies with SD-attenuated IL-15 listed in Table 5 showed dose-dependent binding to MOLM-13 cells with comparable good potencies (Figures 25A and 25B).

[0352] ELISA-based binding assays were employed to evaluate the binding to CD123 and γ9δ2 TCR antigen by exemplary bispecific antibodies targeting CD123 and γδ T cell receptors listed in Table 3 and 5. In this assay, 1 µg / mL recombinant human CD123 protein (Acrobiosystems) or γ9δ2 TCR-Fc antigen was coated on an ELISA plate. Increasing concentrations of the bispecific antibodies were applied on the coated ELISA plate and their binding to the coated antigens were detected by HRP-conjugated anti-human IgG secondary Attorney Docket No.15271.0015-00304 antibody. The exemplary bispecific antibodies targeting CD123 and γδ T cell receptors listed in Table 3 and 5 showed concentration-dependent potent binding to CD123 (Figure 26A) and to γ9δ2 TCR (Figure 26B). Example 17: Activation of γδ T cell receptor by CD123 x γδ TCR bispecific antibodies

[0353] The functional activities of exemplar CD123 x γδ TCR bispecific antibodies in mediating the activating γδ T cell receptors by CD123-expressing tumor cells were assessed by Jurkat cell NFAT reporter assays. In this assay, plasmids expressing combination of γ9 and δ1 subunits or combination of γ9 and δ2 subunits were transiently transfected into Jurkat NFAT reporter cell line (BPS Bioscience) to express γ9δ1TCR or γ9δ2TCR in transfected cells. Exemplar CD123 x γδ TCR bispecific antibodies and null control antibodies were incubated with reporter cells and CD123-expressing tumor cell MOLM-13 or MV-4-11 overnight. The dose- dependent activation of luciferase reporter gene expression in Jurkat NFAT reporter cells transfected with human γ9δ1 TCR and human γ9δ2 TCR by the bispecific antibody mediated ligation with CD123-expressing cells were quantitated. The exemplar CD123 x γδ TCR bispecific antibodies showed concentration-dependent activation of γ9δ1 TCR and γ9δ2 TCR in the presence of CD123 expressing tumor cells, while the null control antibodies failed to activate reporter gene expression in these reporter assays (Figure 27A-27F). Example 18: Cytotoxic degranulation of γδ T cells and cytotoxicity of tumor cells by CD123 x γδ TCR bispecific antibody

[0354] The cytotoxic degranulation of δ1 T cells and δ2 T cells by CD123 x γδ TCR bispecific antibody upon co-culturing with CD123-expressing cells was evaluated in a γδ T cell degranulation assay. In this assay, CD123 expressing tumor cells were preincubated with serial dilutions of antibodies. Thirty minutes later, 1×105δ1 T cells or δ2 T cells were co-cultured with the tumor cells (E:T ratio is 1:1) in a master mix including anti-CD107a antibody and Golgistop (BD Biosciences). After four-hour incubation, the expression of CD107a on γδ T cells was checked by flow cytometry. It was observed that CD123-flotetuzumab x 5A6E9 bispecific antibody dose-dependent activated CD107a expression on γδ T cells when co-cultured with CD123-expressing tumor cells (Figure 28A, 28C, 28E, 28G), while the control antibodies with null arm did not have such activities. Attorney Docket No.15271.0015-00304

[0355] The antibody mediated cytotoxicity of CD123-expressing target cells by δ1 T cells and δ2 T cells were evaluated in a γδ T cell cytotoxicity assay. In this assay, expanded γδ T cells were co-cultured with CD123 expressing tumor cells at 1:1 E:T ratio in the presence of serial dilutions of antibodies for 24 hours. At the end of the incubation time, the ability of γδ T cells to lyse target cells was determined by flow cytometry. The CD123-flotetuzumab x 5A6E9 bispecific antibody concentration-dependently facilitated the killing of CD123-expressing tumor cells by either δ1 T cells or δ2 T cells (Figure 28B, 28D, 28F, 28H), while the control antibodies with null arm lacked such activities due to the absence of crosslinking CD123 expressing cells to γδ T cells. Example 19: IL-15 activities of bispecific antibodies targeting CD123 and γδ T cell receptors with SD-IL15 with attenuation mutations

[0356] The functional IL-15 activities of the SD-attenuated IL-15 component of the bispecific antibodies targeting CD123 and γδ T cell receptors were evaluated in a HEK-Blue IL- 15 reporter assay (Invivogen). Increasing concentrations of CD123-flotetuzumab x 5A6E9 with two, one or no SD-IL15 with S7TI68K mutations were applied to HEK-Blue cells expressing IL- 15 heterodimeric receptor complexes composed of CD122 and CD132 and secreted embryonic alkaline phosphatase (SEAP) reporter construct activated by JAK / STAT5 (Invivogen). It was observed that the reporter gene activation driven by the attenuated IL-15 depended on the copy number of attenuated IL-15 engineered on the molecules (Figure 29A). The effect of copy number of attenuated IL-15 were also studied by a CTLL-2 cell proliferation assay. It was also observed that CD123-flotetuzumab x 5A6E9 molecule engineered with two copies of SD-IL15 with S7TI68K mutations had a better potency in driving CTLL-2 cell proliferation than the one with only one copy (Figure 29B).

[0357] The functional activity of the antibody targeting γδ T cell receptors with SD- IL15 with attenuation mutations in the stimulation of the activation of γ9δ2 T cells was evaluated by a T cell-based assay. In this assay, U-bottom 96-well cell culture plates were coated with serial dilutions of antibodies overnight at 4° C. Next day, wells were washed by PBS twice and 1×105PBMC-derived γ9δ2 T cells were added into each well and incubated overnight at 37 °C. In some wells, Golgistop (BD Biosciences) was added for intracellular cytokine testing. Flow cytometry was then used to determine CD25, CD69, and IFN-γ expression on γδ T cells. The null x 5A6E9 molecule engineered with two copies of SD-IL15 with S7TI68K mutations had a Attorney Docket No.15271.0015-00304 better potency in driving CD25, CD69, and IFN-γ expression on γδ T cells than the construct with only one copy of SD-IL15 with S7TI68K (Figure 30A, 30B, 30C). Example 20: IL-15 enhanced 5A6E9 dependent proliferation and activation of γδ T cells but not αβ T cells from PBMC

[0358] The activation of γδ T cells by bispecific antibodies targeting CD123 and γδ T cell receptors with Sushi domain and attenuated IL-15 upon co-culturing with CD123-expressing cells was evaluated in a γδ T cell activation assay. In this assay, PBMC, including both αβ and γδ T cells, were co-cultured with CD123 expressing MV-4-11 cells (E:T ratio is 10:1) in the presence of tested bispecific antibodies for 7 days. The change of the γδ T cell and αβ T cell population was quantified by flow cytometric counting beads (CountBright Absolute Counting Beads, ThermoFisher). Flow cytometry was also used to determine CD69 and CD25 expression on γδ T cells and αβ T cells.

