Bispecific antibody fusion molecules targeting CD180 and CD3 and methods of use thereof

Abstract

The present disclosure provides bispecific antibodies or antigen binding fragments thereof that specifically bind to CD3 and CD180. The antibodies are optionally fused with a CD58 peptide or portions thereof. Described herein are methods of making and using the bispecific antibodies for treatment of cancers that express CD180. The present disclosure also relates to methods and kits for detecting a predisposition to, determining the risk of, and guiding therapy for CD180 related disorders.

Description

Attorney Docket No. EVIM-006 / 001WO 339103-2023 BISPECIFIC ANTIBODY FUSION MOLECULES TARGETING CD180 AND CD3 AND METHODS OF USE THEREOF RELATED APPLICATIONS

[0001] This application claims the priority to, and benefit of, U.S. Provisional Application No. 63 / 584,166, filed on September 20, 2023, the contents of which are incorporated by reference in its entirety herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (EVIM_006_001WO_SeqList_ST26.xml; Size: 378,759 bytes; and Date of Creation: September 20, 2024) are herein incorporated by reference in its entirety. FIELD

[0003] This invention relates to pharmaceutical compositions of bispecific antibodies that bind CD180 and CD3 and their use. This invention also relates to a method and kits for detecting a predisposition to, determining risk of, and guiding therapy for cancers. BACKGROUND OF THE INVENTION

[0004] CD180 is a type 1 transmembrane toll-like receptor (TLR) with the highest sequence similarity to TLR4 and was initially discovered as a mouse B lymphocyte activation receptor that protects B cells from irradiation and dexamethasone-mediated cell death (Miyake et al., 1995, J. Immunol. 154: 3333–3340; Divanovic et al., 2005, Nat. Immunol. 6: 571–578). CD180 and MD-1, an interacting protein required for CD180 surface expression and function, are found on the surface of B cells and monocytes, but not T lymphocytes. It is also found on other myeloid cells with similar expression in humans and mice (Ohto et al., 2011, J. Mol. Biol. 413: 815–825; Mayeur-Rousse et al., 2016, Cytometry B. Clin. Cytom. 90: 462–466). CD180 / MD-1 is also found on hematological malignancies such as B cell lymphoma (Mayeur-Rousse et al., 2016, Cytometry B. Clin. Cytom. 90: 462–466; Miguet et al., 2013, Leukemia 27: 1748–1750; Mansour et al., 2020, J. Hematopathol.13: 205–211; Fidyt et al., 2022, Blood 140: 10273–10274; Favre et al., 2018, Int. J. Lab. Hematol.40: e59-62), chronicAttorney Docket No. EVIM-006 / 001WO 339103-2023 lymphocytic leukemia (CLL)(Porakishvili et al., 2005, Br. J. Haematol. 131: 313–319; Edwards et al., 2021, Br. J. Haematol.195), and acute myeloid leukemia (AML) (Kramer et al., 2022, Blood 140: 1533–1548; Saito et al., 2010, Sci. Transl. Med. 2) implicating CD180 / MD-1 as a novel target for cancer immunotherapy with differentiated opportunities in relapse and / or refractory B cell malignancies as an alternative B cell lineage antigen, where CD19 and CD20 loss emerge from CD19 / CD20 targeted treatments, and in myeloid malignancies of the monocytic lineage (Marshalek et al., 2022, JCO 40: e19537–e19537; Duell, et al., 2024, Blood 143: 685-696).

[0005] There is a continued need for the development of agents that target CD180 that is efficacious and / or safe for human use. There is a need in the art for agents that target CD180 for the diagnosis and treatment of CD180 associated conditions, such as cancers. Further, there exists a need for methods and kits to identify whether a subject with a CD180 expressing cancer will be responsive to such antibody-based therapies. Provided herein are methods and compositions addressing this need. SUMMARY OF THE INVENTION

[0006] The present disclosure provides a bispecific antibody comprising a first antigen binding region that binds to CD3 and a second antigen binding region that binds to CD180, wherein the first antigen binding region that binds to CD3 comprises three heavy chain complementarity determining regions (CDRH1, CDRH2, CDRH3) and three light chain complementarity determining regions (CDRL1, CDRL2, CDRL3), wherein a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 30; CDRH2 comprises the amino acid sequence of SEQ ID NO: 34; CDRH3 comprises the amino acid sequence of SEQ ID NO: 37; CDRL1 comprises the amino acid sequence of SEQ ID NO: 42; CDRL2 comprises the amino acid sequence of SEQ ID NO: 43; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 45; and wherein the second antigen binding region that binds to CD180 comprises three heavy chain complementarity determining regions (CDRH1, CDRH2, CDRH3) and three light chain complementarity determining regions (CDRL1, CDRL2, CDRL3): i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; CDRH2 comprises the amino acid sequence of SEQ ID NO: 212; CDRH3 comprises the amino acid sequence of SEQ ID NO: 214; CDRL1 comprises the amino acid sequence of SEQ ID NO:Attorney Docket No. EVIM-006 / 001WO 339103-2023 222; CDRL2 comprises the amino acid sequence of SEQ ID NO: 223; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 224.

[0007] In some embodiments, the first antigen binding region that binds to CD3 comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein: a) VH comprises the amino acid sequence of SEQ ID NO: 17 and VL comprises the amino acid sequence of SEQ ID NO: 22; and wherein the second antigen binding region that binds to CD180 comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein: i) VH comprises the amino acid sequence of SEQ ID NO: 197 and VL comprises the amino acid sequence of SEQ ID NO: 196; ii) VH comprises the amino acid sequence of SEQ ID NO: 199 and VL comprises the amino acid sequence of SEQ ID NO: 198; iii) VH comprises the amino acid sequence of SEQ ID NO: 201 and VL comprises the amino acid sequence of SEQ ID NO: 200; iv) VH comprises the amino acid sequence of SEQ ID NO: 203 and VL comprises the amino acid sequence of SEQ ID NO: 202; or v)VH comprises the amino acid sequence of SEQ ID NO: 205 and VL comprises the amino acid sequence of SEQ ID NO: 204.

[0008] In some embodiments, the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1), and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1); and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constantregion (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constantregion 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2), and wherein i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CH1H1is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; iii) the amino acid at position 173 (EU numbering) of the CH1H1 is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and i) the amino acid at position 39 (KabatAttorney Docket No. EVIM-006 / 001WO 339103-2023 numbering) of the VH2 is a D and the amino acid at position 38 (Kabat numbering) of the VL2 is a K; and ii) the amino acid at position 147 (EU numbering) of the CH2H1 is a D and the amino acid at position 180 (EU numbering) of the CL2 is a R.

[0009] In some embodiments, the bispecific antibody comprises: i) the amino acid at position 87 (Kabat numbering) of the VH1 and / or VH2 is a G; and ii) the amino acid at position 45 (Kabat numbering) of the VL1 and / or VL2 is a W.

[0010] In some embodiments, the bispecific antibody comprises: i) the CH1H3 has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3has a C at position 354 and a W at position 366 (EU numbering); ii) the CH2H3has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3has a C at position 354 and a W at position 366 (EU numbering); iii) the CH1H3has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3has a C at position 349 and a W at position 366 (EU numbering); or iv) the CH2H3 has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3 has a C at position 349 and a W at position 366 (EU numbering).

[0011] In some embodiments, the amino acid at position 447 (EU numbering) of the CH1H3 and / or of the CH2H3 is deleted.

[0012] In some embodiments, i) the H1H and / or the H2H has an A at positions 234 and 235 (EU numbering); ii) the H1H and / or the H2H has an A at positions 234, 235 and 237 (EU numbering); or iii) the H1H and / or the H2H has an A at positions 234 and 235 and G at position 329 (EU numbering).

[0013] In some embodiments, i) the CH1H3and / or the CH2H3has an A at position 297 (EU numbering); ii) the CH1H3and / or the CH2H3has a G at position 297 (EU numbering); or iii) the CH1H3and / or the CH2H3has a S at position 297 (EU numbering). In some embodiments, the CH1H3and / or the CH2H3has an S at position 331 (EU numbering).

[0014] In some embodiments, a polypeptide is fused to the N-terminus or the C-terminus of the first heavy chain polypeptide or the second heavy chain polypeptide. In some embodiments, the polypeptide is fused to the C-terminus of the first heavy chain polypeptide or the C-terminus of the second heavy chain polypeptide.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0015] In some embodiments, the polypeptide is fused via a linker peptide. In some embodiments, the linker peptide comprises the amino acid sequence of SEQ ID NO: 53. In some embodiments, the polypeptide comprises a CD58 or a fragment thereof. In some embodiments, the CD58 comprises the amino acid sequence of SEQ ID NO: 49.

[0016] In some embodiments, the bispecific antibody is an IgG1 or an IgG4 antibody. In some embodiments, the bispecific antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody.

[0017] In some embodiments, the first antigen binding region that binds to CD3 comprises a heavy chain (HC) and a light chain (LC), wherein: a) HC comprises the amino acid sequence of SEQ ID NO: 305 and LC comprises the amino acid sequence of SEQ ID NO: 304; b) HC comprises the amino acid sequence of SEQ ID NO: 309 and LC comprises the amino acid sequence of SEQ ID NO: 308; c) HC comprises the amino acid sequence of SEQ ID NO: 313 and LC comprises the amino acid sequence of SEQ ID NO: 312; d) HC comprises the amino acid sequence of SEQ ID NO: 317 and LC comprises the amino acid sequence of SEQ ID NO: 316; or e) HC comprises the amino acid sequence of SEQ ID NO: 321 and LC comprises the amino acid sequence of SEQ ID NO: 320; and wherein the second antigen binding region that binds to CD180 comprises a heavy chain (HC) and a light chain (LC), wherein: i) HC comprises the amino acid sequence of SEQ ID NO: 303 and LC comprises the amino acid sequence of SEQ ID NO: 302; ii) VH comprises the amino acid sequence of SEQ ID NO: 307 and VL comprises the amino acid sequence of SEQ ID NO: 306; iii) VH comprises the amino acid sequence of SEQ ID NO: 311 and VL comprises the amino acid sequence of SEQ ID NO: 310; iv) VH comprises the amino acid sequence of SEQ ID NO: 315 and VL comprises the amino acid sequence of SEQ ID NO: 314; or v) VH comprises the amino acid sequence of SEQ ID NO: 319 and VL comprises the amino acid sequence of SEQ ID NO: 318.

[0018] This disclosure provides a polynucleotide comprising a nucleic acid sequence encoding any one of bispecific antibodies of the disclosure. This disclosure provides a vector comprising any one of the polynucleotides of the disclosure. This disclosure provides a pharmaceutical composition comprising any one of bispecific antibodies of the disclosure. any one of polynucleotides of the disclosure or any one of the vectors of the disclosure and a pharmaceutically acceptable carrier.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0019] This disclosure provides a method of treating a cancer expressing CD180 in a subject in need thereof comprising administering a therapeutically effective amount of any one of the pharmaceutical compositions of the disclosure. The disclosure provides a method of T-cell re-targeting in a subject in need thereof comprising administering a therapeutically effective amount of any one of the pharmaceutical compositions of the disclosure.

[0020] The disclosure provides a method of T-cell activation in a subject in need thereof comprising administering a therapeutically effective amount of any one of the pharmaceutical compositions of the disclosure.

[0021] In some embodiments, the cancer is a lymphoma or leukemia.

[0022] In some embodiments, the lymphoma or leukemia is Hodgkin's lymphoma, non- Hodgkin's lymphoma (NHL), B-cell lymphoma, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's Macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML).

[0023] In some embodiments, the NHL is small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, or bulky disease NHL.

[0024] In some embodiments, the subject in need thereof has a cancer. In some embodiments, the subject is simultaneously administered or previously administered with a therapeutically effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a CAR-T cell therapy, an immune checkpoint inhibitor, a co-stimulatory ligand or a cytokine.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS.1A-1B are non-reduced SDS PAGE analysis for human and cyno CD180- MD1 complex purification (Lane 1) against standard molecular weight markers (Lane 2).

[0027] FIG.2 is the analytical size exclusion chromatography of the purified human and cyno CD180-MD1 complex.

[0028] FIG.3A shows the structural alignment of the modeled structure of Fv domain of murine MHR73-11 antibody (light color) over Fv domain of a structurally similar human antibody (dark color, PDB ID: 4N90) that was preferred as template for CDR grafting. FIG.Attorney Docket No. EVIM-006 / 001WO 339103-2023 3B shows the structural alignment of the modeled structure of Fv domain of murine MHR73-11 antibody (light color) over Fv domain of a structurally less similar human antibody (dark color, PDB ID: 4YE4) that was not preferred as template for CDR grafting.

[0029] FIGS.4A-4E is a series of a schematic of CD180 targeted bispecific fusions with charged pair mutations, disulfide bond repositioning and knob into hole mutations. Grey shaded domains represent a first heavy chain polypeptide (H1) having a heavy chain variable region (VH1), having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1). White shaded domains represent a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2). The + and – symbols between the antigen binding domains represent the charged pair mutations. Lines between the CH1H1 and CL1 domain and CH2H1 and CL2 domain represent disulfide bonds, where a solid line represents an endogenous disulfide bond and a dashed line represents a repositioned disulfide bond. The protrusion and dent between the CH1H3 and CH2H3 domain represent knob into hole mutations. Charged pair mutations, disulfide bond repositioning and knob into hole mutations provide increased heavy chain and light chain heterodimerization, which is advantageous for production and purification of bispecific antibodies of the disclosure. FIG. 4E depicts a schematic diagram of an exemplary bispecific antibody fusion molecule comprising an anti-CD3 arm, an anti-CD180 arm and a CD58 peptide fused to the CH1H3domain.

[0030] FIG. 5A shows preparative chromatograms obtained from tandem purification (protein A elution and size exclusion chromatography) of CD180 targeted bispecific fusions expressed from transient expi293 cells. FIGS. 5B-5F are a series of graphs depicting chromatograms obtained from analytical size exclusion chromatography of CD180 targeted bispecific fusion antibodies. FIG. 5B shows EIP1042. FIG. 5C shows EIP1043. FIG. 5D shows EIP1044. FIG.5E shows EIP1056. FIG.5F shows EIP1057.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0031] FIG. 6 is a differential scanning calorimetry analysis of CD180 targeted bispecific fusions. The samples in PBS were heated from 25 °C to 95 °C with 1 °C / min increment on a nanoDSC system and the data were analyzed using Nanoanalyzer provided by the supplier.

[0032] FIGS.7A-7F are a series of graphs depicting mass spectrometry analysis of intact and reduced masses of the representative CD180 targeted bispecific fusion using Xevo TQ-XS Triple Quadrupole Mass Spectrometer by Waters. FIG.7A Intact mass EIP1042 determined after PNGAse F deglycosylation in non-reduced condition and chromatographic separation using reverse phase C18 column, and FIG. 7B reduced mass analysis of EIP1042 heavy chains and light chains determined after Rapid PNGAse F deglycosylation in reduced condition and chromatographic separation using reverse phase C18 column. FIG. 7C Intact mass EIP1044 determined after PNGAse F deglycosylation in non-reduced condition and chromatographic separation using reverse phase C18 column, and FIG. 7D reduced mass analysis of EIP1044 heavy chains and light chains determined after Rapid PNGAse F deglycosylation in reduced condition and chromatographic separation using reverse phase C18 column. FIG. 7E Intact mass EIP1056 determined after PNGAse F deglycosylation in non-reduced condition and chromatographic separation using reverse phase C18 column, and FIG. 7F reduced mass analysis of EIP1056 heavy chains and light chains determined after Rapid PNGAse F deglycosylation in reduced condition and chromatographic separation using reverse phase C18 column.

[0033] FIGS. 8A-8B are a series of graphs obtained by ELISA depicting binding of light chain pairing bispecific fusion antibodies to recombinant CD180-MD1. FIG. 8A shows binding to human CD180-MD1. FIG.8B shows binding to cyno CD180-MD1.

[0034] FIGS.9A-9B are a series of graphs obtained by ELISA depicting binding of MHR73- 11, humanized bispecific and bispecific fusion antibodies to recombinant CD180-MD1. FIG. 9A shows binding to human CD180-MD1. FIG.9B shows binding to cyno CD180-MD1.

[0035] FIG. 10 is a graph obtained by ELISA depicting binding of the G28.8 antibody and the MHR73-11 antibody to recombinant human CD180-MD1.

[0036] FIGS. 11A-11C are a series of graphs depicting the CD180-targeting biologics binding to a variety of B cell tumor cell lines. FIG.11A shows JeKo-1, FIG.11B shows Raji, and FIG.11C shows Ramos. EIP0051 is CD3xG28.8, EIP0133 is CD3xMHR73-11, EIP0546Attorney Docket No. EVIM-006 / 001WO 339103-2023 is CD3xB12 (non-targeting control), EIP0553 is CD3xhumanized MHR73-11, EIP0554 is CD3xhumanized MHR73-11-CD58.

[0037] FIGS. 12A-12C are a series of line graphs depicting the CD180-targeting biologics mediated cytotoxicity in activated T cells-tumor cell co-culture assays. FIG. 12A shows JeKo-1, FIG. 12B shows Raji, and FIG. 12C shows Ramos. EIP0051 is CD3xG28.8, EIP0133 is CD3xCD180 (MHR73-11), and EIP0209 is non-targeting control. The EC50 of cytotoxicity of each biologics and the EC50 ratio are summarized in the EC50 table.

[0038] FIGS. 13A-13C are a series of graphs depicting CD180 expression and biologic binding on cell lines. FIG.13A shows CD180 levels on the various cell lines (HT, SU-DHL- 10, MV-4-11, and JeKo-1) along with isotype control. FIGS. 13B shows biologics binding to HT and FIG.13C shows biologic binding to MV-4-11.

[0039] FIGS. 14A-14G are a series of graphs depicting functional evaluations of bispecific fusion antibodies (EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) compared to control (EIP0614, non-targeting control) in a tumor cell and PBMC co-culture assay. CellTrace Violet labeled HT tumor cells were treated with PBMC at a 15:1 E:T ratio with a serial dilution of bispecific fusion antibody for 3 days. Cell counts or cell % was calculated by the cell count measured by flow cytometry. FIG.14A shows bispecific fusion antibody mediated cytolysis of tumor cells. FIG. 14B shows the B cell depletion. FIG. 14C shows monocyte. FIG. 14D shows CD4 T cell expansion. FIG. 14E shows CD8 T cell expansion. FIG. 14F shows IFN-Ȗ release. FIG.14G shows IL-2 release.

[0040] FIGS. 15A-15G are a series of graphs depicting functional evaluations of bispecific fusion antibodies (EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) compared to control (EIP0614, non-targeting control) in a tumor cell and PBMC co-culture assay. GFP- expressing-MV-4-11 tumor cells were treated with PBMC at a 15:1 E:T ratio with a serial dilution of bispecific fusion antibody for 3 days. Cell counts or cell % was calculated by the cell count measured by flow cytometry. FIG.15A shows bispecific fusion antibody mediated cytolysis of tumor cells. FIG. 15B shows the B cell depletion. FIG. 15C shows monocyte. FIG. 15D shows CD4 T cell expansion. FIG. 15E shows CD8 T cell expansion. FIG. 15F shows IFN-Ȗ release. FIG.15G shows IL-2 release.Attorney Docket No. EVIM-006 / 001WO 339103-2023 DETAILED DESCRIPTION

[0041] T cell retargeting (or T cell redirecting) bispecific antibodies are a novel class of therapeutics, capable of recruiting T cells to tumor cells and inducing tumor-specific (but MHC-independent) activation of T cell effector activities. The present disclosure is directed toward, T cell retargeting bispecific antibodies containing an antigen binding domain that targets the CD3 portion of the T cell receptor protein complex for T cell recruitment, and an antigen binding domain that targets T cells to a CD180 antigen. This targeting design promotes the recruitment of T cell and positions it in close contact with a CD180-expressing cell, resulting in the formation of an immunological synapse, local T cell activation and the subsequent destruction of the target cell, such as but not limited to a cancer cell, by perforin and granzyme released from T cell cytotoxic granules into the target cell.