[0359] It was observed that CD123-flotetuzumab x 5A6E9 concentration-dependently stimulated the proliferation of γδ T cells by mediating the ligation of tumor cells and the γδ T cells. The presence of either one or two copies of SD-IL15_S7TI68K further enhanced the proliferation of γδ T cells to a similar level (Figure 31A). In contrast, the null x 5A6E9 did not stimulate the proliferation of γδ T cells due to the lack of the ability of linking the tumor cells and the γδ T cells (Figure 31B). However, the presence of either one or two copies of SD- IL15_S7TI68K further enhanced the proliferation of γδ T cells to a similar level due to the binding of 5A6E9 on γδ T cells. Without 5A6E9-mediated γδ T cell ligation, null x 5A6E9 did not mediate tumor cell dependent proliferation of γδ T cells and the engineered SD- IL15_S7TI68K failed to stimulate the proliferation of γδ T cells as well (Figure 31C).

[0360] It was also observed that CD123-flotetuzumab x 5A6E9 concentration- dependently stimulated the activation of γδ T cells (CD69 and CD25 expression) by mediating the ligation of tumor cells and the γδ T cells. The presence of either one or two copies of SD- IL15_S7TI68K further enhanced the activation of γδ T cells to a similar level (Figure 31D and 31G). In contrast, the null x 5A6E9 did not stimulate the activation of γδ T cells due to the lack of the ability of linking the tumor cells and the γδ T cells (Figure 31E and 31H). However, the presence of either one or two copies of SD-IL15_S7TI68K further enhanced the activation of γδ T cells to a similar level due to the binding of 5A6E9 on γδ T cells. Without 5A6E9-mediated γδ Attorney Docket No.15271.0015-00304 T cell ligation, null x 5A6E9 did not mediate tumor cell dependent activation of γδ T cells and the engineered SD-IL15_S7TI68K failed to stimulate the proliferation of γδ T cells as well (Figure 31F and 31I).

[0361] The experiment characterizing the proliferation and activation of αβ T cells demonstrated that CD123-flotetuzumab x 5A6E9 bispecific antibody with or without SD- IL15_S7TI68K domain failed to stimulate the αβ T cell proliferation and activation (Figure 31J, 31K, 31L). Example 21: IL-15 enhanced 5A6E9 dependent cytotoxicity of CD123-expressing tumor cells by γδ T cells from PBMC

[0362] The antibody mediated cytotoxicity of CD123-expressing target cells by γδ T cells from PBMC was evaluated in a cytotoxicity assay. In this assay, PBMC co-cultured with MV-4-11 cells were incubated with serial dilutions of CD123-flotetuzumab x 5A6E9 bispecific antibody and related null control antibodies with one, two, and no SD-IL15_S7TI68K engineered on the molecules for 5 days. The cytotoxicity of the target cells by γδ T cells from PBMC was determined by flow cytometry. It was observed that CD123-flotetuzumab x 5A6E9 concentration-dependently mediated the cytotoxicity of MV-4-11 cells while the engineering of SD-IL15_S7TI68K failed to further enhance the cytotoxicity (Figure 32). In contrast, while the null x 5A6E9 failed to mediate the cytotoxicity of MV-4-11 cells (Figure 32A), the engineering of one or two SD-IL15_S7TI68K enabled null x 5A6E9 with cytotoxicity towards MV-4-11 cells (Figure 32B and 32C), likely due to non-specific tumor cell cytotoxicity by γδ T cells activated by SD-IL15_S7TI68K. However, this non-specific tumor cell cytotoxicity was much weaker than CD123-flotetuzumab x 5A6E9-mediated cytotoxicity (Figure 32B and 32C). Without 5A6E9-mediated γδ T cell ligation, the CD123-flotetuzumab x null failed to mediate tumor cell cytotoxicity no matter the presence of SD-IL15_S7TI68K or not (Figure 32). Example 22: Efficacy of bispecific antibody targeting CD123 and γδ T cell receptors in humanized tumor xenograft model with amplified γδ T cells

[0363] The efficacy of bispecific antibody targeting CD123 and γδ T cell receptors in tumor cell killing was evaluated in a humanized tumor xenograft model with amplified γδ T cells. In this model, CD123-expressing MOLM-13-luc cells were subcutaneously inoculated into NCG mice with 5 mice per study group. Human γ9δ2 T cells expanded from donor PBMC were intravenously injected into the mice at a level of 3 million cells per mouse at day 1, 8, and 15 Attorney Docket No.15271.0015-00304 after tumor cell inoculation. Bispecific antibody targeting CD123 and γδ T cell receptor and control antibodies were dosed intraperitoneally at 0.8 mg / kg twice per week starting at day 1 post tumor cell inoculation. In some groups, 0.3 µg / mouse IL-2 was co-dosed with the test antibodies (Figure 33A). The tumor burden was quantitated by ex vivo live imaging and the weight of mice were measured.

[0364] It was observed that CD123-flotetuzumab x 5A6E9 antibody treatment led to significant MOLM-13 tumor cell growth inhibition compared to the PBS or null control treatment groups (Figure 33B and 33E). The engineering of SD-IL15_S7TI68K on CD123- flotetuzumab x 5A6E9 further decreased tumor burden (Figure 33B) and more significantly extended the survival life of the treated mice (Figure 33D). It was worth noted that the CD123- flotetuzumab-IL15 x 5A6E9-IL15 antibody achieved such efficacy in the absence of added IL-2 while IL-2 was added to all other treatment groups. No drops in body weight were observed for the antibody treatment groups relative to PBS group (Figure 33C). This data demonstrated tumor killing efficacy of bispecific antibody targeting CD123 and γδ T cell receptors, especially when engineered with the attenuated IL-15. Example 23: Efficacy of bispecific antibody targeting CD123 and γδ T cell receptors in humanized tumor xenograft model with PBMC

[0365] The efficacy of bispecific antibody targeting CD123 and γδ T cell receptors in tumor cell killing was also evaluated in a humanized tumor xenograft model with donor PBMC. In this model, CD123-expressing MOLM-13-luc cells were subcutaneously inoculated into NCG mice with 5 mice per study group. Human donor PBMC were intravenously injected into the mice at a level of 5 million cells per mouse at day 1 and 8 after tumor cell inoculation. Bispecific antibody targeting CD123 and γδ T cell receptor and control antibodies were dosed intraperitoneally at 0.8 mg / kg twice per week starting on day 1 post tumor cell inoculation (Figure 34A). The tumor burden was quantitated by ex vivo live imaging and the weight of mice were measured.

[0366] It was observed that CD123-flotetuzumab x 5A6E9 antibody treatment led to significant MOLM-13 tumor cell growth inhibition compared to the PBS treatment group (Figure 34B and 34E). The engineering of SD-IL15_S7TI68K on CD123-flotetuzumab significantly decreased tumor burden no matter the presence of 5A6E9 or not (Figure 34B and 34E). However, relative to the null control, CD123-flotetuzumab-IL15 x 5A6E9 significantly extended Attorney Docket No.15271.0015-00304 the survival life of the treated mice (Figure 34D). No drop in body weights was observed for the antibody treatment groups relative to PBS group (Figure 34C). This data demonstrated tumor killing efficacy of bispecific antibody targeting CD123 and γδ T cell receptors, especially when engineered with the attenuated IL-15. Example 24: Activation of γδ T cell receptor and tumor cell cytotoxicity by CD20 x γδ TCR bispecific antibodies

[0367] The functional activities of exemplar CD20 x γδ TCR bispecific antibodies in mediating the activating γδ T cell receptors by CD20-expressing tumor cells were assessed by Jurkat cell NFAT reporter assays. In this assay, plasmids expressing combination of γ9 and δ1 subunits or combination of γ9 and δ2 subunits were transiently transfected into Jurkat NFAT reporter cell line (BPS Bioscience) to express γ9δ1TCR or γ9δ2TCR in transfected cells. Exemplar CD20 x γδ TCR bispecific antibodies and null control antibodies were incubated with reporter cells and CD20-expressing tumor cell SU-DHL-4 or Raji overnight. The dose-dependent activation of luciferase reporter gene expression in Jurkat NFAT reporter cells transfected with human γ9δ1 TCR and human γ9δ2 TCR by the bispecific antibody mediated ligation with CD20- expressing cells were quantitated. The exemplar CD20-rituximab x γδ TCR bispecific antibody showed concentration-dependent activation of γ9δ1 TCR and γ9δ2 TCR in the presence of CD20 expressing tumor cells in these reporter assays, while the null control antibodies failed to activate reporter gene expression (Figure 35A-35D).

[0368] The antibody mediated cytotoxicity of CD20-expressing target cells by δ2 T cells were evaluated in a γδ T cell cytotoxicity assay. In this assay, expanded γδ T cells were co- cultured with CD20 expressing tumor cells at 1:1 E:T ratio in the presence of serial dilutions of antibodies for 24 hours. At the end of the incubation time, the ability of γδ T cells to lyse target cells was determined by flow cytometry. It was observed that the CD20-rituximab x 5A6E9 bispecific antibody dose-dependently facilitated the killing of CD20-expressing tumor cells by δ2 T cells (Figure 35E), while the control antibodies with the null arm lacked such activities due to the inability to crosslink CD20 expressing cells to γδ T cells. Example 25: Activation of γδ T cell receptor by CDH17 x γδ TCR bispecific antibodies

[0369] The functional activities of exemplar CDH17 x γδ TCR bispecific antibodies in mediating the activating γδ T cell receptors by CDH17-expressing tumor cells were assessed by Attorney Docket No.15271.0015-00304 Jurkat cell NFAT reporter assays. In this assay, plasmids expressing combination of γ9 and δ1 subunits or combination of γ9 and δ2 subunits were transiently transfected into Jurkat NFAT reporter cell line (BPS Bioscience) to express γ9δ1TCR or γ9δ2TCR in transfected cells. Exemplar CDH17 x γδ TCR bispecific antibodies and null control antibodies were incubated with reporter cells and CDH17-expressing tumor cell AsPC-1 overnight. The dose-dependent activation of luciferase reporter gene expression in Jurkat NFAT reporter cells transfected with human γ9δ1 TCR and human γ9δ2 TCR by the bispecific antibody mediated ligation with CDH17-expressing cells were quantitated. The exemplar CDH17-v1 x γδ TCR bispecific antibody showed concentration-dependent activation of γ9δ1 TCR and γ9δ2 TCR in the presence of CDH17 expressing tumor cells in these reporter assays, while the null control antibodies failed to activate reporter gene expression (Figure 36A-36B). Example 26: Identification and characterization of VHH against γδ T cell receptors

[0370] To screen for VHH against γδ T cell receptors, recombinant δ1-specific TCR and δ2-specific TCR antigens were used to immunize Alpaca. The identified hits were subjected to further positive screening and negative screening to identify γδTCR subtype specific VHH molecules. Through this process, VHHs with binding activities to both δ1-TCR and δ2- TCR (pan- γδTCR) were identified. Besides, multiple VHHs with binding affinities to either δ1- TCR or δ2- TCR were also identified.

[0371] ELISA-based binding assays were employed to evaluate representative lead VHH molecules to recombinant γδ T cell receptors. In this assay, 1 µg / mL recombinant human γ9δ1, γ9δ2, γ4δ1, γ4δ2 TCR-Fc antigen was coated on ELISA plate. Increasing concentrations of VHH hits were applied on the coated ELISA plate and their binding to the coated antigens were detected by HRP-conjugated anti-human IgG secondary antibody. The Pan-371 VHH was shown to be a pan-γδTCR molecule with binding affinities with all types of - γδTCR antigens. Multiple VHHs were either δ1-TCR or δ2-TCR specific binders with binding specificity to corresponding subtypes of γδTCR (Figure 37A to 37H). Example 27: Activation of γδ T cell receptor by CD123 x γδ TCR VHH bispecific antibodies

[0372] CD123 x γδTCR bispecific antibodies with anti-CD123 Fab arm and γδ TCR VHH listed in Table 7 were generated by the co-transfection of plasmids expressing the heavy Attorney Docket No.15271.0015-00304 chain and light chain of anti-CD123 antibodies and the γδ TCR VHH-Fc into Expi293F cells following the transfection kit instructions (Thermo Scientific). Cells were spun down five days post transfection, and the supernatant were passed through a 0.2 µm filter. The purifications of expressed bispecific antibodies were conducted by affinity chromatography over mAbSelectSure columns (Cytiva Life Sciences). The purified parental antibodies were buffer exchanged into DPBS, pH7.2 by dialysis, and protein concentrations were determined by UV absorbance at 280 nm.