[0042] As the CD3 binding affinity of the T-cell retargeting bispecific antibodies is crucial for recruitment of T cells, the present invention also relates to the generation of a panel of antibodies that display different binding affinities. The affinity of the CD3 arm of a bispecific antibody can significantly modify the functional activity of the bispecific antibody. Thus, it is desirable and advantageous to have bispecific antibodies with varied affinities.

[0043] Additionally, bispecific antibodies disclosed herein may have a cytokine or costimulatory molecule fusion peptide that acts as antagonist to inhibit or block deleterious interactions or as an agonist to mimic or enhance physiological responses. Physiological responses include but are not limited to T-cell activation, T-cell proliferation, T-cell persistence and prevention of T-cell exhaustion. These properties are advantageous over conventional CD3-bispecific antibodies or tumor targeted co-stimulatory receptor agonists which do not optimally activate T-cells and induce (or promote) T-cell dysfunction. In accordance, cytokine and / or costimulatory fusion peptides are advantageous for enhancing the therapeutic potential of bispecific antibodies. In some embodiments, the costimulatory molecule of the anti- CD180 / anti-CD3 bispecific antibodies of the present disclosure is CD58, or a fragment thereof.

[0044] Bispecific or multispecific T cell retargeting agents of the disclosure share the drug- like properties of human monoclonal antibodies. Further, T-cell retargeting bispecific and multispecific antibodies are advantageous over other existing therapies (e.g. CAR-T therapies) because it provides an off-the-shelf product with a high safety profile (e.g.Attorney Docket No. EVIM-006 / 001WO 339103-2023 mitigation of cytokine release syndrome and reduced levels of tonic signaling leading to T- cell dysfunction) and the possibility of dose titration and escalation.

[0045] ANTIBODY COMPOSITIONS AND STRUCTURES

[0046] The present disclosure provides an antibody comprising the following domain structure: a) a first heavy chain polypeptide (H1) comprising a variable region (VH1), and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and b) a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2). A schematic diagram of the antibody structure of the disclosure is shown in FIGS.4A-4E.

[0047] As used herein, the term “antibody” refers to an immunoglobulin (Ig) molecule and immunologically active portions of an immunoglobulin molecule, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” “or directed against” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 10-6). Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, and chimeric antibodies. The antibody may be from recombinant sources and / or produced in transgenic animals.

[0048] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.

[0049] In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, IgG4 and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. Accordingly,Attorney Docket No. EVIM-006 / 001WO 339103-2023 in one embodiment, the antibody disclosed herein is an IgG antibody.

[0050] Antibodies may be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen, which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28).

[0051] The term “antibody fragment” as used herein is intended to include without limitation, Fv, Fab, Fab’, F(ab’)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, multispecific antibody fragments and Domain Antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab’)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab’)2 fragment can be treated to reduce disulfide bridges to produce Fab’ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab’ and F(ab’)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques.

[0052] Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the disclosure (see e.g., U.S. Patent No.4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse, et al., 1989 Science 246:1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof.

[0053] As used herein, the term “epitope” refers to the site on an antigen that is recognized by the antibodies and fragments disclosed herein. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is < 1 micromolar; e.g., < 100 nM, preferably < 10 nM and more preferably < 1 nM.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0054] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. The present disclosure provides a bispecific antibody having a first antigen binding region that binds to a first antigen (e.g. CD3) and a second antigen binding region that binds to a second antigen (e.g. disease associated antigen).

[0055] Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol.147:60 (1991).

[0056] ANTIBODY VARIANTS

[0057] In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the heavy chain heterodimerization, light chain heterodimerization, binding affinity, and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., light chain heterodimerization, heavy chain heterodimerization, antigen binding).

[0058] Amino acids may be grouped according to common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic (negatively charged): Asp, Glu; (4) basic (positively charged): His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0059] Functional variants of the antibody or antigen-binding fragments described herein are also encompassed by the present disclosure. The term “functional variant” as used herein includes modifications or chemical equivalents of the amino acid and nucleic acid sequences disclosed herein that perform substantially the same function as the polypeptides or nucleic acid molecules disclosed herein in substantially the same way. For example, functional variants of polypeptides disclosed herein include, without limitation, conservative amino acid substitutions.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0060] A “conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue that change an amino acid to a different amino acid with similar biochemical properties (e.g. charge, hydrophobicity and size). Variants of polypeptides also include additions and deletions to the polypeptide sequences disclosed herein. In addition, variant nucleotide sequences include analogs and derivatives thereof. A variant of the binding proteins disclosed herein include proteins that bind to the same antigen or epitope as the binding proteins.

[0061] In some embodiments, the charged amino acid residue is a naturally occurring amino acid or a non-naturally occurring amino acid. In some embodiments, the naturally occurring charged amino acid residue is an arginine, a lysine, a histidine, a glutamic acid or an aspartic acid.

[0062] Light Chain and Heavy Chain Substitution Variants

[0063] To generate a substantially homogeneous population of bispecific antibodies with the correct pairing of heavy chain and light chains (i.e. cognate pairing or heterodimerization of a light chain with the heavy chain necessary to form the variable domain or antigen binding domain of the original antibody), the first heavy chain polypeptide (H1) has a strong preference for binding with the first light chain polypeptide (L1) relative to the second light chain polypeptide (L2); and the second heavy chain polypeptide (H2) has a strong preference for binding with the second light chain polypeptide (L2) relative to first light chain polypeptide (L1). In addition, the first heavy chain polypeptide (H1) and the second heavy chain polypeptide (H2) have a stronger preference for heterodimerization than homodimerization (i.e. heavy chain heterodimerization).

[0064] Antibody variants having one or more amino acid substitutions are provided herein. Exemplary substitutional mutagenesis sites include the charged substitution pairs shown in Tables 1-6.

[0065] Table 1. Kappa Light Chain and Heavy Chain – Constant Domain Mutations PairsAttorney Docket No. EVIM-006 / 001WO 339103-2023All position information is reported using the EU numbering scheme Wild type (WT) indicates the natural amino acid at the indicated position Charge pairs with negative and positive charge residues could be reversed between heavy and light chains, where D or E (negative charge) are replaced by K or R (positive charge) and cognate chain K or R (positive charge) are replaced by D or E (negative charge).

[0066] Table 2. Kappa Light Chain and Heavy Chain – Variable Domain Mutations PairsAll position information is reported using the Kabat numbering scheme Wild type (WT) indicates the natural amino acid at the indicated position Charged pairs with negative and positive charged residues could be reversed between heavy and light chains, where D or E (negative charged) are replaced by K or R (positive charged) and cognate chain K or R (positive charged) are replaced by D or E (negative charged).

[0067] Table 3. Lambda Light Chain and Heavy Chain – Constant Domain Mutations PairsAll position information is reported using the EU numbering scheme Charge pairs with negative and positive charge residues could be reversed between heavy and light chains, where D or E (negative charge) are replaced by K or R (positive charge) and cognate chain K or R (positive charge) are replaced by D or E (negative charge).

[0068] Table 4. Lambda Light Chain and Heavy Chain – Variable Domain Mutations PairsAttorney Docket No. EVIM-006 / 001WO 339103-2023All position information is reported using the Kabat numbering scheme Charge pairs with negative and positive charge residues could be reversed between heavy and light chains, where D or E (negative charge) are replaced by K or R (positive charge) and cognate chain K or R (positive charge) are replaced by D or E (negative charge).

[0069] Table 5. Kappa Constant Chain Cysteine Mutation PairsAll position information is reported using the EU numbering schemeAll position information is reported using the EU numbering scheme

[0071] In certain embodiments, anti-CD3 / anti-CD180 bispecific antibodies of the present disclosure comprise antibody variants comprise substitutions in the variable heavy, variable light, constant heavy or constant light chain domains of the anti-CD3 or the anti-CD180 arms or both, as discussed in PCT Application No. PCT / US2023 / 064728 and PCT Publication No. WO2019 / 104075 A1, which are incorporated by reference herein in their entirety.

[0072] In certain embodiments, the antibody variant comprises the “light chain pairing mutation set D” comprising the following substitutions: a) the heavy chain and light chain of the anti-CD3 arm comprise the following: i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CH1H1is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; iii) the amino acid at position 173 (EU numbering) of the CH1H1is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and b) the heavy chain andAttorney Docket No. EVIM-006 / 001WO 339103-2023 light chain of the anti-CD180 arm comprise the following: i)the amino acid at position 39 (Kabat numbering) of the VH2 is a D and the amino acid at position 38 (Kabat numbering) of the VL2 is a K; and ii) the amino acid at position 147 (EU numbering) of the CH2H1 is a D and the amino acid at position 180 (EU numbering) of the CL2 is a R.

[0073] In certain embodiments, the antibody variant comprises the “light chain pairing mutation set D” comprising the following substitutions: a) the heavy chain and light chain of the anti-CD180 arm comprise the following: i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CH1H1is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; iii) the amino acid at position 173 (EU numbering) of the CH1H1is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and b) the heavy chain and light chain of the anti-CD3 arm comprise the following: i)the amino acid at position 39 (Kabat numbering) of the VH2 is a D and the amino acid at position 38 (Kabat numbering) of the VL2 is a K; and ii) the amino acid at position 147 (EU numbering) of the CH2H1 is a D and the amino acid at position 180 (EU numbering) of the CL2 is a R.

[0074] It can be desirable to modify an antibody disclosed herein with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating diseases and disorders. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement- mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922. (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0075] Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6.737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0076] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0077] In some embodiments, alterations are made in the Fc region that result in altered (i.e., either improved or diminished) C1q binding and / or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551 , WO 99 / 51642, and Idusogie et al. J. Immunol.164: 4178-4184 (2000).

[0078] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No.7,371 ,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821 ; and WO 94 / 29351 concerning other examples of Fc region variants.

[0079] In some embodiments, antibodies may comprise a substitution mutation in the Fc region that reduces effector function. In some embodiments, the substitution mutation is an aglycosylation site mutation. In some embodiments, the aglycosylation site mutation is at amino acid residue 297 and amino acid substitutions at residues 234, 235, 265 and 331 (EU numbering) to disrupt the Fc receptor binding interface. In some embodiments, the aglycosylation site mutation reduces effector function of the antibody.

[0080] In some embodiments, i) the H1H and / or the H2H has an A at positions 234 and 235 (EU numbering); or ii) the H1H and / or the H2H has an A at positions 234, 235 and 237 (EU numbering) iii) the H1H and / or the H2H has an A at positions 234 and 235 and G at position 329 (EU numbering). In some embodiments, i) the CH1H3and / or the CH2H3has an A at position 297 (EU numbering) ii) the CH1H3and / or the CH2H3has a G at position 297 (EU numbering); or iii) the CH1H3 and / or the CH2H3 has a S at position 297 (EU numbering). In some embodiments, the CH1H3 and / or the CH2H3 has an S at position 331 (EU numbering).Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0081] The use of knobs into holes as a method of producing multispecific antibodies is well known in the art. See U.S. Pat. No.5,731,168 granted 24 Mar. 1998 assigned to Genentech, PCT Pub. No. WO2009089004 published 16 Jul. 2009 and assigned to Amgen, and US Pat. Pub. No. 20090182127 published 16 Jul. 2009 and assigned to Novo Nordisk A / S. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9.

[0082] A “protuberance” refers to at least one amino acid side chain which projects from the interface of a first polypeptide and is therefore positionable in a compensatory cavity in the adjacent interface (i.e. the interface of a second polypeptide) so as to stabilize the heteromultimeric antibody, and thereby favor heteromultimeric antibody formation over homomultimeric antibody formation, for example. The protuberance may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the first polypeptide is altered to encode the protuberance. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the first polypeptide is replaced with nucleic acid encoding at least one “import” amino acid residue which has a larger side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the first polypeptide.

[0083] The preferred import residues for the formation of a protuberance are generally naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). Most preferred are tryptophan and tyrosine. In one embodiment, the original residue for the formation of the protuberance has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine. Exemplary amino acid substitutions in the CH1H3or CH2H3domain for forming the protuberance include without limitation the T366W substitution.

[0084] A “cavity” refers to at least one amino acid side chain which is recessed from the interface of a second polypeptide and therefore accommodates a corresponding protuberance on the adjacent interface of a first polypeptide. The cavity may exist in the original interface or may be introduced synthetically (e.g. by altering nucleic acid encoding the interface). Normally, nucleic acid encoding the interface of the second polypeptide is altered to encodeAttorney Docket No. EVIM-006 / 001WO 339103-2023 the cavity. To achieve this, the nucleic acid encoding at least one “original” amino acid residue in the interface of the second polypeptide is replaced with DNA encoding at least one “import” amino acid residue which has a smaller side chain volume than the original amino acid residue. It will be appreciated that there can be more than one original and corresponding import residue. The upper limit for the number of original residues which are replaced is the total number of residues in the interface of the second polypeptide. The side chain volumes of the various amino residues are shown in Table 1 above. The preferred import residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V). Most preferred are serine, alanine or threonine. In one embodiment, the original residue for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan. Exemplary amino acid substitutions in the CH1H3or CH2H3domain for generating the cavity include without limitation the T366S, L368A, Y407A, Y407T and Y407V substitutions. In certain embodiments, the knob half-antibody comprises T366W substitution, and the hole half-antibody comprises the T366S / L368A / Y407V substitutions.

[0085] In certain embodiments, the antibody variant comprises the following substitutions: the CH1H3 has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3 has a C at position 354 and a W at position 366 (EU numbering).

[0086] In certain embodiments, the antibody variant comprises the following substitutions: the CH2H3 has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3 has a C at position 354 and a W at position 366 (EU numbering).

[0087] In certain embodiments, the antibody variant comprises the following substitutions: the CH1H3has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3has a C at position 349 and a W at position 366 (EU numbering).

[0088] In certain embodiments, the antibody variant comprises the following substitutions: the CH2H3 has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3 has a C at position 349 and a W at position 366 (EU numbering).Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0089] CLUSTER OF DIFFERENTIATION 3 (CD3)

[0090] The present disclosure provides a bispecific antibody comprising a first antigen binding domain that binds to a CD3 expressed on a T-cell, and a second antigen binding domain that binds to a CD180 antigen on the surface of a cancer cell. Antibodies of the invention are useful, for example, for treating or delaying the progression of a cell proliferative disorder (e.g., cancer expressing CD180).

[0091] The term “cluster of differentiation 3” or “CD3,” as used herein, refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans, cynomolgus monkey) and rodents (e.g., mice and rats), unless otherwise indicated, including, for example, CD3İ, CD3Ȗ, CD3Į, and CD3ȕ chains. CD3 is a cell surface complex expressed on T cells in association with the T cell receptor. The CD3 complex is required for the activation of CD8+ and CD4+ T lymphocytes. It is formed of three different but highly related chains: one CD3 gamma chain, one CD3 delta chain, and two CD3 epsilon chains, which associate with each other to form a CD3 epsilon / gamma heterodimer, and a CD3 epsilon / delta heterodimer. The two CD3 heterodimers, together with the T cell receptor (TCR) and the signal-transducing zeta chain homodimer form the T cell receptor complex.

[0092] The term encompasses “full-length” unprocessed CD3 (e.g., unprocessed or unmodified CD3İ or CD3Ȗ), as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, including, for example, splice variants or allelic variants. CD3 includes, for example, human CD3İ protein (NCBI RefSeq No. NP_000724), which is 207 amino acids in length.

[0093] In some embodiments, the invention provides isolated antibodies that bind to CD3. In some embodiments, the invention provides antibodies that bind to CD3İ. In some instances, the anti-CD3İ antibody binds to a human CD3İ polypeptide or a cynomolgus monkey (cyno) CD3İ polypeptide. In some instances, the human CD3 polypeptide or the cyno CD3 polypeptide is a human CD3İ polypeptide MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSE ILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYL YLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAG AGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 419) or a cyno CD3İ polypeptideAttorney Docket No. EVIM-006 / 001WO 339103-2023 MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSE AQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVC ENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQR GQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI (SEQ ID NO: 420), respectively. In some instances, the anti-CD3 antibody binds to an epitope within a fragment of CD3İ (e.g., human CD3İ) consisting of amino acid residues 1-26 or amino acid residues 1-27 of human CD3İ (SEQ ID NO: 419).

[0094] A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

[0095] In some embodiments, alanine scanning mutagenesis was performed on the “SP34” anti-CD3İ antibody to produce affinity modulated anti-CD3İ antibodies of the invention.

[0096] In some embodiments, the first antigen binding region that binds to CD3 comprises any one of the VH and VL sequences listed in Table 7. In Table 7, the underlined sequences are CDR sequence according to Kabat and the bolded sequences are CDR sequences according to Chothia.

[0097] In some embodiments, the first antigen binding region comprises a VH region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, or 21.

[0098] In some embodiments, the first antigen binding region comprises a VL region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%,Attorney Docket No. EVIM-006 / 001WO 339103-2023 at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 22, 23, 24, 25, 26, 27, and 28.

[0099] In some embodiments, an anti-CD3 antibody of the disclosure comprises: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VHCDR1), a VH complementarity determining region 2 (VHCDR2) and a VH complementarity determining region 3 (VHCDR3); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLCDR1), a VL complementarity determining region 2 (VLCDR2) and a VL complementarity determining region 3 (VLCDR3). Tables 8 and 9 provide exemplary of CDR sequences of the anti-CD3 antibodies provided herein.

[0100] Table 7. Anti-CD3 Variable Heavy Chain and Variable Light Chain DomainsAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0101] Table 8. Anti-CD3 Heavy Chain CDRsAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0102] Table 9. Anti-CD3 Light Chain CDRsAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0103] Provided herein are bispecific antibodies comprising a first antigen binding domain that binds a first antigen (e.g. CD3) and a second antigen binding domain that binds to a second antigen (e.g. CD180). In some embodiments the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1), and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0104] In some embodiments, the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3) comprising any one of the VH1 and VL1 sequences listed in Table 7. In Table 7, the underlined sequences are CDR sequence according to Kabat and the bolded sequences are CDR sequences according to Chothia.

[0105] In some embodiments, the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3İ) comprising: a) a heavy chain variable region (VH1) comprising a VH complementarity determining region 1 (VH1CDR1), a VH complementarity determining region 2 (VH1CDR2) and a VH complementarity determiningAttorney Docket No. EVIM-006 / 001WO 339103-2023 region 3 (VH1CDR3); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VL1CDR1), a VL complementarity determining region 2 (VL1CDR2) and a VL complementarity determining region 3 (VL1CDR3). Tables 8 and 9 provide exemplary of CDR sequences of the anti-CD3 antibodies provided herein.

[0106] In some embodiments, the bispecific antibody comprises any one of the anti-CD3 antibodies of the disclosure. Exemplary anti-CD3 antibodies of the invention include CD3- 01, CD3-A1, CD3-A2, CD3-A3, CD3-A4, CD3-A5, CD3-A6, CD3-A7, CD3-A8, CD3-A9, CD3-A10, CD3-A11, CD3-A12 and CD3-A13. In some embodiments, the bispecific antibody comprises a “CD3-01” anti-CD3 antibody. In some embodiments, the bispecific antibody comprises a “CD3-A6” anti-CD3 antibody.

[0107] In some embodiments, the binding affinity (KD) of the first antigen binding region of the bispecific antibody that binds to CD3 (e.g., human CD3İ (e.g., (SEQ ID NO: 419) or cynomolgus CD3İ (e.g., (SEQ ID NO: 422)) is about 0.001 nM to about 5000 nM. In some embodiments, the binding affinity to CD3 is about 0.001 nM to about 0.01 nM, about 0.01 to about 0.1 nM or about 0.1 to about 1nM. In some embodiments, the binding affinity is about 1 nM to about 1000 nM, about 10 nM to about 1000 nM or about 100nM to about 1000 nM.

[0108] In some embodiments, the binding affinity is about 50 nM to about 5000 nM, about 50 nM to about 4000 nM, about 50 nM to about 3000 nM, about 50 nM to about 2000 nM, about 50 nM to about 1000 nM, about 50 nM to about 900 nM, about 50 nM to about 800 nM, about 50 nM to about 700 nM, about 50 nM to about 600 nM, about 50 nM to about 500 nM, about 50 nM to about 400 nM, about 50 nM to about 300 nM, about 50 nM to about 200 nM, about 50 nM to about 100 nM or about 50 nM to about 500 nM. In some embodiments, the binding affinity is about 50 nM to about 200 nM.

[0109] In some embodiments, the binding affinity is about 10 nM to about 20 nM, about 20 nM to about 30 nM, about 30 nM to about 40 nM, about 50 nM to about 60 nM, about 60 nM to about 70 nM, about 70 nM to about 80 nM, about 80 nM to about 90 nM, about 90 nM to about 100 nM, about 100 nM to about 110 nM, about 110 nM to about 120 nM, about 120 nM to about 130 nM, about 130 nM to about 140 nM, about 150 nM to about 160 nM, about 160 nM to about 170 nM, about 170 nM to about 180 nM, about 180 nM to about 190 nM, or about 190 nM to about 200 nM.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0110] In some embodiments, the binding affinity is less than about 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 275 nM, 250 nM, 225 nM, 200 nM, 175 nM, 150 nM, 125 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 9.5 nM, 9nM, 8.5 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

[0111] CD180 ANTIGEN

[0112] The present disclosure provides a bispecific antibody comprising a first antigen binding domain that binds to a CD3 expressed on a T-cell, and a second antigen binding domain that binds to a CD180 antigen on the surface of a cancer cell. Antibodies of the invention are useful, for example, for treating or delaying the progression of a cell proliferative disorder (e.g., cancer expressing CD180).

[0113] CD180 is a type 1 transmembrane toll-like receptor (TLR) with the highest sequence similarity to TLR4 and was initially discovered as a mouse B lymphocyte activation receptor that protects B cells from irradiation and dexamethasone-mediated cell death (Miyake et al., 1995, J. Immunol. 154: 3333–3340; Divanovic et al., 2005, Nat. Immunol. 6: 571–578). CD180 and MD-1, an interacting protein required for CD180 surface expression and function, are found on the surface of B cells and monocytes, but not T lymphocytes. It is also found on other myeloid cells with similar expression in humans and mice (Ohto et al., 2011, J. Mol. Biol. 413: 815–825; Mayeur-Rousse et al., 2016, Cytometry B. Clin. Cytom. 90: 462–466). CD180 / MD-1 is also found on hematological malignancies such as B cell lymphoma (Mayeur-Rousse et al., 2016, Cytometry B. Clin. Cytom. 90: 462–466; Miguet et al., 2013, Leukemia 27: 1748–1750; Mansour et al., 2020, J. Hematopathol. 13: 205–211; Fidyt et al., 2022, Blood 140: 10273–10274; Favre et al., 2018, Int. J. Lab. Hematol.40: e59-62), chronic lymphocytic leukemia (CLL)(Porakishvili et al., 2005, Br. J. Haematol. 131: 313–319; Edwards et al., 2021, Br. J. Haematol.195), and acute myeloid leukemia (AML) (Kramer et al., 2022, Blood 140: 1533–1548; Saito et al., 2010, Sci. Transl. Med. 2) implicating CD180 / MD-1 as a novel target for cancer immunotherapy with differentiated opportunities in relapse and / or refractory B cell malignancies as an alternative B cell lineage antigen, where CD19 and CD20 loss emerge from CD19 / CD20 targeted treatments, and in myeloid malignancies of the monocytic lineage (Marshalek et al., 2022, JCO 40: e19537–e19537;Attorney Docket No. EVIM-006 / 001WO 339103-2023 Duell, et al., 2024, Blood 143: 685-696).

[0114] The terms “CD180” and “CD180 antigen” are used interchangeably herein, and include any variants, isoforms and species homologs of human CD180 which are naturally expressed by cells or are expressed on cells transfected with the CD180 gene. Binding of an antibody of the invention to the CD180 antigen mediate the killing of cells expressing CD180 (e.g., a tumor cell) by inactivating CD180. The killing of the cells expressing CD180 may occur by one or more of the following mechanisms: Cell death / apoptosis induction, ADCC and CDC.

[0115] In some embodiments, the invention provides isolated antibodies that bind to CD180. In some instances, the anti-CD3180 antibody binds to a human CD180 polypeptide or a cynomolgus monkey (cyno) CD180 polypeptide. In some instances, the CD180 polypeptide is a human CD180 polypeptide MAFDVSCFFWVVLFSAGCKVITSWDQMCIEKEANKTYNCENLGLSEIPDTLPNTTE FLEFSFNFLPTIHNRTFSRLMNLTFLDLTRCQINWIHEDTFQSHHQLSTLVLTGNPLIF MAETSLNGPKSLKHLFLIQTGISNLEFIPVHNLENLESLYLGSNHISSIKFPKDFPARN LKVLDFQNNAIHYISREDMRSLEQAINLSLNFNGNNVKGIELGAFDSTIFQSLNFGGT PNLSVIFNGLQNSTTQSLWLGTFEDIDDEDISSAMLKGLCEMSVESLNLQEHRFSDIS STTFQCFTQLQELDLTATHLKGLPSGMKGLNLLKKLVLSVNHFDQLCQISAANFPSL THLYIRGNVKKLHLGVGCLEKLGNLQTLDLSHNDIEASDCCSLQLKNLSHLQTLNL SHNEPLGLQSQAFKECPQLELLDLAFTRLHINAPQSPFQNLHFLQVLNLTYCFLDTS NQHLLAGLPVLRHLNLKGNHFQDGTITKTNLLQTVGSLEVLILSSCGLLSIDQQAFH SLGKMSHVDLSHNSLTCDSIDSLSHLKGIYLNLAANSINIISPRLLPILSQQSTINLSHN PLDCTCSNIHFLTWYKENLHKLEGSEETTCANPPSLRGVKLSDVKLSCGITAIGIFFLI VFLLLLAILLFFAVKYLLRWKYQHI (SEQ ID NO: 417) or a cyno CD180 polypeptide MAFDVSCFFWVVLFSAGCKVITSLDQMCIEKEANKTYNCENLGLSEIPDTLPNTTEF LEFSFNFLPTIHNGTFSRLMNLTFLDLTRCQINWIHEDTFQSHHQLNTLVLTGNPLIF MAETSLNGPKSLKHLFLIQTGISNLEFIPVHNLENLESLYLGSNHISSIKFPKDFPARN LKVLDFQNNAIHYLSREDMRSLEQATNLSLNFNGNDVKGIELGAFGSIVFQSLNFG GTLNLSVIFNGLQNSTTQSLWLGTFEDIDDEDISSAMLKGLCEMSVESLNLQKHHFS DFSSTTFQCFTQIQELDLTATHLAGLPSGIKGLNLLKKLVLSVNHFDQLCQISAANFP SLTHLYVRGNVKKLHLGVGCLEKLGNLQTLDLSHNDIEASDCCNLQLRNLSHLQTLAttorney Docket No. EVIM-006 / 001WO 339103-2023 NLSHNELLGLQSQAFKECPRLELLDLAFTRLHINAPQSPFQNLHFLQVLNLTYCFLD TSNQHLLAGLPDLRHLNLKGNHFQDGTIMKTNLLQTVGSLEILILSSCGLLSIDQQA FHGLGKMSHVDLSHNSLTCDSIASVSHFKGIYLNLAANSINIIPPHLLPILSQQSTINFS HNPLDCTCSNIHFLTWYKENLHKLEGSEETTCANPPSLRGVKLSDVKLSCGITAIGV FFLTVFLLLFTILLFFAVKNLLRCKYRHI (SEQ ID NO: 418), respectively.

[0116] The term “anti- CD180 antibody” according to the invention is an antibody that binds specifically to CD180 antigen. Depending on binding properties and biological activities of anti-CD180 antibodies to the CD180 antigen, two types of anti-CD180 antibodies (type I and type II anti-CD180 antibodies) can be distinguished according to Cragg, M. S., et al., Blood 103 (2004) 2738-2743; and Cragg, M. S., et al., Blood 101 (2003) 1045-1052.

[0117] In some instances, CD180 may be expressed in low copy number on the target cell (e.g. tumor cell). For example, in some instances, CD180 is expressed or present at less than 35,000 copies per target cell. In some embodiments, the low copy number cell surface CD180 is present between 100 and 35,000 copies per target cell; between 100 and 30,000 copies per target cell; between 100 and 25,000 copies per target cell; between 100 and 20,000 copies per target cell; between 100 and 15,000 copies per target cell; between 100 and 10,000 copies per target cell; between 100 and 5,000 copies per target cell; between 100 and 2,000 copies per target cell; between 100 and 1,000 copies per target cell; or between 100 and 500 copies per target cell. Copy number of the cell surface CD180 can be determined, for example, using a standard Scratchcard plot.

[0118] This disclosure provides a bispecific antibody comprising a first antigen binding region that binds CD3 and a second binding region that binds CD180.

[0119] In some embodiments the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1), and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0120] For example, the second binding region that binds to CD180 can be derived from the binding regions of an anti-CD180 antibody. Exemplary anti-CD180 antibodies include but are not limited to MHR73-11 and G28-8 monoclonal antibody (BD Biosciences, Thermo Scientific, Sigma Aldrich). The anti-CD180 antibody G28.8 (also referred to as G28-8) is disclosed in U.S. Pat. No. 9,260,529, the contents of which is hereby incorporated by reference in its entirety.

[0121] In some embodiments, a second antigen binding region that binds CD180 comprises any one of the VH and VL sequences listed in Table 10. In Table 10, the underlined sequences are CDR sequences according to Kabat and the bolded sequences are CDR sequences according to Chothia.

[0122] In some embodiments, the second antigen binding region that binds CD180 comprises a VH region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, or 205.

[0123] In some embodiments, the second antigen binding region that binds CD180 comprises a comprises a VL region that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence set forth in any one of SEQ ID NO: 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, or 204.

[0124] In some embodiments, the second binding region that binds to CD180 comprises: a) a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VHCDR1), a VH complementarity determining region 2 (VHCDR2) and a VH complementarity determining region 3 (VHCDR3); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VLCDR1), a VL complementarity determining region 2 (VLCDR2) and a VL complementarity determining region 3 (VLCDR3). Tables 11 and 12 provide exemplary of CDR sequences of the anti-CD180 antibodies.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0125] Table 10. CD180 Variable Heavy Chain and Variable Light Chain DomainsAttorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0126] Table 11. Anti-CD180 Heavy Chain CDRsAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0127] Table 12. Anti-CD180 Light Chain CDRsAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0128] In some embodiments, the second antigen binding region comprises a) a heavy chain variable region (VH) comprising a i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 210, ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 212, iii) and a VH complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO: 214.

[0129] In some embodiments, the second antigen binding region comprises a) a heavy chain variable region (VH) comprising a i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 210, ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 216, iii) and a VH complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO: 214.

[0130] In some embodiments, the second antigen binding region comprises a) a heavy chain variable region (VH) comprising a i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 218, ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 220, iii) and a VH complementarity determining region 3 (VHCDR3) comprising the amino acidAttorney Docket No. EVIM-006 / 001WO 339103-2023 sequence of SEQ ID NO: 221.

[0131] In some embodiments, the second antigen binding region comprises i) a light chain variable region (VL) comprising a i) a VL complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 222, ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO: 223, iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 224.

[0132] In some embodiments, the second antigen binding region comprises i) a light chain variable region (VL) comprising a i) a VL complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 222, ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO: 225, iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 224.

[0133] In some embodiments, the second antigen binding region comprises i) a light chain variable region (VL) comprising a i) a VL complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 430, ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO: 431, iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 432.

[0134] In some embodiments, the second antigen binding region comprises i) a light chain variable region (VL) comprising a i) a VL complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 433, ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO: 434, iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 435.

[0135] In some embodiments, the second antigen binding region comprises a) a heavy chain variable region (VH) comprising a i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 210, ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 212, iii) a VH complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO: 214; and b) a light chain variable region (VL) comprising a i) a VLAttorney Docket No. EVIM-006 / 001WO 339103-2023 complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 222, ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO: 223, iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 224.

[0136] In some embodiments, the second antigen binding region comprises a) a heavy chain variable region (VH) comprising a i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 210, ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 216, iii) a VH complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO: 214; and b) a light chain variable region (VL) comprising a i) a VL complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 222, ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO: 225, iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 224.

[0137] In some embodiments, the second antigen binding region comprises a) a heavy chain variable region (VH) comprising a i) a VH complementarity determining region 1 (VHCDR1) comprising the amino acid sequence of SEQ ID NO: 210, ii) a VH complementarity determining region 2 (VHCDR2) comprising the amino acid sequence of SEQ ID NO: 216, iii) a VH complementarity determining region 3 (VHCDR3) comprising the amino acid sequence of SEQ ID NO: 214; and b) a light chain variable region (VL) comprising a i) a VL complementarity determining region 1 (VLCDR1) comprising the amino acid sequence of SEQ ID NO: 222, ii) a VL complementarity determining region 2 (VLCDR2) comprising the amino acid sequence of SEQ ID NO: 223, iii) a VL complementarity determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 224.

[0138] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 197 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 196.

[0139] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 199 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 198.

[0140] In some embodiments, the second antigen binding region comprises a VH regionAttorney Docket No. EVIM-006 / 001WO 339103-2023 comprising the amino acid sequence shown in SEQ ID NO: 201 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 200.

[0141] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 203 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 202.

[0142] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 205 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 204.

[0143] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 161 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 160.

[0144] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 165 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 164.

[0145] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 167 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 166.

[0146] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 169 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 168.

[0147] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 171 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 170.

[0148] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 173 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 172.

[0149] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 175 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 174.

[0150] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 177 and a VL region comprisingAttorney Docket No. EVIM-006 / 001WO 339103-2023 the amino acid sequence shown in SEQ ID NO: 176.

[0151] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 179 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 178.

[0152] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 181 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 180.

[0153] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 183 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 182.

[0154] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 185 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 184.

[0155] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 187 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 186.

[0156] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 189 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 188.

[0157] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 191 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 190.

[0158] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 193 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 192.

[0159] In some embodiments, the second antigen binding region comprises a VH region comprising the amino acid sequence shown in SEQ ID NO: 195 and a VL region comprising the amino acid sequence shown in SEQ ID NO: 194.

[0160] EXEMPLARY BISPECIFIC ANTIBODIES THAT BIND TO CD3 AND CD180

[0161] Provided herein are bispecific antibodies comprising a first antigen binding domain that binds a first antigen (e.g. CD3İ) and a second antigen binding domain that binds to aAttorney Docket No. EVIM-006 / 001WO 339103-2023 CD180. In some embodiments the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1), and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1), and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2).

[0162] In some embodiments, the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3İ) comprising: a) a heavy chain variable region (VH1) comprising a VH complementarity determining region 1 (VH1CDR1), a VH complementarity determining region 2 (VH1CDR2) and a VH complementarity determining region 3 (VH1CDR3); and b) a light chain variable region (VL) comprising a VL complementarity determining region 1 (VL1CDR1), a VL complementarity determining region 2 (VL1CDR2) and a VL complementarity determining region 3 (VL1CDR3); and a second antigen binding domain (e.g. binding to CD180) comprising: a) a heavy chain variable region (VH2) comprising a VH complementarity determining region 1 (VH2CDR1), a VH complementarity determining region 2 (VH2CDR2) and a VH2 complementarity determining region 3 (VH2CDR3); and b) a light chain variable region (VL2) comprising a VL complementarity determining region 1 (VL2CDR1), a VL complementarity determining region 2 (VL2CDR2) and a VL complementarity determining region 3 (VL2CDR3). Tables 8 and 9 provide exemplary of CDR sequences of the anti-CD3İ antibodies provided herein. Tables 11 and 12 provide exemplary of CDR sequences of the anti-CD180 antibodies provided herein.

[0163] In some embodiments, the bispecific antibody of the disclosure comprises a first antigen binding domain (e.g. binding to CD3İ) comprising any one of the VH1 and VL1 sequences listed in Table 7 and a second antigen binding domain (e.g. binding to CD180) comprising any one of the VH2 and VL2 sequences listed in Table 10.

[0164] In some embodiments, the bispecific antibody of the disclosure comprises a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and a second heavyAttorney Docket No. EVIM-006 / 001WO 339103-2023 chain polypeptide (H2) and a second light chain polypeptide (L2) comprising any one of the sequences listed in Table 13 and Table 16. The italicized sequences are the heavy chain variable regions and the light chain variable regions. The underlined sequences are CDRs according to Kabat and the bolded sequences are CDRs according to Chothia.

[0165] In some embodiments, the bispecific antibody of the disclosure provided herein comprises a H1 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16. In some embodiments, the H1 comprises a C-terminal lysine (K) residue.

[0166] In some embodiments, the bispecific antibody of the disclosure provided herein comprises a L1 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16.

[0167] In some embodiments, the bispecific antibody of the disclosure provided herein comprises a H2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16. In some embodiments, the H1 comprises a C-terminal lysine (K) residue.

[0168] In some embodiments, the bispecific antibody of the disclosure provided herein comprises a L2 that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% identical to the amino acid sequence of the sequences listed in Table 13 and Table 16.

[0169] In some embodiments, the H1 amino acid sequence is numbered in accordance with SEQ ID NO: 229. In some embodiments the L1 amino acid sequence is numbered in accordance with SEQ ID NO: 228. In some embodiments, the H2 amino acid sequence is numbered in accordance with SEQ ID NO: 227. In some embodiments, the L2 amino acid sequence is numbered in accordance with SEQ ID NO: 226.

[0170] Table 13. Exemplary Bispecific Antibodies that bind CD3İ and CD180Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0171] Exemplary CD3İ x CD180 bispecific antibodies of the invention include EIP1870, EIP1871, EIP1872, EIP1873, EIP1874, EIP0696, EIP0706, EIP0710, EIP0698, EIP0708, EIP0745 and EIP0751.

[0172] Bispecific antibodies EIP1870, EIP1871, EIP0872, EIP1873, EIP1874 and EIP0698, comprise a first antigen binding domain that binds CD3İ which comprising a VH1,Attorney Docket No. EVIM-006 / 001WO 339103-2023 comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 30; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 45.

[0173] Bispecific antibody EIP0696 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VL1CDR1having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3having the amino acid sequence of SEQ ID NO: 45.

[0174] Bispecific antibody EIP0706 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 44; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 45.