[0373] The functional activities of exemplar CD123-flotetuzumab paired with representative γδTCR VHH with different subtype specificity in mediating the activating γδ T cell receptors by CD123-expressing tumor cells were assessed by Jurkat cell NFAT reporter assays. In this assay, plasmids expressing combination of γ9 and δ1 subunits or combination of γ9 and δ2 subunits were transiently transfected into Jurkat NFAT reporter cell line (BPS Bioscience) to express γ9δ1TCR or γ9δ2TCR in transfected cells. Exemplar CD123 x γδTCR VHH bispecific antibodies were incubated with reporter cells and CD123-expressing tumor cell MOLM-13 overnight. The dose-dependent activation of luciferase reporter gene expression in Jurkat NFAT reporter cells transfected with human γ9δ1 TCR and human γ9δ2 TCR by the bispecific antibody mediated ligation with CD123-expressing cells were quantitated. The CD123-flotetuzumab x Pan-371VHH and CD123-flotetuzumab x 5A6E9 bispecific antibodies showed concentration-dependent activation of both γ9δ1 TCR and γ9δ2 TCR in the presence of CD123 expressing tumor cells in these reporter assays, while CD123-flotetuzumab paired with γδTCR VHH with binding specificity to either γ9δ1 TCR or γ9δ2 TCR only activated Jurkat NFAT reporter cells transfected with corresponding subtypes of γδ TCR (Figure 38A and 38B).

[0374] CD123 x γδTCR VHH bispecific antibodies with different anti-CD123 Fab arms with or without SD-IL15_S7TI68K paired with Pan-371 VHH listed in Table 7 were also generated and characterized. ELISA-based binding assays were employed to evaluate their binding to CD123 and γ9δ2 TCR antigens. All the tested CD123 x γδTCR VHH bispecific antibodies showed concentration-dependent binding to CD123 (Figure 39A) and to γ9δ2 TCR (Figure 39B) with comparable good potencies. The functional activities of the tested CD123 x γδTCR VHH bispecific antibodies in mediating the activating γδ T cell receptors by CD123- expressing tumor cells were also assessed by Jurkat cell NFAT reporter assays. All the tested Attorney Docket No.15271.0015-00304 CD123 x γδTCR VHH bispecific antibodies showed concentration-dependent activation of both γ9δ1 TCR and γ9δ2 TCR in the presence of CD123 expressing tumor cells in these reporter assays (Figure 39C and 39D).