[0175] Bispecific antibody EIP0710 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3having the amino acid sequence of SEQ ID NO: 39; and a VL1, comprising a VL1CDR1having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3having the amino acid sequence of SEQ ID NO: 47.

[0176] Bispecific antibody EIP0708 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 35; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 38; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the aminoAttorney Docket No. EVIM-006 / 001WO 339103-2023 acid sequence of SEQ ID NO: 43; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 47.

[0177] Bispecific antibody EIP0745 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 36; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 40; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3having the amino acid sequence of SEQ ID NO: 47.

[0178] Bispecific antibody EIP0751 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3having the amino acid sequence of SEQ ID NO: 41; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 47.

[0179] Bispecific antibodies EIP1870, EIP1871, EIP1872, EIP1873, EIP1874, EIP0696, EIP0706, EIP0710, EIP0698, EIP0708, EIP0745 and EIP0751 comprise a second antigen binding domain that binds CD180 comprising a VH2, comprising VH2CDR1 having the amino acid sequence of SEQ ID NO: 210; a VH2CDR2 having the amino acid sequence of SEQ ID NO: 212; and a VH2CDR3 having the amino acid sequence of SEQ ID NO: 214; and a VL2, having VL2CDR1 having the amino acid sequence of SEQ ID NO: 222; a VL2CDR2 having the amino acid sequence of SEQ ID NO: 223; and a VL2CDR3having the amino acid sequence of SEQ ID NO: 224.

[0180] In some embodiments, the bispecific antibody EIP1870 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 197, and a VL2 comprising the amino acid sequence of SEQ ID NO: 196.

[0181] In some embodiments, the bispecific antibody EIP1870 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 229, a L1 comprising the amino acid sequence ofAttorney Docket No. EVIM-006 / 001WO 339103-2023 SEQ ID NO: 228, a H2 comprising the amino acid sequence of SEQ ID NO: 227, and a L2 comprising the amino acid sequence of SEQ ID NO: 226.

[0182] In some embodiments, the bispecific antibody EIP1871 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 199, and a VL2 comprising the amino acid sequence of SEQ ID NO: 198.

[0183] In some embodiments, the bispecific antibody EIP1871 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 233, a L1 comprising the amino acid sequence of SEQ ID NO: 232, a H2 comprising the amino acid sequence of SEQ ID NO: 231, and a L2 comprising the amino acid sequence of SEQ ID NO: 230.

[0184] In some embodiments, the bispecific antibody EIP1872 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 201, and a VL2 comprising the amino acid sequence of SEQ ID NO: 200.

[0185] In some embodiments, the bispecific antibody EIP1872 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 237, a L1 comprising the amino acid sequence of SEQ ID NO: 236, a H2 comprising the amino acid sequence of SEQ ID NO: 235, and a L2 comprising the amino acid sequence of SEQ ID NO: 234.

[0186] In some embodiments, the bispecific antibody EIP1873 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0187] In some embodiments, the bispecific antibody EIP1873 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 241, a L1 comprising the amino acid sequence of SEQ ID NO: 240, a H2 comprising the amino acid sequence of SEQ ID NO: 239, and a L2 comprising the amino acid sequence of SEQ ID NO: 238.

[0188] In some embodiments, the bispecific antibody EIP1874 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 205, and a VL2 comprising the amino acid sequence of SEQ ID NO: 204.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0189] In some embodiments, the bispecific antibody EIP1874 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 245, a L1 comprising the amino acid sequence of SEQ ID NO: 244, a H2 comprising the amino acid sequence of SEQ ID NO: 243, and a L2 comprising the amino acid sequence of SEQ ID NO: 242.

[0190] In some embodiments, the bispecific antibody EIP0696 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0191] In some embodiments, the bispecific antibody EIP0696 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 249, a L1 comprising the amino acid sequence of SEQ ID NO: 248, a H2 comprising the amino acid sequence of SEQ ID NO: 247, and a L2 comprising the amino acid sequence of SEQ ID NO: 246.

[0192] In some embodiments, the bispecific antibody EIP0706 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 27, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0193] In some embodiments, the bispecific antibody EIP0706 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 257, a L1 comprising the amino acid sequence of SEQ ID NO: 256, a H2 comprising the amino acid sequence of SEQ ID NO: 255, and a L2 comprising the amino acid sequence of SEQ ID NO: 254.

[0194] In some embodiments, the bispecific antibody EIP0710 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 16, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0195] In some embodiments, the bispecific antibody EIP0710 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 265, a L1 comprising the amino acid sequence of SEQ ID NO: 264, a H2 comprising the amino acid sequence of SEQ ID NO: 263, and a L2 comprising the amino acid sequence of SEQ ID NO: 262.

[0196] In some embodiments, the bispecific antibody EIP0698 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acidAttorney Docket No. EVIM-006 / 001WO 339103-2023 sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0197] In some embodiments, the bispecific antibody EIP0698 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 273, a L1 comprising the amino acid sequence of SEQ ID NO: 272, a H2 comprising the amino acid sequence of SEQ ID NO: 271, and a L2 comprising the amino acid sequence of SEQ ID NO: 270.

[0198] In some embodiments, the bispecific antibody EIP0708 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 18, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0199] In some embodiments, the bispecific antibody EIP0708 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 281, a L1 comprising the amino acid sequence of SEQ ID NO: 280, a H2 comprising the amino acid sequence of SEQ ID NO: 279, and a L2 comprising the amino acid sequence of SEQ ID NO: 278.

[0200] In some embodiments, the bispecific antibody EIP0745 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 19, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0201] In some embodiments, the bispecific antibody EIP0745 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 289, a L1 comprising the amino acid sequence of SEQ ID NO: 288, a H2 comprising the amino acid sequence of SEQ ID NO: 287, and a L2 comprising the amino acid sequence of SEQ ID NO: 286.

[0202] In some embodiments, the bispecific antibody EIP0751 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 20, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0203] In some embodiments, the bispecific antibody EIP0751 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 297, a L1 comprising the amino acid sequence of SEQ ID NO: 296, a H2 comprising the amino acid sequence of SEQ ID NO: 295, and a L2 comprising the amino acid sequence of SEQ ID NO: 294.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0204] Any one of the bispecific antibodies shown above in Table 13 can be further modified by substituting any one of the anti-CD3İ antigen binding regions with any one of the anti-CD3İ binding regions shown in Tables 7-9. For example, the anti-CD3İ antigen binding regions of bispecific antibody “EIP1224” can be substituted with any one of the anti- CD3İ binding regions shown in Tables 7-9 to produce the bispecific antibodies of the invention. Exemplary antibodies are shown in Table 13.

[0205] FUSION PEPTIDES

[0206] Provided herein is an antibody (e.g. monospecific antibody or bispecific antibody) that has a fusion peptide fused to the N-terminus or the C-terminus of the first heavy chain polypeptide or the second heavy chain polypeptide.

[0207] Critical to the initial T cell response is the capacity for T cells to detect foreign and mutated proteins through their T cell receptor. This response, often referred to as signal 1 of T cell activation, occurs when the T cell receptor engages a cell that displays a foreign or mutated protein fragment or antigen in a specific protein complex called the Major Histocompatibility Complex I (MHCI). The activation of the T cell receptor is by itself both activating and auto-regulatory to T cells. Strong binding of the TCR to an MHCI complex creates chronic activation of the TCR. This form of signal is associated with T cells that are reactive to self-antigens. T cells are programed to inactivate when they experience this form activation. T cells with TCR that bind weaker, but sufficient for activation, experience acute signaling with the potential to remain active and differentiate into memory T cells. This is emerging as important consideration in the design the T cell therapeutics.

[0208] T cell cytokine activation, often referred to signal 3, is important in T cell transitions, either from non-dividing to a state of rapid cell division or from one phenotypic state to another. T cell cytokine receptors bind to cytokines that are produced by immune and non- immune cells and depending on the cytokine and the state of the T cell at the time of receiving the cytokine signal can induce cell proliferation, can sustain vitality, or can induce differentiation of T cells into a specialized cell state appropriate for sustained activation or inactivation following infection.

[0209] One example is the transition that naïve cells experience through cytokines which can induce naïve T cells to proliferate and promote T cell differentiation into memory T cells.Attorney Docket No. EVIM-006 / 001WO 339103-2023 Exemplary cytokines include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18 and IL-21.

[0210] Costimulatory receptor activation, referred to as signal 2 provides a context specific cell-to-cell reinforcement of T activation. The most recognized form of costimulation occurs when T cells interact with activated antigen presenting cells through the T cell costimulatory receptor CD28 with CD80 and CD86 ligands found on APCs. These interactions can “prime” specific T cells armed with T cell receptors responsive to pathogen or cancer proteins.

[0211] Less appreciated is costimulation induced at the site of infection and malignancies. This includes costimulation that acts through CD2 and NKG2D receptors responsive to ligands like CD58 and UL16 binding proteins (e.g. ULBP2 / 5 / 6) that are induced in immune cells and epithelial cells upon viral infection. These signals provide not only reinforcement of T activation, but confirmation that the T cell’s lethal effector activities are targeted with single cell accuracy. While many costimulatory receptors have been discovered, the importance of each receptor’s specific context and the impact of concurrent signaling of multiple costimulatory receptors remains largely unknown and an area to greatly advance our understanding of T cell biology and creating possibilities for novel tumor-targeted T cell therapeutic development.

[0212] Costimulatory ligands include but are not limited to CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL or ICOSLG.

[0213] CD58 is advantageous over other costimulatory ligands in that it is the primary costimulatory pathway available at the tumor site as tumor infiltrating T lymphocytes often lose expression of other costimulatory receptors like CD28, or due to the low immunogenicity of tumor cells, tumor cells do not sufficiently activate T cell, thus limiting the potential of inducible costimulatory receptors like 41BB.

[0214] As described previously, the anti-CD3İ antibodies of the disclosure induce varying levels of T cell receptor activation that confer alteration in T cell vitality and cytokine production. Accordingly, a fusion of the costimulatory ligand CD58 to the anti-CD3İ bispecific antibody provides integrated costimulatory T cell activation for optimal T cell activation.

[0215] In some embodiments, the bispecific antibody has a peptide fused to the N-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody hasAttorney Docket No. EVIM-006 / 001WO 339103-2023 a peptide fused to the C-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a polypeptide fused to the N-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the second heavy chain polypeptide (H2). Exemplary peptides include but are not limited to IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21 or portions thereof. Exemplary peptides include but are not limited to CD48, CD58, CD86, TNFSF9, OX40L, 4- 1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG or portions thereof. Exemplary peptide sequences that are fused to the bispecific antibodies include but are not limited to those listed in Table 14.1 and Table 14.2.

[0216] Table 14.1. Exemplary Fusion Peptide Sequences

[0217] Table 14.2. Exemplary Fusion Peptide SequencesAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0218] In some embodiments the polypeptide is fused directly to the bispecific antibody. In some embodiments, the polypeptide is fused indirectly through a linker. In some embodiments, the bispecific antibody fused with a peptide comprises a linker sequence. Exemplary linker sequences include but are not limited to those listed in Table 15.1 and Table 15.2.

[0219] Table 15.1. Exemplary Linker Sequences

[0220] Table.15.2 Exemplary Linker SequencesAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0221] In some embodiments, the CD3 x CD180 bispecific antibodies of the invention have a CD58 fusion peptide (SEQ ID NO: 49) fused indirectly at the C-terminus of the first heavy chain polypeptide (H1) using linker-1 (SEQ ID NO: 52). In some embodiments, the CD3 x CD180bispecific antibodies of the invention have a CD58v* fusion peptide (SEQ ID NO: 50) fused indirectly at the C-terminus of the first heavy chain polypeptide (H1) using linker-1 (SEQ ID NO: 52).

[0222] In some embodiments, the CD3 x CD180 bispecific antibodies of the invention have a CD58 fusion peptide (SEQ ID NO: 49) fused indirectly at the C-terminus of the first heavy chain polypeptide (H1) using linker-2 (SEQ ID NO: 53). In some embodiments, the CD3 xAttorney Docket No. EVIM-006 / 001WO 339103-2023 CD180 bispecific antibodies of the invention have a CD58v* fusion peptide (SEQ ID NO: 50) fused indirectly at the C-terminus of the first heavy chain polypeptide (H1) using linker- 2 (SEQ ID NO: 53).

[0223] In some embodiments, the CD3 x CD180 bispecific antibodies of the invention have a CD58 fusion peptide (SEQ ID NO: 49) fused indirectly at the C-terminus of the first heavy chain polypeptide (H1) using linker-3 (SEQ ID NO: 54). In some embodiments, the CD3 x CD180 bispecific antibodies of the invention have a CD58v* fusion peptide (SEQ ID NO: 50) fused indirectly at the C-terminus of the first heavy chain polypeptide (H1) using linker- 3 (SEQ ID NO: 54).

[0224] Exemplary CD3 x CD180 bispecific antibodies having a CD58 fusion are shown in Table 16.

[0225] Table 16. Exemplary Bispecific Antibodies that bind to CD3İ and CD180 with a C-terminus CD58 fusion peptideAttorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0226] Exemplary, CD3İ x CD180 bispecific antibodies with a C-terminus fusion peptide of the invention include EIP1042, EIP1043, EIP1044, EIP1056, EIP1057, EIP0716, EIP0707, EIP0717, EIP0699, EIP0709, EIP0746 and EIP0752.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0227] Bispecific antibodies with a CD58 fusion peptide EIP1042, EIP1043, EIP1044, EIP1056, EIP1057, and EIP0699 comprise a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 30; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 45.

[0228] Bispecific antibody with a CD58 fusion peptide EIP0716 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VL1CDR1having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3having the amino acid sequence of SEQ ID NO: 45.

[0229] Bispecific antibody with a CD58 fusion peptide EIP0707 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 37; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 44; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 45.

[0230] Bispecific antibody with a CD58 fusion peptide EIP0717 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3having the amino acid sequence of SEQ ID NO: 39; and a VL1, comprising a VL1CDR1having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3having the amino acid sequence of SEQ ID NO: 47.

[0231] Bispecific antibody with a CD58 fusion peptide EIP0709 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQAttorney Docket No. EVIM-006 / 001WO 339103-2023 ID NO: 35; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 38; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 47.

[0232] Bispecific antibody with a CD58 fusion peptide EIP0746 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1 having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 36; and a VH1CDR3having the amino acid sequence of SEQ ID NO: 40; and a VL1, comprising a VL1CDR1having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3having the amino acid sequence of SEQ ID NO: 47.

[0233] Bispecific antibody with a CD58 fusion peptide EIP0752 comprises a first antigen binding domain that binds CD3İ which comprising a VH1, comprising a VH1CDR1having the amino acid sequence of SEQ ID NO: 29; a VH1CDR2 having the amino acid sequence of SEQ ID NO: 34; and a VH1CDR3 having the amino acid sequence of SEQ ID NO: 41; and a VL1, comprising a VL1CDR1 having the amino acid sequence of SEQ ID NO: 42; a VL1CDR2 having the amino acid sequence of SEQ ID NO: 43; and a VL1CDR3 having the amino acid sequence of SEQ ID NO: 47.

[0234] Bispecific antibodies with a CD58 fusion peptide EIP1042, EIP1043, EIP1044, EIP1056, EIP1057, EIP0716, EIP0707, EIP0717, EIP0699, EIP0709, EIP0746 and EIP0752 comprise a second antigen binding domain that binds CD180 comprising a VH2,comprising VH2CDR1 having the amino acid sequence of SEQ ID NO: 210; a VH2CDR2 havingthe amino acid sequence of SEQ ID NO: 212; and a VH2CDR3having the amino acid sequence of SEQ ID NO: 214; and a VL2, having VL2CDR1having the amino acid sequence of SEQ ID NO: 222; a VL2CDR2having the amino acid sequence of SEQ ID NO: 223; and a VL2CDR3having the amino acid sequence of SEQ ID NO: 224.

[0235] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1042 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 197, and a VL2 comprising the amino acid sequence of SEQ ID NO: 196.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0236] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1042 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 305, a L1 comprising the amino acid sequence of SEQ ID NO: 304, a H2 comprising the amino acid sequence of SEQ ID NO: 303, and a L2 comprising the amino acid sequence of SEQ ID NO: 302.

[0237] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1043 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 199, and a VL2 comprising the amino acid sequence of SEQ ID NO: 198.

[0238] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1043 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 309, a L1 comprising the amino acid sequence of SEQ ID NO: 308, a H2 comprising the amino acid sequence of SEQ ID NO: 307, and a L2 comprising the amino acid sequence of SEQ ID NO: 306.

[0239] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1044 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 201, and a VL2 comprising the amino acid sequence of SEQ ID NO: 200.

[0240] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1044 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 313, a L1 comprising the amino acid sequence of SEQ ID NO: 312, a H2 comprising the amino acid sequence of SEQ ID NO: 311, and a L2 comprising the amino acid sequence of SEQ ID NO: 310.

[0241] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1056 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0242] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1056 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 317, a L1 comprising the amino acid sequence of SEQ ID NO: 316, a H2 comprising the amino acid sequence of SEQ ID NO: 315, and a L2 comprising the amino acid sequence of SEQ ID NO: 314.

[0243] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1057 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprisingAttorney Docket No. EVIM-006 / 001WO 339103-2023 the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 205, and a VL2 comprising the amino acid sequence of SEQ ID NO: 204.

[0244] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP1057 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 321, a L1 comprising the amino acid sequence of SEQ ID NO: 320, a H2 comprising the amino acid sequence of SEQ ID NO: 319, and a L2 comprising the amino acid sequence of SEQ ID NO: 318.

[0245] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0716 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0246] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0716 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 329, a L1 comprising the amino acid sequence of SEQ ID NO: 328, a H2 comprising the amino acid sequence of SEQ ID NO: 327, and a L2 comprising the amino acid sequence of SEQ ID NO: 326.

[0247] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0707 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 13, a VL1 comprising the amino acid sequence of SEQ ID NO: 27, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0248] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0707 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 337, a L1 comprising the amino acid sequence of SEQ ID NO: 336, a H2 comprising the amino acid sequence of SEQ ID NO: 335, and a L2 comprising the amino acid sequence of SEQ ID NO: 334.

[0249] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0717 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 16, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0250] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0717 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 345, a L1 comprising the amino acid sequence of SEQ ID NO: 344, a H2 comprising the amino acid sequence of SEQ ID NO: 343, and a L2 comprising the amino acid sequence of SEQ ID NO: 342.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0251] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0699 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 17, a VL1 comprising the amino acid sequence of SEQ ID NO: 22, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0252] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0699 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 354, a L1 comprising the amino acid sequence of SEQ ID NO: 353, a H2 comprising the amino acid sequence of SEQ ID NO: 352, and a L2 comprising the amino acid sequence of SEQ ID NO: 351.

[0253] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0709 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 18, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0254] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0709 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 363, a L1 comprising the amino acid sequence of SEQ ID NO: 362, a H2 comprising the amino acid sequence of SEQ ID NO: 361, and a L2 comprising the amino acid sequence of SEQ ID NO: 360.

[0255] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0746 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 19, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0256] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0746 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 372, a L1 comprising the amino acid sequence of SEQ ID NO: 371, a H2 comprising the amino acid sequence of SEQ ID NO: 370, and a L2 comprising the amino acid sequence of SEQ ID NO: 369.

[0257] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0752 comprises a VH1 comprising the amino acid sequence of SEQ ID NO: 20, a VL1 comprising the amino acid sequence of SEQ ID NO: 26, a VH2 comprising the amino acid sequence of SEQ ID NO: 203, and a VL2 comprising the amino acid sequence of SEQ ID NO: 202.

[0258] In some embodiments, the bispecific antibody with a CD58 fusion peptide EIP0752 comprises a H1 comprising the amino acid sequence of SEQ ID NO: 381, a L1 comprisingAttorney Docket No. EVIM-006 / 001WO 339103-2023 the amino acid sequence of SEQ ID NO: 380, a H2 comprising the amino acid sequence of SEQ ID NO: 379, and a L2 comprising the amino acid sequence of SEQ ID NO: 368.

[0259] Any one of the anti-CD3 / anti-CD180 bispecific antibodies with a CD58 fusion peptide shown above in Table 16 can be further modified by substituting any one of the anti- CD3İ antigen binding regions with any one of the anti-CD3İ binding regions shown in Tables 7-9. For example, the anti-CD3İ antigen binding regions of bispecific antibody “EIP1042” can be substituted with any one of the anti-CD3İ binding regions shown in Tables 7-9 to produce the bispecific antibodies of the invention. Exemplary antibodies are shown in Table 13 and Table 16.

[0260] METHODS OF PRODUCTION

[0261] Various procedures known within the art may be used for the production of polyclonal or monoclonal antibodies directed against a given target, such as, for example, CD180, a disease associated antigen or other target, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0262] Antibodies are purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen, which is the target of the immunoglobulin sought, or an epitope thereof, may be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol.14, No.8 (April 17, 2000), pp.25-28).

[0263] In some embodiments, the antibodies of the invention are monoclonal antibodies. Monoclonal antibodies are generated, for example, by using the procedures set forth in the Examples provided herein. Antibodies are also generated, e.g., by immunizing BALB / c mice with combinations of cell transfectants expressing high levels of a given target on their surface. Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to the selected target.

[0264] Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method,Attorney Docket No. EVIM-006 / 001WO 339103-2023 a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0265] The immunizing agent will typically include the protein antigen, a fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.

[0266] Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of monoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp.51-63)).

[0267] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) orAttorney Docket No. EVIM-006 / 001WO 339103-2023 enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.

[0268] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0269] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0270] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non- immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variableAttorney Docket No. EVIM-006 / 001WO 339103-2023 domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0271] Monoclonal antibodies of the invention include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without engendering an immune response by the human against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that are principally comprised of the sequence of a human immunoglobulin, and contain minimal sequence derived from a non-human immunoglobulin. Humanization is performed, e.g., by following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. (See also U.S. Patent No.5,225,539). In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies also comprise, e.g., residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody includes substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also includes at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0272] Fully human antibodies are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Monoclonal antibodies can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad SciAttorney Docket No. EVIM-006 / 001WO 339103-2023 USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0273] In addition, human antibodies can also be produced using additional techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol.1365-93 (1995).

[0274] Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal’s endogenous antibodies in response to challenge by an antigen. (See PCT publication WO94 / 02602). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host’s genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. An example of such a nonhuman animal is a mouse termed the XenomouseTMas disclosed in PCT publications WO 96 / 33735 and WO 96 / 34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered andAttorney Docket No. EVIM-006 / 001WO 339103-2023 expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.

[0275] An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No.5,939,598. It can be obtained by a method, which includes deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.

[0276] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.

[0277] In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen and a correlative method for selecting an antibody that binds specifically to the relevant epitope with high affinity are disclosed in PCT publication WO 99 / 53049.

[0278] The antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

[0279] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA. gene gun, catheters, etc. Vectors include chemical conjugates such as described in WO 93 / 64701, which has targeting moiety (e.g., a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g., polylysine), viral vector (e.g., a DNA or RNA viral vector), fusion proteins such as described in PCT / US 95 / 02140 (WO 95 / 22618) which is a fusion protein containing a target moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., a protamine), plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal or synthetic.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0280] Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. These vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

[0281] Pox viral vectors introduce the gene into the cell’s cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno- associated virus vectors and herpes simplex virus (HSV) vectors are preferred for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g., infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, (Ca)2(PO4)3 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0282] The vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors (e.g., adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and may be useful in delivering the vector to the target cell. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0283] Bispecific antibodies are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a first target such as CD3İ or any fragment thereof. The second binding target is a disease associated antigen such as CD180 or any fragment thereof.

[0284] Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93 / 08829, published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0285] Bispecific and / or monovalent antibodies of the invention can be made using any of a variety of art-recognized techniques, including those disclosed in co-pending application WO 2012 / 023053, filed August 16, 2011, the contents of which are hereby incorporated by reference in their entirety. The methods described in WO 2012 / 023053 generate bispecific antibodies that are identical in structure to a human immunoglobulin. This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant Kappa domain and second light chain variable region fused to a constant Lambda domain. Each combining site displays a different antigen specificity to which both the heavy and light chain contribute. The light chain variable regions can be of the Lambda or Kappa family and are preferably fused to a Lambda and Kappa constant domains, respectively. This is preferred in order to avoid the generation of non-natural polypeptide junctions. However, it is also possible to obtain bispecific antibodies of the invention by fusing a Kappa light chain variable domain to a constant Lambda domain for a first specificity and fusing a Lambda light chain variable domain to a constant Kappa domain for the second specificity. The bispecific antibodies described in WO 2012 / 023053 are referred to as IgG^^ antibodies or “^^ bodies,” a new fully human bispecific IgG format. This ^^-body format allows the affinity purification of a bispecific antibody that is undistinguishable from a standard IgG molecule with characteristics that areAttorney Docket No. EVIM-006 / 001WO 339103-2023 undistinguishable from a standard monoclonal antibody and, therefore, favorable as compared to previous formats.

[0286] An essential step of the method is the identification of two antibody Fv regions (each composed by a variable light chain and variable heavy chain domain) having different antigen specificities that share the same heavy chain variable domain. Numerous methods have been described for the generation of monoclonal antibodies and fragments thereof. (See, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference). Fully human antibodies are antibody molecules in which the sequence of both the light chain and the heavy chain, including the CDRs 1 and 2, arise from human genes. The CDR3 region can be of human origin or designed by synthetic means. Such antibodies are termed “human antibodies”, or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by using the trioma technique; the human B-cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96).

[0287] Monoclonal antibodies are generated, e.g., by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells. A variety of techniques are well-known in the art for producing xenogenic non-human animals. For example, see U.S. Pat. No.6,075,181 and No.6,150,584, which is hereby incorporated by reference in its entirety.

[0288] Alternatively, the antibodies are obtained by screening a library that contains antibody or antigen binding domain sequences for binding to the target antigen. This library is prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNAAttorney Docket No. EVIM-006 / 001WO 339103-2023 sequences contained within the phage particles (i.e., “phage displayed library”). Alternatively, a library can be prepared in yeast as protein or peptide fusions to a cell wall protein on the surface of yeast cells and encoding DNA sequences contained within the yeast cells (i.e. “yeast display library”).

[0289] Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to the target antigen. Monoclonal antibodies are prepared, for example, using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0290] Although not strictly impossible, the serendipitous identification of different antibodies having the same heavy chain variable domain but directed against different antigens is highly unlikely. Indeed, in most cases the heavy chain contributes largely to the antigen binding surface and is also the most variable in sequence. In particular the CDR3 on the heavy chain is the most diverse CDR in sequence, length and structure. Thus, two antibodies specific for different antigens will almost invariably carry different heavy chain variable domains.

[0291] The methods disclosed in co-pending application WO 2012 / 023053 overcomes this limitation and greatly facilitates the isolation of antibodies having the same heavy chain variable domain by the use of antibody libraries in which the heavy chain variable domain is the same for all the library members and thus the diversity is confined to the light chain variable domain. Such libraries are described, for example, in co-pending applications WO 2010 / 135558 and WO 2011 / 084255, each of which is hereby incorporated by reference in its entirety. However, as the light chain variable domain is expressed in conjunction with the heavy variable domain, both domains can contribute to antigen binding. To further facilitate the process, antibody libraries containing the same heavy chain variable domain and either a diversity of Lambda variable light chains or Kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach enables the identification of two antibodies having a common heavy chain but one carrying a Lambda light chain variable domain and the other a Kappa light chain variable domain that can beAttorney Docket No. EVIM-006 / 001WO 339103-2023 used as building blocks for the generation of a bispecific antibody in the full immunoglobulin format of the invention. The bispecific antibodies of the invention can be of different Isotypes and their Fc portion can be modified in order to alter the bind properties to different Fc receptors and in this way modify the effectors functions of the antibody as well as it pharmacokinetic properties. Numerous methods for the modification of the Fc portion have been described and are applicable to antibodies of the invention. (see for example Strohl, WR Curr Opin Biotechnol 2009 (6):685-91; U.S. Pat. No.6,528,624; PCT / US2009 / 0191199 filed Jan 9, 2009). The methods of the invention can also be used to generate bispecific antibodies and antibody mixtures in a F(ab’)2 format that lacks the Fc portion.

[0292] The common heavy chain and two different light chains are co-expressed into a single cell to allow for the assembly of a bispecific antibody of the invention. If all the polypeptides get expressed at the same level and get assembled equally well to form an immunoglobulin molecule then the ratio of monospecific (same light chains) and bispecific (two different light chains) should be 50%. However, it is likely that different light chains are expressed at different levels and / or do not assemble with the same efficiency. Therefore, a means to modulate the relative expression of the different polypeptides is used to compensate for their intrinsic expression characteristics or different propensities to assemble with the common heavy chain. This modulation can be achieved via promoter strength, the use of internal ribosome entry sites (IRES) featuring different efficiencies or other types of regulatory elements that can act at transcriptional or translational levels as well as acting on mRNA stability. Different promoters of different strength could include CMV (Immediate- early Cytomegalovirus virus promoter); EF1-1Į (Human elongation factor 1Į-subunit promoter); Ubc (Human ubiquitin C promoter); SV40 (Simian virus 40 promoter). Different IRES have also been described from mammalian and viral origin. (See e.g., Hellen CU and Sarnow P. Genes Dev 200115: 1593–612). These IRES can greatly differ in their length and ribosome recruiting efficiency. Furthermore, it is possible to further tune the activity by introducing multiple copies of an IRES (Stephen et al. 2000 Proc Natl Acad Sci USA 97: 1536-1541). The modulation of the expression can also be achieved by multiple sequential transfections of cells to increase the copy number of individual genes expressing one or the other light chain and thus modify their relative expressions. The Examples provided herein demonstrate that controlling the relative expression of the different chains is critical forAttorney Docket No. EVIM-006 / 001WO 339103-2023 maximizing the assembly and overall yield of the bispecific antibody.

[0293] The co-expression of the heavy chain and two light chains generates a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter has to be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies of the invention. This is in sharp contrast to specific purification methods that have to be developed and optimized for each bispecific antibodies derived from quadromas or other cell lines expressing antibody mixtures. Indeed, if the biochemical characteristics of the different antibodies in the mixtures are similar, their separation using standard chromatography technique such as ion exchange chromatography can be challenging or not possible at all.

[0294] Other suitable purification methods include those disclosed in co-pending application PCT / IB2012 / 003028, filed on October 19, 2012, published as WO2013 / 088259, the contents of which are hereby incorporated by reference in their entirety.

[0295] In other embodiments of producing bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co- transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0296] According to another approach described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface includes at least a part of the CH3 region of an antibody constant domain. In this method, one or more smallAttorney Docket No. EVIM-006 / 001WO 339103-2023 amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0297] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0298] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VLand VHdomains of another fragment, thereby forming two antigen- binding sites. Another strategy for making bispecific antibody fragments by the use of single- chain Fv (sFv) dimers has also been reported. See, Gruber et al., J. Immunol.152:5368 (1994).

[0299] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol.147:60 (1991).

[0300] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7),Attorney Docket No. EVIM-006 / 001WO 339103-2023 or Fc receptors for IgG (FcȖR), such as FcȖRI (CD64), FcȖRII (CD32) and FcȖRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

[0301] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (see U.S. Patent No.4,676,980), and for treatment of HIV infection (see WO 91 / 00360; WO 92 / 200373; EP 03089). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0302] It can be desirable to modify the antibody of the invention with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer and / or other diseases and disorders associated with aberrant CD180 expression and / or activity. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191- 1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[0303] The invention also pertains to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0304] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include212Bi,131I,131In,90Y, and186Re.

[0305] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See WO94 / 11026).

[0306] Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies of the invention. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0307] Coupling may be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding and complexation. The preferred binding is, however, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodiesAttorney Docket No. EVIM-006 / 001WO 339103-2023 of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

[0308] Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N- hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2- pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2- pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo-LC- SPDP (sulfosuccinimidyl 6 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0309] The linkers described above contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.

[0310] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc.Attorney Docket No. EVIM-006 / 001WO 339103-2023 Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No.5,013,556.

[0311] Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

[0312] METHODS OF USE

[0313] Any of the bispecific antibodies of the disclosure (e.g. anti-CD3İ and anti-CD180 antibody; anti-CD3İ and anti-CD180 antibody with CD58 fusion peptide) may be used in therapeutic methods. In one aspect, a bispecific antibody of the disclosure may be for use as a medicament is provided. In further aspects, a bispecific antibody of the disclosure may be for use in treating or delaying progression of a cell proliferative disorder (e.g., cancer). In certain embodiments, a bispecific antibody for use in a method of treatment is provided. In certain embodiments, the invention provides a bispecific antibody for use in a method of treating an individual having a cell proliferative disorder comprising administering to the individual an effective amount of the bispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In further embodiments, the invention provides a bispecific antibody for use in enhancing immune function in an individual having a cell proliferative disorder. In certain embodiments, the invention provides a bispecific antibody for use in a method of enhancing immune function in an individual having a cell proliferative disorder comprising administering to the individual an effective of the bispecific antibody to activate effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell population, and / or kill a target cell (e.g., target tumor cell). An "individual" according to any of the above embodiments may be a human.

[0314] In a further aspect, the invention provides for the use of bispecific antibodies of the disclosure (e.g. anti-CD3İ and anti-CD180 antibody; anti-CD3İ and anti-CD180 antibody with CD58 fusion peptide) in the manufacture or preparation of a medicament. In oneAttorney Docket No. EVIM-006 / 001WO 339103-2023 embodiment, the medicament is for treatment of a cell proliferative disorder (e.g., cancer, e.g., esophageal cancer or an adenocarcinoma). In a further embodiment, the medicament is for use in a method of treating a cell proliferative disorder or an autoimmune disorder comprising administering to an individual having a cell proliferative disorder or an autoimmune disorder an effective amount of the medicament. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. In a further embodiment, the medicament is for activating effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expanding (increasing) an effector cell population, reducing a target cell (e.g., a cell expressing CD180) population, and / or killing target cells (e.g., target tumor cells) in the individual. In a further embodiment, the medicament is for use in a method of enhancing immune function in an individual having a cell proliferative disorder or an autoimmune disorder comprising administering to the individual an amount effective of the medicament to activate effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell (e.g., a cell expressing CD180) population, and / or kill a target cell (e.g., target tumor cell). An "individual" according to any of the above embodiments may be a human.

[0315] In a further aspect, the invention provides a method for treating a cell proliferative disorder (e.g., cancer). In one embodiment, the method comprises administering to an individual having such a cell proliferative disorder an effective amount of a bispecific antibody. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, for example, as described below. An "individual" according to any of the above embodiments may be a human. In a further aspect, the invention provides a method for enhancing immune function in an individual having a cell proliferative disorder or an autoimmune disorder in an individual having a cell proliferative disorder or an autoimmune disorder. In one embodiment, the method comprises administering to the individual an effective amount of a bispecific antibody to activate effector cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells), expand (increase) an effector cell population, reduce a target cell (e.g., a cell expressing CD180) population, and / or kill a target cell (e.g., target tumor cell).Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0316] In some embodiments, the invention provides a method for treating a cancer that expresses CD180. In some embodiments, the method of treating a cancer comprises administering an effective amount of the bispecific antibody of the disclosure (e.g. anti-CD3İ and anti-CD180 antibody; anti-CD3İ and anti-CD180 antibody with CD58 fusion peptide).

[0317] In some embodiments, the cancer expresses CD180 at a high level. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is at least one of lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non- cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma. In some embodiments, the cancer is Waldenstrom's Macroglobulinemia. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML). In some embodiments, any of the types of cancers provided herein can be treated with a CD180 antibody.

[0318] In some embodiments, the CD180 expression is induced by the addition of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a BCL- 2 inhibitor. Exemplary BCL-2 inhibitors include but are not limited to Venclexta / Venclyxto / venetoclax. In some embodiments, the additional therapeutic agent is a FLT-3 inhibitor. Exemplary FLT-3 inhibitors include but are not limited to Rydapt / midostaurin, Xospata / gilteritinib, Vanflyta / quizartinib, and crenolanib. In some embodiments, the additional therapeutic agent is a IDH1 / 2 inhibitor. Exemplary IDH1 / 2 inhibitors include but are not limited to Tibsovo / ivosidenib, Idhifa / enasidenib, and Rezlidhia / olutasidenib. In some embodiments, the additional therapeutic agent is a chemotherapy and / or hypomethylating agent. Exemplary chemotherapies and / or hypomethylating agents include but are not limited to Vyxeos / daunorubicin + cytarabine liposome, Onureg / CC-486 / oral azacitidine, and Inqovi / Inaqovi / ASTX727. In some embodiments, the additional therapeutic agent is a Hedgehog inhibitor. Exemplary Hedgehog inhibitors include but are not limited to Daurismo / glasdegib. In some embodiments, the additional therapeutic agent is an antibody-drug conjugate. Exemplary antibody-drugAttorney Docket No. EVIM-006 / 001WO 339103-2023 conjugates include but are not limited to Mylotarg / gemtuzumab ozogamicin. In some embodiments, the additional therapeutic agent is a radioimmunoconjugate. Exemplary radioimmunoconjugates include but are not limited to Iomab-B / 131I apamistamab, and Actimab-A / lintuzumab-Ac225.

[0319] In some embodiments, the additional therapeutic agent is CAR T-cell therapy. Exemplary CAR T-cell therapies include but are not limited to Yescarta / axicabtagene ciloleucel, Breyanzi / lisocabtagene maraleucel, Kymriah / tisagenlecleucel, and Tecartus / brexucabtagene autoleucel. In some embodiments, the additional therapeutic agent is a T cell-engaging bispecific antibody. Exemplary T cell-engaging bispecific antibodies include but are not limited to Lunsumio / mosunetuzumab, Epkinly / Tepkinly / epcoritamab, Columvi / glofitamab, and Ordspono / odronextamab. In some embodiments, the additional therapeutic agent is an antibody-drug conjugate. Exemplary antibody-drug conjugates include but are not limited to Polivy / polatuzumab vedotin, and Zynlonta / loncastuximab tesirine. In some embodiments, the additional therapeutic agent is a monoclonal antibody. Exemplary monoclonal antibodies include but are not limited to rituximab, Gazyva / obinutuzumab, Monjuvi / Minjuvi / tafasitamab, and magrolimab. In some embodiments, the additional therapeutic agent is a BTK inhibitor. Exemplary BTI inhibitors include but are not limited to Brukinsa / Zanubrutinib, Calquence / acalabrutinib, Imbruvica / ibrutinib, Jaypirca / pirtobrutinib, Nemtabrutinib, NX-5948, NX-2127, and BGB-16673. In some embodiments, the additional therapeutic agent is a combination of treatments. Exemplary combinations of treatments include but are not limited to R-CHOP (Rituximab, Cyclophosphamide, Doxorubicin Hydrochloride / Hydroxydaunomycin, Vincristine Sulfate / Oncovin, and Prednisone), R- EPOCH (Rituximab, Etoposide Phosphate, Prednisone, Vincristine Sulfate / Oncovin Cyclophosphamide, and Doxorubicin Hydrochloride / Hydroxydaunomycin), R-GemOx (rituximab, gemcitabine, and oxaliplatin), and R-GVCP (rituximab, gemcitabine, cyclophosphamide, vincristine, and prednisolone). In some embodiments, the additional therapeutic agent is an angiogenesis inhibitor. Exemplary angiogenesis inhibitors include but are not limited to Revlimid / lenalidomide.