[0375] Provided herein is a representative list of certain sequences included in embodiments provided herein. Table 8 Sequences SEQ ID Description Sequence Attorney Docket No.15271.0015-00304 Anti-CD123 IPSNGA flotetuzumab HCDR2 Attorney Docket No.15271.0015-00304 Anti-CD20-rituximab YPGNGD HCDR2 Attorney Docket No.15271.0015-00304 Anti-γδTCR D1- 130VHH CDR2 IGDDN Attorney Docket No.15271.0015-00304 Anti-γδTCR D2- TSAGDP 142VHH CDR2 P T N N P M S N P M S K Y Attorney Docket No.15271.0015-00304 Anti-γδTCR 5A6E9 DIQMTQSTSSLSASVGDRVTITCRASQDISNYLNWYQQKPGK hum2LC light chain VVKLLIYYTSRLHSGVPSRFSGSGSGTDYTLTISSLQPEDVAT G K Q A L W L P P M V S S K Y Q V T Attorney Docket No.15271.0015-00304 Anti-CD20-rituximab QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSP light chain variable KPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYY Q S V P M G G N V EI TT IS E I G S G N C G RI D IT Y P T Attorney Docket No.15271.0015-00304 with L234A, L235A, KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA F405L mutations LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK A K T S N N F H V IS T A Q N F H V IS T A Q P M S G T L R E Attorney Docket No.15271.0015-00304 DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK S F H V IS T A Q N P T D R IS V G P M S G T L R E W S F H V IS T A Attorney Docket No.15271.0015-00304 VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK K Y T R K G K R G G K R K G K R Q S N A L K L P T F K Attorney Docket No.15271.0015-00304 DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTW YQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSL Anti-CD123 P E S P L P T F K P M P S N T P I S T T V S P S A P T Attorney Docket No.15271.0015-00304 PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK E N S P A P N A W V K Y R Q V K R T N V P H N L L Y A S E Attorney Docket No.15271.0015-00304 EVQLQQSVAELVKPGASVKMSCKVSGYTLTDHTIHWMKQRPEQGLEWIG YIYPRDGITGYNEKFKGKATLTADTSSSTAYMQLNSLTSEDSAVYFCARWGY K IC L R P F P W W P M G R E W G N V R IS V G G S C E K G N S Attorney Docket No.15271.0015-00304 T366S, L368A, HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT Y407V mutations VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV N A A L K L P T G P L P T G P M P S N T P I P R S A P T Y K Attorney Docket No.15271.0015-00304 AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN S P A P N A N T L K T L K T L K C P T A K A K Y S Y L N Attorney Docket No.15271.0015-00304 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNFGMNWVRQAP GKGLEWVAWINTNTGEPRYAEEFKGRFTISRDNAKNSLYLQ T A K A K Y S K I P T A K A K Y S Y S N N F H V IS T A Q G Attorney Docket No.15271.0015-00304 KAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGSGGGGSGG GSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHP S N F H V IS T A Q G G P S N F H V IS T A Q G G P S P M S G T L R E W Attorney Docket No.15271.0015-00304 QQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSG GGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGF S V L P M S G T L R E W G S V L P M S G T L R E W G S V L S F H V IS T Attorney Docket No.15271.0015-00304 ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQ G G P S S F H V IS T A Q G G P S S F H V IS T A Q G G P S C K T Attorney Docket No.15271.0015-00304 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT ECVLNKATNVAHWTTPSLKCIRGGGSGGGGSGGGSGGGGSL K C Y K N P N A W T Y K N Y K N Y K N C T A C T T A T Attorney Docket No.15271.0015-00304 CAAGGACTACTTCCCCGAACCCGTGACAGTCAGCTGGAAT AGCGGCGCCCTCACAAGCGGCGTGCACACATTTCCCGCCG C G G C A C A A C T A G G A G G G T G G G A A A A T G C G G A Attorney Docket No.15271.0015-00304 CCCTGGACCTTCGGTCAAGGCACTAAGGTGGAAATCAAAC GTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCT C G G C C G C C C C A T G C G C G T A A G G T C T A G A C C T A Attorney Docket No.15271.0015-00304 GGTTAAGTCATACTCACTTTATTCTCGCGAGAGATACATAT GTAACAGCGGGTTTAAACGGAAAGCAGGTACTTCAAGTCT T T G A T T A A T T T A C T C T C T A G G C G N P T D R IS V G G G S Attorney Docket No.15271.0015-00304 CKVTAMKCFLLELQVISLESGDASIHDTVENLIKLANNSLSSN GNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS P M S G T L R E W G S V L A L K L P T F K C T A T P L P T F K Attorney Docket No.15271.0015-00304 SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGG GGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYIC T A T P M P S N T P I S G S S A P T Y K N S C G S P A P N Attorney Docket No.15271.0015-00304 STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEW V G K N I T V YI T Y P S G A S V R Q S N F V S C K R G A L K L P Attorney Docket No.15271.0015-00304 mutations with SD- SNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPK IL15_S7TI68K DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT P G I K I P L P T F K C T A T P M P S N T P I S G S Attorney Docket No.15271.0015-00304 QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPP GKGLEWMGYINFRGSTNYNPSLKSRVTISRDTSKNQFSLKLS A P T Y K N S C G S P A P N A W V G K N References Adams, E. J., S. Gu and A. M. Luoma (2015). "Human gamma delta T cells: Evolution and ligand recognition." Cell Immunol 296(1): 31-40. Akinleye, A. and Z. Rasool (2019). "Immune checkpoint inhibitors of PD-L1 as cancer therapeutics." J Hematol Oncol 12(1): 92. Bolt, S., E. Routledge, I. Lloyd, L. Chatenoud, H. Pope, S. D. Gorman, M. Clark and H. Waldmann (1993). "The generation of a humanized, non-mitogenic CD3 monoclonal antibody which retains in vitro immunosuppressive properties." Eur J Immunol 23(2): 403-411. Attorney Docket No.15271.0015-00304 Cano, C. E., C. Pasero, A. De Gassart, C. Kerneur, M. Gabriac, M. Fullana, E. Granarolo, R. Hoet, E. Scotet, C. Rafia, T. Herrmann, C. Imbert, L. Gorvel, N. Vey, A. Briantais, A. C. le Floch and D. Olive (2021). "BTN2A1, an immune checkpoint targeting Vgamma9Vdelta2 T cell cytotoxicity against malignant cells." Cell Rep 36(2): 109359. Chothia, C. and A. M. Lesk (1987). "Canonical structures for the hypervariable regions of immunoglobulins." J Mol Biol 196(4): 901-917. Chu, S. Y., I. Vostiar, S. Karki, G. L. Moore, G. A. Lazar, E. Pong, P. F. Joyce, D. E. Szymkowski and J. R. Desjarlais (2008). "Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies." Mol Immunol 45(15): 3926-3933. Dall'Acqua, W. F., P. A. Kiener and H. Wu (2006). "Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)." J Biol Chem 281(33): 23514-23524. Dempke, W. C. M., K. Fenchel, P. Uciechowski and S. P. Dale (2017). "Second- and third- generation drugs for immuno-oncology treatment-The more the better?" Eur J Cancer 74: 55-72. Diebolder, C. A., F. J. Beurskens, R. N. de Jong, R. I. Koning, K. Strumane, M. A. Lindorfer, M. Voorhorst, D. Ugurlar, S. Rosati, A. J. Heck, J. G. van de Winkel, I. A. Wilson, A. J. Koster, R. P. Taylor, E. O. Saphire, D. R. Burton, J. Schuurman, P. Gros and P. W. Parren (2014). "Complement is activated by IgG hexamers assembled at the cell surface." Science 343(6176): 1260-1263. Donia, M., E. Ellebaek, M. H. Andersen, P. T. Straten and I. M. Svane (2012). "Analysis of Vdelta1 T cells in clinical grade melanoma-infiltrating lymphocytes." Oncoimmunology 1(8): 