[0320] In some embodiments, the additional therapeutic agent is a BCL-2 inhibitor. Exemplary BCL-2 inhibitors include but are not limited to Venclexta / Venclyxto / venetoclax, Sonrotoclax / BGB-11417, and lisaftoclax. In some embodiments, the additional therapeuticAttorney Docket No. EVIM-006 / 001WO 339103-2023 agent is CAR T-cell therapy. Exemplary CAR T-cell therapies include but are not limited to MB-106, and Breyanzi / lisocabtagene maraleucel.

[0321] In some embodiments, a method of identifying a subject to receive a CD180 therapy is provided. The method comprises testing a cancer sample for a high level of expression of CD180. In some embodiments, the cancer is one or more of a lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma. In some embodiments, In some embodiments, the cancer is Waldenstrom's Macroglobulinemia. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML).

[0322] The method further comprises comparing a level of CD180 expression in the cancer sample to a level of CD180 expression in healthy / control / reference tissue to determine if the level of CD180 expression in the cancer sample is higher. If the level of CD180 expression is higher in the cancer sample, the subject receives a CD180-targeting agent. In some embodiments, the CD180-targeting agent is one or more of the antibodies provided herein. In some embodiments, any method for measuring the level of CD180 can be employed. In some embodiments, this is any amount greater than a negative amount in a staining assay. In some embodiments, this is any amount above a level present in surrounding healthy / control / reference tissue or corresponding tissue from a healthy / control / reference subject. In some embodiments, high levels of expression of CD180 is defined in comparison to an expression level of CD180 in a non-cancer sample.

[0323] In some embodiments, the expression level of CD180 in the cancer sample is at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% higher than the expression level of CD180 in the non-cancer sample. In some embodiments, the expression level of CD180 in the cancer sample is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% higher than the expression level of CD180 in the non-cancer sample. In some embodiments, the expression level of CD180 in the cancer sample is at least 15%, 20%, 25%, 30%, 35%, 40%,Attorney Docket No. EVIM-006 / 001WO 339103-2023 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475% or 500% higher than the expression level of CD180 in the non-cancer sample. In some embodiments, the expression level of CD180 in the cancer sample is at least 2 fold, 3, fold, 4 fold, 5, fold, 6 fold, 7, fold 8, fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold or 20 fold higher than the expression level of CD180 in the non-cancer sample.

[0324] In some embodiments, the healthy / control / reference sample is a sample from a normal tissue. In some embodiments, the normal tissue is a tissue that is an adjacent tissue to the cancer in the subject. For example, when assaying for expression via staining, the levels of the protein are above those understood to be “negative” to one of skill in the art for the particular assay. Exemplary assays include but are not limited to RNA based assays, flow cytometry, FACS, and IHC, each with their appropriate levels of positive and negative results. Any method of detecting the level of a protein in a sample is contemplated. One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of proteins being detected. Nonlimiting exemplary such methods include immunohistochemistry, ELISA, Western blotting, multiplex analyte detection (using, for example, Luminex technology), mass spectrometry, etc. Similarly, any method of detecting the level of an mRNA in a sample is contemplated. One skilled in the art can select a suitable method depending on the type of sample being analyzed and the identity and number of mRNAs being detected. Nonlimiting exemplary such methods include RT-PCR, quantitative RT-PCR and microarray-based methods, etc.

[0325] A CD180-positive cancer according to any of the above embodiments may be, e.g., CD180-positive B cell lymphoma, CD180-positive MDS / AML, and CD180-positive CLL. In some embodiments, an CD180-positive cancer is a cancer that receives an anti-CD180 immunohistochemistry (IHC) or in situ hybridization (ISH) score greater than “0,” which corresponds to very weak or no staining in >90% of tumor cells. In another embodiment, a CD180-positive cancer expresses CD180 at a 1+, 2+ or 3+ level. In some embodiments, a CD180-positive cancer is a cancer that expresses CD180 according to a reverse-transcriptase PCR (RT-PCR) assay that detects CD180 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR. In some embodiments, the RT-PCR is quantitative RT-PCR. In someAttorney Docket No. EVIM-006 / 001WO 339103-2023 embodiments, a CD180-positive cancer is a cancer that expresses CD180 according to a flow cytometry assay that detects CD180 expression on the tumor cell surface.

[0326] In a further aspect, the invention provides pharmaceutical formulations comprising any of the bispecific antibodies provided herein, e.g., for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the bispecific antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the bispecific antibodies provided herein and at least one additional therapeutic agent, for example, as described herein.

[0327] Antibodies of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody of the invention may be co-administered with at least one additional therapeutic agent. In certain embodiments, an additional therapeutic agent is a chemotherapeutic agent, growth inhibitory agent, cytotoxic agent, agent used in radiation therapy, anti-angiogenesis agent, apoptotic agent, anti-tubulin agent, or other agent, such as a epidermal growth factor receptor (EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1 / EGFR inhibitor (e.g., erlotinib (Tarceva™), platelet derived growth factor inhibitor (e.g., Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferon, cytokine, antibody other than the anti-CD3 antibody of the invention, such as an antibody that bind to one or more of the following targets ErbB2, ErbB3, ErbB4, PDGFR- beta, BlyS, APRIL, GPRC5D, CD20, CD19, BCMA VEGF, or VEGF receptor(s), TRAIL / Apo2, PD-1 (e.g. Nivolumab, Pembrolizumab, Cemiplimab), PD-L1 (Atezolizumab, Avelumab, Durvalumab), PD-L2, or another bioactive or organic chemical agent. In some embodiments, the invention provides a method wherein the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0328] In some aspects, the additional therapeutic agent is a monoclonal anti-CD180 antibody. In some embodiments, the additional therapeutic agent is a CD180 antibody linked to another antigen. In some aspects, antigen-linked CD180 antibodies are used as therapeutic vaccines. Exemplary antigen-linked CD180 antibodies are described in PCT publication WO2019099624A1, which is incorporated herein in its entirety.

[0329] In some aspects, the additional therapeutic agent is a checkpoint inhibitor. The term “inhibition” or “inhibitor” includes a reduction in a certain parameter, e.g., an activity, of a given molecule, e.g., an immune checkpoint inhibitor. For example, inhibition of an activity,Attorney Docket No. EVIM-006 / 001WO 339103-2023 e.g., an activity of, e.g., PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta, of at least 5%, 10%, 20%, 30%, 40% or more is included by this term. The level of inhibition need not be 100%.

[0330] In some aspects, the checkpoint inhibitor is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor is an anti-PD1 antibody. In some aspects, the PD-1 inhibitor is an anti PD-1 monoclonal antibody. Exemplary anti-PD-1 monoclonal antibodies include, but are not limited to cemiplimab (Libtayo), nivolumab (Opdivo), pembrolizumab (Keytruda). In some aspects, the checkpoint inhibitor is a PD-L1 inhibitor. Exemplary PD-L1 inhibitors include but are not limited to avelumab (Bavencio), durvalumab (Imfinzi) and atezolizumab (Tecentriq).

[0331] Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the antibody of the invention can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent or agents. In one embodiment, administration of the bispecific antibody and administration of an additional therapeutic agent occur within about one month, or within about one, two or three weeks, or within about one, two, three, four, five, or six days, of each other. Bispecific antibodies of the disclosure can also be used in combination with radiation therapy.

[0332] In some aspects, the additional therapeutic agent is a chimeric antigen receptor (CAR) T cell therapy. In some embodiments, the CAR-T cell therapy specifically binds CD19. Exemplary CAR-T cell therapies that specifically bind CD19 include but are not limited to BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™ (axicabtagene ciloleucel), ABECMA® (idecabtagene vicleucel), or CARVYKTI™ (ciltacabtagene autoleucel).

[0333] An antibody of the invention (and / or any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibody is administered by subcutaneous administration. In someAttorney Docket No. EVIM-006 / 001WO 339103-2023 embodiments, an anti-CD3İ antibody administered by subcutaneous injection exhibits a less toxic response in a patient than the same anti-CD3İ antibody administered by intravenous injection. Dosing can be by any suitable route, for example, by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0334] Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate.

[0335] For the prevention or treatment of disease, the appropriate dosage of an antibody of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0336] As a general proposition, the therapeutically effective amount of the bispecific anti- CD3İ and anti-CD180 antibody administered to human will be in the range of about 0.01 to about 100 mg / kg of patient body weight whether by one or more administrations. In some embodiments, the antibody used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25Attorney Docket No. EVIM-006 / 001WO 339103-2023 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg administered daily, for example. In one embodiment, an bispecific antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21 -day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, every week or every three weeks (e.g., such that the patient receives from about two to about twenty, or, for example, about six doses of the bispecific antibody). An initial higher loading dose, followed by one or more lower doses may be administered. The progress of this therapy is easily monitored by conventional techniques and assays.

[0337] In some embodiments, the methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery, chemotherapy, gene therapy, DMA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymatic inhibitor or anti- metastatic agent. In some embodiments, the additional therapy is the administration of side- effect limiting agents (e.g., agents intended to lessen the occurrence and / or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be a separate administration of one or more of the therapeutic agents described above.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0338] It will be appreciated that administration of therapeutic entities in accordance with the invention will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. A multitude of appropriate formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug, Seymour, therein. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsions carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Any of the foregoing mixtures may be appropriate in treatments and therapies in accordance with the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable with the route of administration. See also Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery-some emerging concepts.” J Pharm Sci. 89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information related to formulations, excipients and carriers well known to pharmaceutical chemists.

[0339] Therapeutic formulations of the invention, which include an antibody of the invention, are used to treat or alleviate a symptom associated with a cancer, such as, by way of non-limiting example, leukemias, lymphomas, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung & bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, urinary bladder cancer, kidney and renal pelvis cancer, oral cavity & pharynx cancer, uterine corpus cancer, and / or melanoma The present invention also provides methods of treating or alleviating a symptom associated with a cancer. A therapeutic regimen is carried out by identifying a subject, e.g., a human patient suffering from (or at risk of developing) a cancer, using standard methods.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0340] Efficaciousness of treatment is determined in association with any known method for diagnosing or treating the particular immune-related disorder. Alleviation of one or more symptoms of the immune-related disorder indicates that the antibody confers a clinical benefit.

[0341] Methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme linked immunosorbent assay (ELISA) and other immunologically mediated techniques known within the art.

[0342] Antibodies directed against a target such as CD3İ, CD180, or a combination thereof (or a fragment thereof), may be used in methods known within the art relating to the localization and / or quantitation of these targets, e.g., for use in measuring levels of these targets within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific any of these targets, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to hereinafter as “Therapeutics”).

[0343] An antibody of the invention can be used to isolate a particular target using standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies of the invention (or a fragment thereof) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, ȕ-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include125I,131I,35S or3H.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0344] Antibodies of the invention, including polyclonal, monoclonal, humanized and fully human antibodies, may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a disease or pathology associated with aberrant expression or activation of a given target in a subject. An antibody preparation, preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Administration of the antibody may abrogate or inhibit or interfere with the signaling function of the target. Administration of the antibody may abrogate or inhibit or interfere with the binding of the target with an endogenous ligand to which it naturally binds.

[0345] A therapeutically effective amount of an antibody of the invention relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies may range, for example, from twice daily to once a week.

[0346] Antibodies or a fragment thereof of the invention can be administered for the treatment of a variety of diseases and disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol.4), 1991, M. Dekker, New York.

[0347] Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemicallyAttorney Docket No. EVIM-006 / 001WO 339103-2023 and / or produced by recombinant DNA technology. (See, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended.

[0348] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

[0349] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0350] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and Ȗ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.

[0351] An antibody according to the invention can be used as an agent for detecting the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable label. Antibodies are polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. TheAttorney Docket No. EVIM-006 / 001WO 339103-2023 term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term “biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in “ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; “Immunoassay”, E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0352] PHARMACEUTICAL COMPOSITIONS

[0353] The antibodies of the invention (also referred to herein as “active compounds”), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents,Attorney Docket No. EVIM-006 / 001WO 339103-2023 and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0354] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0355] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propyleneAttorney Docket No. EVIM-006 / 001WO 339103-2023 glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0356] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0357] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0358] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0359] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0360] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0361] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No.4,522,811.

[0362] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect toAttorney Docket No. EVIM-006 / 001WO 339103-2023 be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0363] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0364] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0365] DEFINITIONS

[0366] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0367] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0368] The term “CD180-positive cancer” refers to a cancer comprising cells that express CD180 on their surface. In some embodiments, expression of CD180 on the cell surface isAttorney Docket No. EVIM-006 / 001WO 339103-2023 determined, for example, using antibodies to CD180 in a method such as immunohistochemistry, FACS, etc. Alternatively, CD180 mRNA expression is considered to correlate to CD180 expression on the cell surface and can be determined by a method selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).

[0369] The term “CD180-positive cell” refers to a cell that expresses CD180 on its surface.

[0370] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non- Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (including triple negative (ERí / PRí / Her2í) breast cancer), colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0371] The phrase “reference sample”, “reference cell”, or “reference tissue”, denote a sample with at least one known characteristic that can be used as a comparison to a sample with at least one unknown characteristic. In some embodiments, a reference sample can be used as a positive or negative indicator. A reference sample can be used to establish a level of protein and / or mRNA that is present in, for example, healthy tissue, in contrast to a level of protein and / or mRNA present in the sample with unknown characteristics. In some embodiments, the reference sample comes from the same subject, but is from a different part of the subject than that being tested. In some embodiments, the reference sample is from a tissue area surrounding or adjacent to the cancer. In some embodiments, the reference sample is not from the subject being tested, but is a sample from a subject known to have, or not to have, a disorder in question (for example, a particular cancer or CD180 related disorder). In some embodiments, the reference sample is from the same subject, but from a point in time before the subject developed cancer. In some embodiments, the reference sample is from a benign cancer sample (for example, benign breast cancer sample), from the same or a differentAttorney Docket No. EVIM-006 / 001WO 339103-2023 subject. When a negative reference sample is used for comparison, the level of expression or amount of the molecule in question in the negative reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is no and / or a low level of the molecule. When a positive reference sample is used for comparison, the level of expression or amount of the molecule in question in the positive reference sample will indicate a level at which one of skill in the art will appreciate, given the present disclosure, that there is a level of the molecule.

[0372] The A “disease” or “disorder” as used herein refers to a condition where treatment is needed and / or desired.

[0373] The term “tumor cell”, “cancer cell”, “cancer”, “tumor”, and / or “neoplasm”, unless otherwise designated, are used herein interchangeably and refer to a cell (or cells) exhibiting an uncontrolled growth and / or abnormal increased cell survival and / or inhibition of apoptosis which interferes with the normal functioning of bodily organs and systems. Included in this definition are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. The terms “cancer” and “tumor” encompass solid and hematological / lymphatic cancers and also encompass malignant, pre-malignant, and benign growth, such as dysplasia. Also, included in this definition are cells having abnormal proliferation that is not impeded (e g immune evasion and immune escape mechanisms) by the immune system (e.g. virus infected cells). Exemplary tumor cells include, but are not limited to: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system;Attorney Docket No. EVIM-006 / 001WO 339103-2023 vulval cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B- cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non- cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs' syndrome.

[0374] In some embodiments, the term cancer can encompass: a lung cancer, a breast cancer, a head and neck cancer, an ovarian cancer, and / or an endometrial cancer. When desired, the difference between a “cancer” and a “cancer cell” can be denoted by the use of the explicit use of the phrase “cancer cell”; however, the term “cancer” will encompass concepts such as the subject having cancer, and multicellular tumors, as well as single cancer cells.

[0375] An “increase or decrease” refers to a statistically significant increase or decrease respectively. As will be clear to the skilled person, “modulating” can also involve effecting a change (which can either be an increase or a decrease) in affinity, avidity, specificity and / or selectivity of a target or antigen, for one or more of its ligands, binding partners, partners for association into a homomultimeric or heteromultimeric form, or substrates; effecting a change (which can either be an increase or a decrease) in the sensitivity of the target or antigen for one or more conditions in the medium or surroundings in which the target or antigen is present (such as pH, ion strength, the presence of co-factors, etc.), compared to the same conditions but without the presence of an antibody, bispecific or multispecific polypeptide agent. This can be determined in any suitable manner and / or using any suitable assay known per se or described herein, depending on the target involved.

[0376] As used herein, “an immune response” is meant to encompass cellular and / or humoral immune responses that are sufficient to inhibit or prevent onset or ameliorate the symptoms of disease (for example, cancer or cancer metastasis). “An immune response” can encompass aspects of both the innate and adaptive immune systems.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0377] As used herein, “treatment” is an approach for obtaining beneficial or desired clinical results. “Treatment” as used herein, covers any administration or application of a therapeutic for disease in a mammal, including a human. For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, any one or more of: alleviation of one or more symptoms, diminishment of extent of disease, preventing or delaying spread (for example, metastasis, for example metastasis to the lung or to the lymph node) of disease, preventing or delaying recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, and remission (whether partial or total). Also encompassed by “treatment” is a reduction of pathological consequence of a proliferative disease. The methods provided herein contemplate any one or more of these aspects of treatment. In-line with the above, the term treatment does not require one-hundred percent removal of all aspects of the disorder.

[0378] “Ameliorating” means a lessening or improvement of one or more symptoms as compared to not administering a CD180 antibody. “Ameliorating” also includes shortening or reduction in duration of a symptom.

[0379] The term “biological sample” means a quantity of a substance from a living thing or formerly living thing. Such substances include, but are not limited to, blood, (for example, whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes and spleen.

[0380] The term “control” refers to a composition known to not contain an analyte (“negative control”) or to contain analyte (“positive control”). A positive control can comprise a known concentration of analyte. “Control,” “positive control,” and “calibrator” may be used interchangeably herein to refer to a composition comprising a known concentration of analyte. A “positive control” can be used to establish assay performance characteristics and is a useful indicator of the integrity of reagents (for example, analytes).

[0381] “Predetermined cutoff” and “predetermined level” refer generally to an assay cutoff value that is used to assess diagnostic / prognostic / therapeutic efficacy results by comparing the assay results against the predetermined cutoff / level, where the predetermined cutoff / level already has been linked or associated with various clinical parameters (for example, severity of disease, progression / nonprogression / improvement, etc.). While the present disclosure mayAttorney Docket No. EVIM-006 / 001WO 339103-2023 provide exemplary predetermined levels, it is well-known that cutoff values may vary depending on the nature of the immunoassay (for example, antibodies employed, etc.). It further is well within the skill of one of ordinary skill in the art to adapt the disclosure herein for other immunoassays to obtain immunoassay-specific cutoff values for those other immunoassays based on this disclosure. Whereas the precise value of the predetermined cutoff / level may vary between assays, correlations as described herein (if any) may be generally applicable.

[0382] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and / or amount as compared to a reference. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater. In some embodiments, the amount noted above is inhibited or decreased over a period of time, relative to a control dose (such as a placebo) over the same period of time.

[0383] As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0384] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. Unless otherwise specified, the terms “reduce”, “inhibit”, or “prevent” do not denote or require complete prevention over all time.

[0385] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, an antibody whichAttorney Docket No. EVIM-006 / 001WO 339103-2023 suppresses tumor growth reduces the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the antibody.

[0386] The term "antibody binding region" refers to a region of the antigen, which comprises the epitope to which the antibody binds. An antibody binding region may be determined by epitope binning using biolayer interferometry, by alanine scan, or by domain shuffle assays (using antigen constructs in which regions of the antigen are exchanged with that of another species and determining whether the antibody still binds to the antigen or not). The amino acids within the antibody binding region that are involved in the interaction with the antibody may be determined by hydrogen / deuterium exchange mass spectrometry and / or by crystallography of the antibody bound to its antigen.

[0387] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. By “specifically bind” or “immunoreacts with” or “immunospecifically bind” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at much lower affinity (Kd > 10-6). Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain, Fab, Fab’ and F(ab')2 fragments, scFvs, and an Fab expression library.

[0388] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgG1, IgG2, IgG4 and others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain.

[0389] The term “monoclonal antibody” (MAb) or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a uniqueAttorney Docket No. EVIM-006 / 001WO 339103-2023 heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.

[0390] The term “antigen binding region” or “antigen-binding site” or “binding portion” refers to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as “hypervariable regions,” are interposed between more conserved flanking stretches known as “framework regions,” or “FRs”. Thus, the term “FR” refers to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as “complementarity-determining regions,” or “CDRs.” Various methods are known in the art for numbering the amino acids sequences of antibodies and identification of the complementary determining regions. For example, the Kabat numbering system (See Kabat, E.A., et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (See IMGT®, the international ImMunoGeneTics information system®. Available online: http: / / www.imgt.org / ). The IMGT numbering system is routinely used and accepted as a reliable and accurate system in the art to determine amino acid positions in coding sequences, alignment of alleles, and to easily compare sequences in immunoglobulin (IG) and T-cell receptor (TR) from all vertebrate species. The accuracy and the consistency of the IMGT data are based on IMGT-ONTOLOGY, the first, and so far unique, ontology for immunogenetics and immunoinformatics (See Lefranc. M.P. et al., Biomolecules, 2014 Dec; 4(4), 1102-1139). IMGT tools and databases run against IMGT reference directories built from a large repository of sequences. In the IMGT system the IG V-DOMAIN and IG C-DOMAIN are delimited taking into account the exon delimitation,Attorney Docket No. EVIM-006 / 001WO 339103-2023 whenever appropriate. Therefore, the availability of more sequences to the IMGT database, the IMGT exon numbering system can be and “is used” by those skilled in the art reliably to determine amino acid positions in coding sequences and for alignment of alleles. Additionally, correspondences between the IMGT unique numbering with other numberings (i.e., Kabat) are available in the IMGT Scientific chart (See Lefranc. M.P. et al., Biomolecules, 2014 Dec; 4(4), 1102-1139).

[0391] The term "hypervariable region" or “variable region” refers to the amino acid residues of an antibody that are typically responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g., around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the VHwhen numbered in accordance with the Kabat numbering system; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or those residues from a "hypervariable loop" (e.g., residues 24- 34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and 26-32 (HI), 52-56 (H2) and 95-101 (H3) in the VH when numbered in accordance with the Chothia numbering system; Chothia and Lesk, J. Mol. Biol.196:901-917 (1987)); and / or those residues from a "hypervariable loop" VCDR (e.g., residues 27-38 (LI), 56-65 (L2) and 105-120 (L3) in the VL, and 27-38 (HI), 56-65 (H2) and 105-120 (H3) in the VH when numbered in accordance with the IMGT numbering system; Lefranc, M.P. et al. Nucl. Acids Res.27:209-212 (1999), Ruiz, M. e al. Nucl. Acids Res.28:219-221 (2000)). Optionally, the antibody has symmetrical insertions at one or more of the following points 28, 36 (LI), 63, 74-75 (L2) and 123 (L3) in the VL, and 28, 36 (HI), 63, 74-75 (H2) and 123 (H3) in the VHwhen numbered in accordance with AHo; Honneger, A. and Plunkthun, A. J. Mol. Biol.309:657-670 (2001)).

[0392] As used herein, the term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin, an scFv, or a T-cell receptor. The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies may be raised against N-terminal or C-terminal peptides of a polypeptide. AnAttorney Docket No. EVIM-006 / 001WO 339103-2023 antibody is the to specifically bind an antigen when the dissociation constant is ^ 1 μM; e.g., ^ 100 nM, preferably ^ 10 nM and more preferably ^ 1 nM.

[0393] As used herein, the terms “immunological binding,” and “immunological binding properties” refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller Kd represents a greater affinity. Immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). The ratio of Koff / Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present invention is the to specifically bind to its target, when the equilibrium binding constant (Kd) is ≤1 μM, e.g., ≤ 100 nM, preferably ≤ 10 nM, and more preferably ≤ 1 nM, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0394] The term “isolated polynucleotide” as used herein shall mean a polynucleotide of genomic, cDNA, or synthetic origin or some combination thereof, which by virtue of its origin the “isolated polynucleotide” (1) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (2) is operably linked to a polynucleotide which it is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides in accordance with the invention include the nucleic acid molecules encoding the heavy chain immunoglobulin molecules, and nucleic acid molecules encoding the light chain immunoglobulin molecules described herein.

[0395] The term “isolated protein” referred to herein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin, or source of derivation, the “isolated protein” (1) is not associated with proteins found in nature, (2) isAttorney Docket No. EVIM-006 / 001WO 339103-2023 free of other proteins from the same source, e.g., free of marine proteins, (3) is expressed by a cell from a different species, or (4) does not occur in nature.

[0396] The term “polypeptide” is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein fragments, and analogs are species of the polypeptide genus. Polypeptides in accordance with the invention comprise the heavy chain immunoglobulin molecules, and the light chain immunoglobulin molecules described herein, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.

[0397] The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature, and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring.

[0398] The term “operably linked” as used herein refers to positions of components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0399] The term “control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression and processing of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence in eukaryotes, generally, such control sequences include promoters and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. The term “polynucleotide” as referred to herein means a polymeric boron of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0400] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology - A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers (e.g., D- amino acids) of the twenty conventional amino acids, unnatural amino acids such as Į-, Į-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4 hydroxyproline, Ȗ-carboxyglutamate, İ-N,N,N-trimethyllysine, İ -N- acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3-methylhistidine, 5- hydroxylysine, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4- hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

[0401] As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

[0402] Preferably, residue positions which are not identical differ by conservative amino acid substitutions.

[0403] Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic- hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine valine, glutamic- aspartic, and asparagine-glutamine.

[0404] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention,Attorney Docket No. EVIM-006 / 001WO 339103-2023 providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99%. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al. Science 253:164 (1991). Thus, the foregoing examples demonstrate thatAttorney Docket No. EVIM-006 / 001WO 339103-2023 those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the invention.

[0405] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally- occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).

[0406] As used herein, the terms “label” or “labeled” refers to incorporation of a detectable marker, e.g., by incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or calorimetric methods). In certain situations, the label or marker can also be therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g.,3H,14C,15N,35S,90Y,99Tc,111In,125I,131I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, p- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance. The term “pharmaceutical agent or drug” as used herein refers to aAttorney Docket No. EVIM-006 / 001WO 339103-2023 chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[0407] Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw- Hill, San Francisco (1985)).

[0408] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.

[0409] Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0410] The term patient includes human and veterinary subjects. EXAMPLES

[0411] EXAMPLE 1: Design and Analysis of anti-CD3 / CD180 bispecific antibody fusion molecules

[0412] A schematic of the anti-CD3 / anti-CD180 targeted bispecific antibodies of the present disclosure is represented in FIG. 4 and is composed of the following: (1) a first antigen binding region that binds CD3 comprising SP34 antibody variants (“anti-CD3” or “anti-CD3 arm”), (2) a second antigen binding region that binds CD180 (“anti-CD180” or “anti-CD180 arm”), (3) knob-in-hole mutations in the heavy chain (4) mutations that enforce light chain heterodimerization, (5) a protein linker, and (6) full length CD58 peptide fused to the C- terminal of the heavy chain of the CD3 binding arm.

[0413] Antibody sequencing

[0414] Antibody sequencing of mouse monoclonal MHR73-11 antibody clone was performed at Bioinformatics Solutions Inc. (Waterloo, ON, Canada). Briefly the antibody was reduced with DTT and alkylated using iodoacetamide, then digested into overlappingAttorney Docket No. EVIM-006 / 001WO 339103-2023 peptides using Asp N, chymotrypsin, elastase, trypsin, and pepsin proteases followed by reversed phase chromatography cleanup. Intact mass analysis with DTT reduction and PNGase F deglycosylation was performed on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer. For LC-MS / MS analysis, peptides were analyzed on an Orbitrap analyzer (Orbitrap Fusion Lumos, Thermo Fisher Scientific). Both full MS scans and MS2 scans were acquired in the high resolution Orbitrap mass analyzer. MS2 data were acquired using HCD and ETD followed by HCD (EThcD) fragmentation methods. All raw data files were used for data analysis including Ile / Leu differentiation using the PEAKS AB 2.0 software (Bioinformatics Solutions Inc.).

[0415] Modeling mouse antibody structure

[0416] The homology model of variable region of mouse anti-CD180 antibody MHR73-11 was generated using “Antibody structure prediction” tool in BioLuminate software (Schrödinger, LLC, New York, NY, 2021). Based on the murine VH and VL sequences of the MHR73-11, the following templates were selected from the curated antibody database: (1) PDB structure 6J5D was used as template for light chain, (2) PDB structure 1XIW was used as template for the heavy chain, and (3) 6J5D was also used as a common template for aligning heavy and light chain frameworks. Based on the sequence similarity of loop clusters, the templates for the loops were automatically determined. The generated model was visualized and refined for resolving any structural problems like steric clash, missing atom etc. This was done by using “protein preparation workflow” in Bioluminate by performing a restrained minimization using OPLS_2005 forcefield.

[0417] Antibody humanization Humanization of murine MHR73-11 antibody was performed using BioLuminate (Schrodinger). Briefly, the murine MHR73-11 homology model was compared against a human antibody structure database to identify human antibody framework with highest level of sequence and structural similarity to the mouse antibody. The CDR loops were then grafted onto the human frameworks. Structural guided mutations were introduced to eliminate steric clashes and maximize the stem geometry original geometry of the CDR regions.

[0418] In total 16 proposed humanized antibodies were obtained. These proposed 16 humanized antibodies came from a combination of 7 heavy chain and 7 light chain sequences.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0419] Small scale antibody Expression and Purification

[0420] The heavy and light chain DNA sequences obtained above were synthesized in pDT5 vector (ATUM, Newark CA). All the proposed humanized variants were expressed in small scale by transfecting the Expi293 cells with plasmid DNA (1 μg / ml) (ThermoFisher) according to manufacturer’s protocols. Cells were grown in flasks with rotation (125 rpm) at 37 °C with 8 % CO2. Five days post-transfection, the conditioned media was harvested from the cells by centrifugation (3000 x g) for 30 minutes and filtered using 0.45 μm filter. Antibodies were then purified using ProteinA magnetic agarose beads (from Pierce Cat #78610). Bound antibodies to magnetic beads were eluted with 0.1 M citrate buffer pH 3.0 and neutralized with 1M Tris pH 8.0. Antibodies were buffer exchanged to PBS, pH 7.4 using Zeba spin desalting column (Thermofisher catalog # 89892) and stored at 4 °C.

[0421] Enzyme Linked Immunosorbent Assay (ELISA)

[0422] The humanized variants were analyzed by ELISA for binding to human and cyno CD180-MD1. Maxisorp plates (Nunc) were coated with proteins at a concentration of 4μg / ml in PBS, pH 7.2 for 16 hours at 4 °C. All subsequent steps were performed at room temperature. The plates were then blocked with blocking buffer (PBS, pH 7.2, 3% BSA) for 1 hour. The plates were then washed using PBS pH 7.2, 0.1% Tween. Humanized variants were diluted in blocking buffer at a 1 in 4 dilution starting at 100 nM and incubated with blocked wells for 1 hour. Plates were then washed and incubated with Goat anti-human HRP conjugated IgG, FcȖ fragment specific antibody (Jackson Immune Research Labs, Code: 109- 035-098) at a 1 in 800 dilution. Following a final wash, the luminescence substrate was added according to manufacturer’s instructions (SeraCare). Luminescence was measured using Ensight plate reader (Perkin Elmer).

[0423] Surface Plasmon Resonance (SPR) Biacore 8K kinetic binding analysis

[0424] Exemplary bispecific antibodies were advanced for further analysis via surface plasma resonance (SPR) for binding to recombinant human CD180-MD1 heterodimer. Analysis was performed using the Biacore 8K system (Cytiva). Briefly, bispecific antibodies were diluted in 10 mM sodium acetate, pH 5.0, at 10 ug / mL, and immobilized on a CM5 sensor chip by standard amine coupling recommended by Cytiva. CD180-MD1 samples at different concentrations diluted in the running buffer (10 mM HEPES, 150 mM NaCl, 0.05% v / v Surfactant P20, pH 7.4) were then injected to each channel at a flow rate of 30 ^L / minAttorney Docket No. EVIM-006 / 001WO 339103-2023 for the multi-cycle kinetics analysis. The binding kinetics were carried out at 25 °C. The association contact and dissociation times were 300 and 600 seconds, respectfully. The chip surface was regenerated by injecting 10 mM glycine, pH 2.0, at a flow rate of 30 ^L / min for 30 sec. The kinetic and affinity constants were determined by fitting the data to a 1:1 binding model using the Biacore Insight Evaluation Software (Cytiva).

[0425] Stable cell line development:

[0426] CD180 protein expression and purification

[0427] Stable cell lines were prepared by manufacturer’s protocol (Lonza Group AG). For co-expression of human CD180 and MD1, a double gene vector (DGV) was created wherein the single gene vectors (SGV) with CD180 and MD1 respectively were combined.

[0428] For human CD180, the sequence of extracellular domain, Uniprot accession no. Q99467; residues 24-626 (SEQ ID NO: 418) were utilized and added a C-terminal Twin- Strep-tag for protein purification. For human MD1, Uniprot accession no. O95711, residues 21-162 SEQ ID NO: 417, were taken and added a C-terminal (His)6 tag.

[0429] Similarly, a double gene vector for co-expression of Cynomolgus monkey CD180- MD1 complex was constructed. For this, the sequence of extracellular domain of Cynomolgus monkey CD180 (Uniprot accession no. A0A2K5VMS6, residues 24-626) with a C-terminal Twin-Strep-tag (SEQ ID NO: 416) and Cynomolgus monkey-MD1 sequence, Uniprot accession no. A0A2K5UVX3, residues 21-162 with a C-terminal (His)6 tag were utilized (SEQ ID NO: 415).

[0430] The prepared plasmid was then transfected by electroporation of the CHOK1SV GS- KO host cell line (part of Lonza’s GS Xceed system). Once the cells were recovered, these were scaled up for fed-batch shake-flask culture according to manufacturer’s protocol. At the end of 12 day fed-batch culture, the media was subjected to centrifugation and filtration using 0.2 μM filter in preparation for purification

[0431] Protein expression and Purification

[0432] The filtered supernatant was incubated with the Biolock (IBA life sciences, 2-0205- 050) for 1hr to sequester any biotin that could be present in the media. CD180-MD1 complex were purified by Strep-Tactin®XT 4Flow® high capacity FPLC column (IBA life sciences, 2-5027-001) and eluted using elution buffer (0.1M Tris-Cl, 0.15M NaCl and 1mM EDTA, 0.25mM desthiobiotin at pH 8). The molecular weight and integrity of the CD180-MD1Attorney Docket No. EVIM-006 / 001WO 339103-2023 complex was confirmed by SDS PAGE and analytical size exclusion chromatography respectively (FIGS.1A & 1B and FIG.2).

[0433] Large Scale Antibody Expression and Purification

[0434] The heavy and light chain DNA sequences obtained above were synthesized in pDT5 vector (ATUM, Newark CA). Plasmid DNA (1 μg / ml) were transfected into Expi293 cells (ThermoFisher) according to manufacturer’s protocols. Cells were grown in flasks with rotation (125 rpm) at 37 °C with 8 % CO2. Five days post-transfection, the conditioned media was harvested from the cells by centrifugation (3000 x g) for 30 minutes and filtered using 0.45 μm filter. Antibodies were then purified using an AKTA Avant chromatography system (Cytiva) and a tandem purification method using HiTrap Mabselect Protein A and HiLoad Superdex 200 pg columns (Cytiva). Antibodies were stored in PBS, pH 7.4 at 4 °C following purification and prior to analysis.

[0435] Differential Scanning Calorimetry

[0436] Differential Scanning Calorimetry or DSC was performed using Nano DSC instrument from TA Instruments – Waters LLC. Protein samples were prepared in PBS at 1 mg / mL concentration, loaded into sample cell and scanned at 1 °C / min increment from 25 to 95 °C. Data was analyzed using NanoAnalyzer program subtracting PBS buffer background from each individual scan.

[0437] Mass Spectrometry

[0438] The intact mass of the purified fusion proteins and antibody chains was confirmed by Xevo G2-XS QTof Quadrupole Time-of-Flight Mass Spectrometry coupled to a Acquity UHPLC system (Waters) equipped with a Protein BEH C4 (300 Å 1.7 μm) column. Antibody samples were deglycosylated first using rapid PNGase F enzyme (New England Biolabs) in reducing and non-reducing conditions following supplier’s protocols. Reaction mixture was diluted to 1:10 in 50% Acetonitrile containing 0.1% Formic acid and 2 ^L of which was injected into LC-MS. The total ion chromatogram and m / z data of the proteins were acquired with gradient run of 10 to 70 mL HPLC grade acetonitrile over 12 min. Mass of the protein samples was deconvoluted from total ion chromatogram using BYOS software from Protein Metrics.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0439] Hematological cancer cell lines

[0440] Hematological cancer cell lines were ordered from ATCC: HT (DLBCL), SU-DHL- 10 (DLBCL), MV-4-11-GFP-luc (AML), JeKo-1 (mantle cell lymphoma), Raji (Burkitt’s lymphoma), Ramos (Burkitt’s lymphoma). MV-4-11-GFP-luc (AML) was obtained from Cellomics Technology. WSU-DLCL2 (DLBLC) was ordered from BioIVT. MOLM-13 and OCI-AML-2 were ordered from AcceGen. JeKo-1 cells were engineered to overexpress GFP in-house.

[0441] Target expression evaluation and biologic binding to tumor cell lines

[0442] A BD Biosciences FACSymphony A3 instrument was used for analytical flow cytometry. For the CD180 surface expression evaluation, the cells lines were stained with Zombie NIR™ Fixable Viability Kit (BioLegend). Then the cells were incubated with Human TruStain FcX Fc blocking solution (BioLegend), followed by staining with either an antibody against CD180 (MHR73-11 clone) or an isotype antibody (MOPC-21) conjugated to APC (Biolegend) to evaluate the target expression on cell surface.