1297-1304. Gentles, A. J., A. M. Newman, C. L. Liu, S. V. Bratman, W. Feng, D. Kim, V. S. Nair, Y. Xu, A. Khuong, C. D. Hoang, M. Diehn, R. B. West, S. K. Plevritis and A. A. Alizadeh (2015). "The prognostic landscape of genes and infiltrating immune cells across human cancers." Nat Med 21(8): 938-945. Gunasekaran, K., M. Pentony, M. Shen, L. Garrett, C. Forte, A. Woodward, S. B. Ng, T. Born, M. Retter, K. Manchulenko, H. Sweet, I. N. Foltz, M. Wittekind and W. Yan (2010). "Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG." J Biol Chem 285(25): 19637-19646. Attorney Docket No.15271.0015-00304 Han, C. and B. S. Kwon (2018). "Chimeric antigen receptor T-cell therapy for cancer: a basic research-oriented perspective." Immunotherapy 10(3): 221-234. Hinton, P. R., J. M. Xiong, M. G. Johlfs, M. T. Tang, S. Keller and N. Tsurushita (2006). "An engineered human IgG1 antibody with longer serum half-life." J Immunol 176(1): 346-356. Kinder, M., A. R. Greenplate, K. D. Grugan, K. L. Soring, K. A. Heeringa, S. G. McCarthy, G. Bannish, M. Perpetua, F. Lynch, R. E. Jordan, W. R. Strohl and R. J. Brezski (2013). "Engineered protease-resistant antibodies with selectable cell-killing functions." J Biol Chem 288(43): 30843-30854. Labrijn, A. F., J. I. Meesters, B. E. de Goeij, E. T. van den Bremer, J. Neijssen, M. D. van Kampen, K. Strumane, S. Verploegen, A. Kundu, M. J. Gramer, P. H. van Berkel, J. G. van de Winkel, J. Schuurman and P. W. Parren (2014). "Efficient generation of stable bispecific IgG1 by controlled Fab-arm exchange." Proc Natl Acad Sci U S A 110(13): 5145-5150. Lefranc, M. P., C. Pommie, M. Ruiz, V. Giudicelli, E. Foulquier, L. Truong, V. Thouvenin- Contet and G. Lefranc (2003). "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains." Dev Comp Immunol 27(1): 55-77. Lu, H., W. Dai, J. Guo, D. Wang, S. Wen, L. Yang, D. Lin, W. Xie, L. Wen, J. Fang and Z. Wang (2020). "High Abundance of Intratumoral gammadelta T Cells Favors a Better Prognosis in Head and Neck Squamous Cell Carcinoma: A Bioinformatic Analysis." Front Immunol 11: 573920. Luoma, A. M., C. D. Castro, T. Mayassi, L. A. Bembinster, L. Bai, D. Picard, B. Anderson, L. Scharf, J. E. Kung, L. V. Sibener, P. B. Savage, B. Jabri, A. Bendelac and E. J. Adams (2013). "Crystal structure of Vdelta1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human gammadelta T cells." Immunity 39(6): 1032-1042. Mangan, B. A., M. R. Dunne, V. P. O'Reilly, P. J. Dunne, M. A. Exley, D. O'Shea, E. Scotet, A. E. Hogan and D. G. Doherty (2013). "Cutting edge: CD1d restriction and Th1 / Th2 / Th17 cytokine secretion by human Vdelta3 T cells." J Immunol 191(1): 30-34. Maude, S. L., N. Frey, P. A. Shaw, R. Aplenc, D. M. Barrett, N. J. Bunin, A. Chew, V. E. Gonzalez, Z. Zheng, S. F. Lacey, Y. D. Mahnke, J. J. Melenhorst, S. R. Rheingold, A. Shen, D. T. Teachey, B. L. Levine, C. H. June, D. L. Porter and S. A. Grupp (2014). "Chimeric antigen receptor T cells for sustained remissions in leukemia." N Engl J Med 371(16): 1507-1517. Attorney Docket No.15271.0015-00304 Mellman, I., G. Coukos and G. Dranoff (2011). "Cancer immunotherapy comes of age." Nature 480(7378): 480-489. Meraviglia, S., E. Lo Presti, M. Tosolini, C. La Mendola, V. Orlando, M. Todaro, V. Catalano, G. Stassi, G. Cicero, S. Vieni, J. J. Fournie and F. Dieli (2017). "Distinctive features of tumor- infiltrating gammadelta T lymphocytes in human colorectal cancer." Oncoimmunology 6(10): e1347742. Nussbaumer, O. and M. Koslowski (2019). "The emerging role of gammadelta T cells in cancer immunotherapy." Immunooncol Technol 1: 3-10. Petkova, S. B., S. Akilesh, T. J. Sproule, G. J. Christianson, H. Al Khabbaz, A. C. Brown, L. G. Presta, Y. G. Meng and D. C. Roopenian (2006). "Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease." Int Immunol 18(12): 1759-1769. Ridgway, J. B., L. G. Presta and P. Carter (1996). "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization." Protein Eng 9(7): 617-621. Saura-Esteller, J., M. de Jong, L. A. King, E. Ensing, B. Winograd, T. D. de Gruijl, P. Parren and H. J. van der Vliet (2022). "Gamma Delta T-Cell Based Cancer Immunotherapy: Past-Present- Future." Front Immunol 13: 915837. Shields, R. L., A. K. Namenuk, K. Hong, Y. G. Meng, J. Rae, J. Briggs, D. Xie, J. Lai, A. Stadlen, B. Li, J. A. Fox and L. G. Presta (2001). "High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R." J Biol Chem 276(9): 6591-6604. Silva-Santos, B., K. Serre and H. Norell (2015). "gammadelta T cells in cancer." Nat Rev Immunol 15(11): 683-691. Stavenhagen, J. B., S. Gorlatov, N. Tuaillon, C. T. Rankin, H. Li, S. Burke, L. Huang, S. Johnson, S. Koenig and E. Bonvini (2008). "Enhancing the potency of therapeutic monoclonal antibodies via Fc optimization." Adv Enzyme Regul 48: 152-164. Tanaka, Y., C. T. Morita, Y. Tanaka, E. Nieves, M. B. Brenner and B. R. Bloom (1995). "Natural and synthetic non-peptide antigens recognized by human gamma delta T cells." Nature 375(6527): 155-158. Tosolini, M., F. Pont, M. Poupot, F. Vergez, M. L. Nicolau-Travers, D. Vermijlen, J. E. Sarry, F. Dieli and J. J. Fournie (2017). "Assessment of tumor-infiltrating TCRVgamma9Vdelta2 Attorney Docket No.15271.0015-00304 gammadelta lymphocyte abundance by deconvolution of human cancers microarrays." Oncoimmunology 6(3): e1284723. Vafa, O., G. L. Gilliland, R. J. Brezski, B. Strake, T. Wilkinson, E. R. Lacy, B. Scallon, A. Teplyakov, T. J. Malia and W. R. Strohl (2014). "An engineered Fc variant of an IgG eliminates all immune effector functions via structural perturbations." Methods 65(1): 114-126. Waldman, A. D., J. M. Fritz and M. J. Lenardo (2020). "A guide to cancer immunotherapy: from T cell basic science to clinical practice." Nat Rev Immunol 20(11): 651-668. Wang, J., C. Lin, H. Li, R. Li, Y. Wu, H. Liu, H. Zhang, H. He, W. Zhang and J. Xu (2017). "Tumor-infiltrating gammadeltaT cells predict prognosis and adjuvant chemotherapeutic benefit in patients with gastric cancer." Oncoimmunology 6(11): e1353858. Worn, A. and A. Pluckthun (2001). "Stability engineering of antibody single-chain Fv fragments." J Mol Biol 305(5): 989-1010. Wu, T. T. and E. A. Kabat (1970). "An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity." J Exp Med 132(2): 211-250. Xu, D., M. L. Alegre, S. S. Varga, A. L. Rothermel, A. M. Collins, V. L. Pulito, L. S. Hanna, K. P. Dolan, P. W. Parren, J. A. Bluestone, L. K. Jolliffe and R. A. Zivin (2000). "In vitro characterization of five humanized OKT3 effector function variant antibodies." Cell Immunol 200(1): 16-26. Zalevsky, J., A. K. Chamberlain, H. M. Horton, S. Karki, I. W. Leung, T. J. Sproule, G. A. Lazar, D. C. Roopenian and J. R. Desjarlais (2010). "Enhanced antibody half-life improves in vivo activity." Nat Biotechnol 28(2): 157-159. Zhang, D., A. A. Armstrong, S. H. Tam, S. G. McCarthy, J. Luo, G. L. Gilliland and M. L. Chiu (2017). "Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody multimerization." MAbs 9: 1129-1142. Zhao, Y., C. Niu and J. Cui (2018). "Gamma-delta (gammadelta) T cells: friend or foe in cancer development?" J Transl Med 16(1): 3.

Claims

Attorney Docket No.15271.0015-00304 We Claim:

1. An anti-γδTCR antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 is selected from: SEQ ID NOs: 7, 43, 46, 49, 52, 55, 58, 61, 64, 67, and 70; the HCDR2 is selected from: SEQ ID NOs: 8, 44, 47, 50, 53, 56, 59, 62, 65, 68, and 71; and the HCDR3 is selected from: SEQ ID NOs: 9, 45, 48, 51, 54, 57, 60, 63, 66, 69, and 72.

2. The anti-γδTCR antibody or antigen-binding fragment of claim 1, wherein the HCDR1, HCDR2, and HCDR3 comprise: SEQ ID NOs: 7, 8, and 9; SEQ ID NOs: 43, 44, and 45; SEQ ID NOs: 46, 47, and 48; SEQ ID NOs: 49, 50, and 51; SEQ ID NOs: 52, 53, and 54; SEQ ID NOs: 55, 56, and 57; SEQ ID NOs: 58, 59, and 60; SEQ ID NOs: 61, 62, and 63; SEQ ID NOs: 64, 65, and 66; SEQ ID NOs: 67, 68, and 69; or SEQ ID NOs: 70, 71, and 72; respectively; 3. The anti-γδTCR antibody or antigen-binding fragment of any one of claims 1-2, wherein the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 103-109 and 129-138, and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 103-109 and 129-138.

4. The anti-γδTCR antibody or antigen-binding fragment of any one of claims 1-3, further comprisingAttorney Docket No.15271.0015-00304 a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1 is SEQ ID NO: 10; the LCDR2 is SEQ ID NOs: 11; and the LCDR3 is SEQ ID NOs:

12.

5. The anti-γδTCR antibody or antigen-binding fragment of claim 4, wherein the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 110-114 and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 110-114.

6. The anti-γδTCR antibody or antigen-binding fragment of any one of claims 1-3, wherein the antibody or antigen-binding fragment is a VHH antibody or antigen-binding fragment.

7. A bispecific antibody comprising: a first arm that is capable of targeting a disease associated antigen, and a second arm comprising the anti-γδTCR antibody or antigen-binding fragment of any one of claims 1-6.

8. The bispecific antibody of claim 7, wherein the bispecific antibody is capable of targeting a γδ T cell receptor and is capable of directing γδ T cells to kill target cells expressing the disease associated antigen, and wherein the bispecific antibody optionally comprises an immune modulator component that is capable of stimulating the proliferation and / or activation of γδ T cells.

9. The bispecific antibody of any one of claims 7-8, wherein the optional immune modulator component comprises an attenuated IL-15 and / or the sushi domain of IL-15Ra (SD- IL15).

10. The bispecific antibody of claims 8-9, wherein the attenuated IL-15 and the sushi domain of IL-15Ra (SD-IL15) comprises amino acid sequence SEQ ID NO: 301, 302, or 303.Attorney Docket No.15271.0015-00304 11. The bispecific antibody of any one of claims 7-10, wherein the first arm comprises an antibody or antibody fragment targeting 5T4, CD123, CD20, or CDH17.

12. The bispecific antibody of any one of claims 7-11, wherein the first arm comprises: a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise sequences chosen from SEQ ID NOs: 1 to 3, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise sequences chosen from SEQ ID NOs: 4 to 6, respectively.

13. The bispecific antibody of any one of claims 7-11, wherein the first arm comprises: a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise sequences chosen from SEQ ID NOs: 13-15, 19-21, and 25-27, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise sequences chosen from SEQ ID NOs: 16-18, 22-24, and 28-30, respectively.

14. The bispecific antibody of any one of claims 7-11, wherein the first arm comprises: a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise sequences chosen from SEQ ID NOs: 31-33 and 37-39, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise sequences chosen from SEQ ID NOs: 34-36 and 40-42, respectively.

15. The bispecific antibody of any one of claims 7-14, wherein the second arm comprises:Attorney Docket No.15271.0015-00304 a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 comprise SEQ ID NOs: 7, 8, and 9, respectively; and a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1, LCDR2, and LCDR3 comprise SEQ ID NOs: 10, 11, and 12, respectively.

16. The bispecific antibody of any one of claims 7-14, wherein the second arm comprises: a VHH variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 is selected from: SEQ ID NOs: 43, 46, 49, 52, 55, 58, 61, 64, 67, and 70; the HCDR2 is selected from: SEQ ID NOs: 44, 47, 50, 53, 56, 59, 62, 65, 68, and 71; and the HCDR3 is selected from: SEQ ID NOs: 45, 48, 51, 54, 57, 60, 63, 66, 69, and 72; respectively.

17. The bispecific antibody of any one of claims 7-11, wherein the first arm comprises: a heavy chain variable region selected from SEQ ID NOs: 101, 115, 117, 118, 121, 122, 125, 127, and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 101, 115, 117, 118, 121, 122, 125, and 127, and a light chain variable region chosen from SEQ ID NOs: 102, 116, 119, 120, 123, 124, 126, 128, and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 102, 116, 119, 120, 123, 124, 126, and 128.

18. The bispecific antibody of any one of claims 7-11 and 17, wherein the second arm comprises: a heavy chain variable region selected from SEQ ID NOs: 103-109 and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 103-109, and a light chain variable region selected from SEQ ID NOs: 110-114 and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 110-114.Attorney Docket No.15271.0015-00304 19. The bispecific antibody of any one of claims 7-11 and 17, wherein the second arm comprises a VHH variable region selected from SEQ ID NOs: 129-138 and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 129-138.

20. The bispecific antibody of any one of claims 7-11, wherein the first arm comprises: a heavy chain amino acid sequence chosen from SEQ ID NOs: 201, 215, 217, 218, 221, 222, 225, 227, 233, 234, 235, 236, 237, and an amino acid sequence having at least 85% identity to SEQ ID NO: 201, 215, 217, 218, 221, 222, 225, 227, 233, 234, 235, 236, and 237, and a light chain amino acid sequence chosen from SEQ ID NOs: 202, 216, 219, 220, 223, 224, 226, 228, and an amino acid sequence having at least 85% identity to SEQ ID NOs: 202, 216, 219, 220, 223, 224, 226, and 228.

21. The bispecific antibody of any one of claims 7-11 and 20, wherein the second arm comprises: a heavy chain amino acid sequence selected from SEQ ID NOs: 203-209, and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 203-209, and a light chain amino acid sequence selected from SEQ ID NOs: 210-214 and an amino acid sequence having at least 85% identity to SEQ ID NOs: 210-214.

22. The bispecific antibody of any one of claims 7-11 and 20, wherein the second arm comprises a VHH amino acid sequence selected from SEQ ID NOs: 229, 230, 231, 232, and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 229, 230, 231, and 232.

23. The bispecific antibody of any one of claims 7-8, wherein the first arm comprises: a heavy chain amino acid sequence chosen from SEQ ID NOs: 304 to 306, 330 to 341, and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 304 to 306, and 330 to 341, and a light chain sequence chosen from SEQ ID NOs: 202, 216, 219, 220, 223, 224, 226, 228, and an amino acid sequence having at least 85% identity to SEQ ID NO: 202, 216, 219, 220, 223, 224, 226, and 228.Attorney Docket No.15271.0015-00304 24. The bispecific antibody of any one of claims 7-8 and 23, wherein the second arm comprises: a heavy chain amino acid sequence chosen from SEQ ID NOs: 307 to 315, 328, 329, and an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 307-315, 328, and 329, and a light chain sequence chosen from SEQ ID NOs: 210 to 214, and an amino acid sequence having at least 85% identity to SEQ ID NOs: 210 to 214.

25. An anti-CD123 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising complementarity determining region (CDR) sequences HCDR1, HCDR2, and HCDR3, wherein the HCDR1 is selected from: SEQ ID NOs: 19 and 25; the HCDR2 is selected from: SEQ ID NOs: 20 and 26; and the HCDR3 is selected from: SEQ ID NOs: 21 and 27; and / or a light chain variable region comprising complementarity determining region (CDR) sequences LCDR1, LCDR2, and LCDR3, wherein the LCDR1 is selected from: SEQ ID NOs: 22 and 28; the LCDR2 is selected from: SEQ ID NOs: 23 and 29; and the LCDR3 is selected from: SEQ ID NOs: 24 and 30.

26. The anti-CD123 antibody or antigen-binding fragment of claim 25, wherein the HCDR1, HCDR2, and HCDR3 comprise: SEQ ID NOs: 19, 20, and 21; or SEQ ID NOs: 25, 26, and 27; respectively; the LCDR1, LCDR2, and LCDR3 comprise: SEQ ID NOs: 22, 23, and 24; or SEQ ID NOs: 28, 29, and 30; respectively.

27. The anti-CD123 antibody or antigen-binding fragment of any one of claims 25-26, whereinAttorney Docket No.15271.0015-00304 the heavy chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 117-118 and 121-122 or an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 117-118 and 121-122; and / or the light chain variable region comprises an amino acid sequence selected from SEQ ID NOs: 119-120 and 123-124 or an amino acid sequence having at least 85% identity to any one of SEQ ID NOs: 119-120 and 123-124.

28. The anti-CD123 antibody or antigen-binding fragment of claim 25, comprising: heavy chain variable region SEQ ID NO: 117 and light chain variable region SEQ ID NO: 119; heavy chain variable region SEQ ID NO: 117 and light chain variable region SEQ ID NO: 120; heavy chain variable region SEQ ID NO: 118 and light chain variable region SEQ ID NO: 119; heavy chain variable region SEQ ID NO: 118 and light chain variable region SEQ ID NO: 120; heavy chain variable region SEQ ID NO: 121 and light chain variable region SEQ ID NO: 123; heavy chain variable region SEQ ID NO: 121 and light chain variable region SEQ ID NO: 124; heavy chain variable region SEQ ID NO: 122 and light chain variable region SEQ ID NO: 123; or heavy chain variable region SEQ ID NO: 122 and light chain variable region SEQ ID NO: 124; respectively.

29. A nucleic acid encoding one or more of the polypeptides of the antibody or antigen- binding fragment of any one of claims 1-6 and 25-28 or of the bispecific antibody of any one of claims 7-24.

30. A vector comprising the nucleic acid of claim 29.

31. A cell comprising the vector of claim 30.

32. A method for preparing the antibody or antigen-binding fragment of any one of claims 1-6 and 25-28 or the bispecific antibody of any one of claims 7-24, comprising: culturing the cell of claim 31, and obtaining the antibody, antigen-binding fragment or the bispecific antibody from the culture, optionally using controlled Fab arm exchange of culture supernatants.Attorney Docket No.15271.0015-00304 33. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-6 and 25-28 or the bispecific antibody of any one of claims 7-24 and a pharmaceutical carrier.

34. A method for treating or preventing a cancer in a subject, comprising administering to the subject a pharmaceutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-6 and 25-28 or the bispecific antibody of any one of claims 7-24 or the pharmaceutical composition of claim 33.

35. The method for treating or preventing a cancer according to claim 34, wherein the cancer is selected from pancreatic cancer, triple negative breast cancer, lung cancer, and any cancer with tumor associated antigen expression.

36. The method for treating or preventing a cancer according to claim 34, further comprising administering chemotherapy to the subject.

37. A method for treating or preventing an autoimmune disease with a disease associated antigen expression in a subject, comprising administering to the subject a pharmaceutically effective amount of the antibody or antigen-binding fragment of any one of claims 1-6 and 25-28 or the bispecific antibody of any one of claims 7-24 or the pharmaceutical composition of claim 33.

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