[0443] Biding of the antibodies (EIP0051, EIP0133, EIP0209, EIP0553, EIP0554, EIP0546, EIP1042, EIP1043, EIP0614, EIP1056, EIP1057) to the tumor cell lines were determined by flow cytometry using BD Biosciences FACSymphony A3. The cells lines were stained with Zombie NIR™ Fixable Viability Kit (BioLegend) or based on scatter to facilitate gating of live cells for analysis, followed by incubation with serial dilution of biologics. The surface bound bispecific antibody was detected by incubating the cells with R-Phycoerythrin (PE) of Alexa Fluor 488-labeled anti-human IgG FcȖ antibody (Jackson ImmunoResearch). Thereafter, the cells were analyzed by flow cytometry or fixed in 2% PFA in PBS prior to analysis by flow cytometry. FlowJo software (BD) was used to process the data and obtain geometric mean fluorescence (MFI) of stained live cells.

[0444] Activated T cells Mediated Cytotoxicity Assays

[0445] The efficacy of the bispecific antibodies to mediate tumor cell lysis was evaluated in activated T cells – tumor cell line co-culture assays (JeKo-1-GFP, Raji, Ramos). Pan CD3 T cells were enriched from healthy human donor PBMC using EasySep Human T cell Isolation kit (Stemcell Technologies). To activate T cells, Dynabeads coated with ĮCD3 and ĮCD28 antibodies (Invitrogen) were added to the T cells at a ratio of 25 μL beads per million cells and incubated at 37°C in 5% CO2 for 48-72 hours. Following removal of Dynabeads, T cellsAttorney Docket No. EVIM-006 / 001WO 339103-2023 were incubated with IL-7 (10 ng / ml) in supplemented in media for an additional 7 days. The supplemented media was replaced with freshly thawed cytokine every 48-72 hours. Expanded T cells were seeded at 100,000 per well along with 20,000 either GFP-expressing JeKo-1 cells or CellTrace Violet-labeled Raji or Ramos (E:T=5:1) in the presence of a serial dilutions of antibodies and incubate for 2 days at 37oC. The cells were stained with Zombie NIR™ Fixable Viability Kit (BioLegend), followed by analyses by flow cytometry. Live tumor cells were analyzed in FlowJo software. Cytolysis of tumor cells curve was generated by normalized cell counts versus bispecific antibodies concentration using Prism software (GraphPad). Four-parameter non-linear regression analysis was done by Prism software (GraphPad).

[0446] Pan CD3 T cells Mediated Cytotoxicity Assays

[0447] The efficacy of the bispecific antibodies to mediate tumor cell lysis was evaluated in pan CD3 T cells – JeKo-1 co-culture assays. Pan CD3 T cells were enriched from healthy human donor PBMC using EasySep Human T cell Isolation kit (Stemcell Technologies), and were seeded at 75,000 per well along with 10,000 GFP-expressing JeKo-1 (E:T=7.5=1) in the presence of a serial dilutions of CD180-targeting bispecific antibodies for 3 days incubate at

[0448] 37oC. The cells were stained with Zombie NIR™ Fixable Viability Kit (BioLegend), followed by analyses by flow cytometry. Live tumor cells were analyzed in FlowJo software. Cytolysis of tumor cells curve was generated by normalized cell counts versus bispecific antibodies concentration using Prism software (GraphPad). Four-parameter non-linear regression analysis was used to obtain EC50s and maximal killing using Prism software (GraphPad).

[0449] PBMC Mediated Cytotoxicity Assays

[0450] The efficacy of the bispecific antibodies to mediate tumor cell lysis was evaluated in PBMC-tumor cell co-culture assays. PBMC from healthy human donors were added at 150,000-200,000 per well in 96-well cell-repellent plates (Greiner Bio-One), and CellTrace Violet (Invitrogen) - labeled or GFP-luc expressing human tumor cells were seeded at 8,000- 15,000 cells per well, depends on the E:T ratio, in the presence or absence of serial dilution of antibodies and incubated at 37oC. Epcoritamab Research Grade Biosimilar (ProSci, catalog: 10-978) was purchased from ProSci Incorporated. After 3 days, the plate was centrifugated and supernatant was collected and frozen for further cytokine analysis. The cellAttorney Docket No. EVIM-006 / 001WO 339103-2023 pellets were stained with Zombie NIR™ Fixable Viability Kit (BioLegend), followed by Fc receptor blocking and surface marker staining. Flow antibodies again surface cell markers (CD3, CD4, CD8, CD19, CD14, CD33) were purchased from BioLegend and BD Bioscience, and stained cells were analyzed by flow cytometry. Numbers of tumor cells, CD4 T cells, CD8 T cells, B cells, and monocytes were analyzed in FlowJo software. Cytolysis of tumor cells, depletion of B cells and monocytes, CD4 T cells and CD8 T cells proliferation curves were generated by either plotting the cell counts or normalize cell counts versus bispecific antibodies concentration using Prism software (GraphPad). Four-parameter non-linear regression analysis was used to obtain EC50s and maximal killing using Prism software (GraphPad).

[0451] Quantification of cytokine release by Enzyme Linked Immunosorbent Assay (ELISA)

[0452] Cytokine (IFN-Ȗ, IL-2, IL-6) were measured using LEGEND MAX ELISA kit (BioLegend) and LumiGlo Peroxidase Chemiluminescent Substrate Kit (SeraCare) following the manufacturers’ protocols. Luminescence was measured using Ensight plate reader (Perkin Elmer). The cytokine values were interpolated using Excel. Four-parameter non-linear regression analysis was used to obtain EC50s and maximal cytokine release using Prism software (GraphPad).

[0453] CD180 antibody binding capacity measurement of tumor cell lines

[0454] A BD Biosciences FACSymphony A3 instrument was used for analytical flow cytometry. For the CD180 surface expression evaluation, the cell lines were stained with Zombie Violet™ Fixable Viability Kit (BioLegend). Then the cells were incubated with Human TruStain FcX™ Fc blocking solution (BioLegend), followed by staining with either an antibody against CD180 (MHR73-11 clone) or an isotype antibody (MOPC-21) conjugated to APC (Biolegend) to evaluate the target expression on cell surface. The Quantum™ Simply Cellular®anti-Mouse IgG beads (Bangs Laboratories, Inc) were used following the manufacturer’s protocol to determine the CD180 antibody binding capacity of the tumor cell lines. The average and standard deviation of the antibody binding capacity were used when applicable.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0455] CD180 antibody binding capacity measurement of AML blast in AML PBMC

[0456] The frozen AML PBMC was thawed, and the cells were stained with BD Horizon™ Fixable Viability Stain 780. Followed by staining with fluorochrome-labeled flow antibodies against human surface protein including CD45, CD3, CD4, CD8, CD14, CD33, CD20, CD25, CD69, CD123, and CD180 (Biolegend or BD). Within the live single-cell population, the AML blast was defined as CD45+CD33+ cells, B cells were defined as CD45+CD33-CD20+, and monocytes were defined as CD45+CD33-CD14+. The Quantum™ Simply Cellular®anti- Mouse IgG beads (Bangs Laboratories, Inc) were used following the manufacturer’s protocol to determine the CD180 antibody binding capacity on the AML blast, B cells, and monocytes.

[0457] Ex vivo culture of AML PBMC to evaluate bispecific fusion proteins cytotoxicity towards AML blast

[0458] The frozen AML PBMC was thawed and cultured in a 12-well plate, and treated with bispecific fusion proteins at the indicated concentration or control biologic for 6 days. On day 6, the cells were harvested, followed by the staining of the cells with BD Horizon™ Fixable Viability Stain 780 and flow antibody cocktail targeting human surface protein including CD45, CD3, CD4, CD8, CD14, CD33, CD20, CD25, CD69, CD123, and CD180 (Biolegend or BD). Within the live single-cell population, the AML blast was defined as CD45+CD33+ cells, T cells were defined as CD45+CD33-CD3+, CD4 T cells were defined as CD45+CD33- CD3+CD4+ and CD8 T cells were defined as CD45+CD33-CD3+CD8+. The remaining AML blast percentage was normalized compared to the non-targeting control treated wells, and the AML blast reduction = 100% - remaining AML blast compared to non-targeting control treated wells (%). The T cell fold expansion (%), CD4 T cell expansion, and CD8 T cell expansion were normalized to the corresponding non-targeting control treated wells.

[0459] Result and Discussion:

[0460] MHR73-11 is a murine monoclonal antibody that recognizes the human CD180 or RP105 (radioactive protein 105) (Miura et al. Blood. 1998 Oct 15;92(8):2815-22). The complete sequence of the variable heavy chain (SEQ ID NO: 161) and variable light chain (SEQ ID NO: 160) the mouse monoclonal anti-human CD180 antibody, MHR73-11, was determined by de novo protein sequencing using mass spectrometry. Following elucidation of the antibody sequence, we humanized MHR73-11 using CDR grafting approach. In the absence of the known crystal structure available for this antibody, a homology-basedAttorney Docket No. EVIM-006 / 001WO 339103-2023 structural model of the Fv region of MHR73-11 (FIG.3) was utilized to query a database of human antibody frameworks for structural homology and sequence similarity. Figure 3A emphasize on choosing a template based on higher structural and sequence similarity for successful CDR grafting to retain binding to recombinant CD180-MD1 complex over a template with lower structural and sequence similarity (Figure 3B). Murine CDR regions including the Vernier zone residues, the canonical structural residues, and the interface residue were all grafted onto candidate human frameworks. Structural guided back mutations were introduced to eliminate steric clashes and maximize the stem geometry of the CDR regions. The resulting humanized structures were checked for any steric clashes and were resolved by back mutation. Another set of humanized variants were prepared by choosing the closest human germline sequences as framework templates. In this case, the mouse MHR73- 11 structure is mutated to the chosen template sequence and any insertions or deletions were re-built.

[0461] Together, we got a list of humanized MHR73-11 variant sequences for heavy and light chains. These humanized variants were expressed and purified on a small scale as mentioned in Materials and Method section. Purified humanized variants and mouse MHR73- 11 antibodies were tested by sandwich ELISA for binding against recombinant human and cyno CD180-MD1 protein expressed and purified by stable cell line (as mentioned in Material and method section) (Table 17).

[0462] Table 17. Binding of humanized MHR73-11 variants and mouse MHR73-11 (EIP0464) against recombinant human CD180-MD1 protein obtained by ELISA.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0463] Variants that exhibited binding to human CD180-MD1 similar or better than mouse MHR73-11 antibody were then expressed at a larger scale and purified by SEC purification to further characterize their biophysical characterization. These variants were then used to produce CD180 targeted bispecific fusion molecules.

[0464] A schematic of CD180 targeted bispecific fusions described here is represented in FIG.4 and is composed of the following: (1) the CD180 targeting antibody, (2) CD3 binding antibody consisting of previously described SP34 antibody variants (Table 18), (3) knob-in- hole mutations in the heavy chain as previously described in WO2023178357A1, the contents of which are incorporated herein in its entirety, (4) mutations corresponding to set D the enforce light chain heterodimerization (Table 19) as described previously (Ref Platform patent), protein linker, and full length CD58.

[0465] Table 18. Nomenclature and mutations associated with CD3 variants

[0466] Table 19. Light chain mutation set D.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0467] CD180 targeted bispecific and bispecific fusion antibodies were produced in transient Expi293 system. Shown in FIG. 5 is the preparative size exclusion chromatogram of several bispecific fusion proteins following protein A purification. Purified variants were also validated for their integrity and purity by analytical size exclusion chromatography (aSEC), FIGS.5B- 5F.

[0468] The thermal stability of CD180 targeted bispecific fusions were assessed using differential scanning calorimetry (DSC). As shown in FIG. 6 and Table 20, all proteins show first major melting transition between 69-70 degrees Celsius corresponding to the CH2 domain and CH3 domain with knob in hole mutations. A second major melting transition corresponding to the Fab domain can be observed following first transition.

[0469] Table 20: Differential scanning calorimetry analysis for CD180-targeted bispecific fusions. The tabulated Tm values are provided for each variant.

[0470] Mass spectrometry was used to assess the degree of mispairing of peptide chains for each bispecific fusion. The expected mass for a correctly paired bispecific fusion was observed by non-reduced intact antibody mass analysis for EIP1042 (FIG.7A), for EIP1044 (FIG. 7C) and for EIP1056 (FIG. 7E). Four distinct polypeptide chains corresponding to two light chains and two heavy chains of the expected mass were observed upon reduction for EIP1042 (FIG. 7B), for EIP1044 (FIG.7D) and for EIP1056 (FIG.7E).

[0471] The binding for human and cyno CD180-MD1 of CD180-targeted bispecific fusions is summarized in Table 21 and FIGS.8A and 8B.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0472] Table 21. EC50 values for humanized CD180-targeted bispecific fusions against recombinant human and cyno CD180-MD1 complex obtained by ELISA

[0473] Exemplary bispecific antibodies were advanced for further analysis via surface plasma resonance (SPR) for binding to recombinant human CD180-MD1 heterodimer. The kinetics and affinity constants (ka, kd, and KD) are shown in Table 22 for human CD180-MD1 heterodimer.

[0474] Table 22: Biacore surface plasma resonance values for binding of CD180 targeted bispecific fusions to human CD180-MD1 heterodimer complex.

[0475] Murine antibody MHR73-11 (EIP0464) was compared against humanized bispecific MHR73-11 antibodies, EIP0698 and EIP0699 for their binding to human and cyno CD180- MD1 protein complex. As shown in Figure 9A & 9B and Table 23, the humanized bispecific (EIP0698) as well as bispecific fusion molecule (EIP0699) when compared to murine MHR73-11 antibody (EIP0464) show comparable binding affinity to human and cyno CD180-MD1 protein complex. This exhibits the humanized molecules in bispecific fusion format retain the affinity towards the antigen similar to the murine CD180 antibody, MHR73-11.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0476] Table 23. Comparing EC50 values for murine MHR73-11 antibody (EIP0464) and humanized CD180- targeted bispecific (EIP0698) and bispecific fusion (EIP0699) against recombinant human and cyno CD180-MD1 complex obtained by ELISA .

[0477]

[0478] For proof-of-concept studies, we compared MHR73-11 antibody (EIP0464) against G28.8 antibody (EIP0100) (U.S. Patent No.9,260,529.). As shown in Figure 10, MHR73-11 has higher binding affinity to recombinant CD180-MD1 as compared to G28.8.

[0479] Table 24. EC50 for binding of MHR73-11 (EIP0464) antibody against G28.8 (EIP0100) antibody against recombinant human CD180-MD1 obtained by ELISA.

[0480]

[0481] Further, we generated chimeric antibody using G28.8 or MHR73-11 in CD3- bispecific format with and without CD58 fusion by engrafting mouse derived mouse sequences or humanized MHR73-11 as tumor targeting arm in human IgG backbone: EIP0051 is CD3xG28.8, EIP0133 is CD3xMHR73-11, EIP0546 is CD3xB12 (non-targeting control), EIP0553 is CD3xhumanized MHR73-11, EIP0554 is CD3xhumanized MHR73-11- CD58. We evaluated these biologics binding to CD180-expressing cell lines: JeKo-1 (FIG. 11A), Raji (FIG. 11B), and Ramos (FIG. 11C), and consistently we observed mouse MHR73-11 (EIP0133) and humanized MHR73-11 (EIP0553, EIP0664) derived antibodies showed similar binding to the tumor cells while the mouse G28.8 derived bispecific (EIP0051) showed weaker binding. Then we further tested bispecific antibodies in activated T cell-tumor cell co-culture assay to evaluate cytolytic activities (EIP0051, EIP0133), and EIP0133 (MHR73-11-based) showed greater potency than EIP0051 (G28.8-based): JeKo-1 (FIG. 12A), Raji (FIG.12B), and Ramos (FIG.12C). The EIP0133 showed around 5 to 10Attorney Docket No. EVIM-006 / 001WO 339103-2023 fold improvement in tumor killing potency compared to EIP0051 reflected by the ratio of EC50.

[0482] We selected a subset of the B cell malignancies (HT, SU-DHL-10, JeKo-1) and acute myeloid leukemia (MV-4-11) based on the CD180 expression level determined by RNA-seq (CCLE database) and confirmed the surface CD180 expression (MFI) by flow cytometry (Table 25, FIG 13A).

[0483] Table 25. CD180 expression quantified at transcription level (RNA-seq, CCLE database) and at protein level (flow cytometry).

[0484] To evaluate the bispecific fusion biologics binding to CD180-expressing cell lines, we evaluated a series of bispecific fusion antibodies (EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) binding to HT (FIG. 13B) and MV-4-11-GFP-luc (FIG. 13C) cell lines. We observed that all antibodies showed comparable binding curve to cells, but EIP1042, EIP1044, EIP1056 might show relatively stronger binding than EIP1043 and EIP1057 in both cell line models, as reflected by the higher maximal MFI of CD180.

[0485] To determine the CD3 affinity-tuning and CD58 fusion impact on the bispecific antibody’s activity, we generated a series of the CD180-targeting biologics (same tumor antigen targeting arm, various CD3 affinity) and evaluated their functions in pan CD3 T cell- JeKo-1 co-culture (Table 26) and PBMC-JeKo-1 co-culture assays (Table 27). Consistently, the bispecific fusion biologics outperformed the corresponding bispecific: most of the tested biologics achieved 90%+ tumor killing, except EIP0751 (very weak CD3 affinity bispecific) reached 87.2% (pan CD3 T cells) and 75% (PBMC); CD58 fusion bispecific showed greater cytolytic activity as indicated by EC50. The cytokines measurement from PBMC-JeKo-1 co- culture also showed that CD58 fusion bispecific antibodies tend to induce comparable (IL-6) or higher (IFN-Ȗ, IL-2) maximal cytokine production at lower concentrations compared to theAttorney Docket No. EVIM-006 / 001WO 339103-2023 bispecific designs. We could modulate the therapeutic windows of bispecific antibodies by varying CD3 affinity, as defined by the cytotoxicity and cytokine release.

[0486] Table 26. In vitro tumor killing and cytokine release EC50s, maximal killing, and maximal cytokine release in naïve T cells-JeKo-1 co-culture assays.

[0487] Table 27. In vitro tumor killing and cytokine release EC50s, maximal killing, and maximal cytokine release in PBMC-JeKo-1 co-culture assays.Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0488] The activity of the bispecific fusion antibodies (same CD3 arm, different tumor antigen targeting arm, with CD58 fusion; EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) was evaluated in a PBMC-tumor cell line co-culture assay using two model cell lines: HT (B cell malignancies, FIGS. 14A-14G) and MV-4-11 (AML FIGS. 15A-15G). In PBMC-HT co-culture assay, all five antibodies showed comparable performance in tumor cell depletion (FIG.14A), B cells and monocyte reduction (FIGS.14B-14C). When compared with a CD20 targeting bispecific Epcoritamab Research Grade Biosimilar (ProSci, catalog: 10-978), and CD180-targeting biologics showed greater potency in depletion tumor cells and less potency in B cell reduction. No monocyte depletion in Epcoritamab Research Grade Biosimilar (ProSci, catalog: 10-978) -treated conditions was observed.

[0489] We observed similar T cell expansion as a function of bispecific fusion antibodies concentration (FIGS.14D-14E) among all the five variants, and EIP1043 induced relatively lower T cell expansion than other CD180-targeting bispecific antibodies but still higher than Epcoritamab Research Grade Biosimilar (ProSci, catalog: 10-978). CD180-targeing bispecific antibodies induced higher levels of cytokine release (IFN-Ȗ and IL-2, FIGS. 14F- 14G) than Epcoritamab Research Grade Biosimilar (ProSci, catalog: 10-978), and EIP1044 and EIP1056 induced greater IL-2 with higher biologic concentration (FIG.15G). In PBMC- MV-4-11 co-culture experiment, all five variants showed similar potency in tumor cell depletion (FIG. 15A), B cell and monocyte reduction (FIGS. 15B-15C), CD4 and CD8 T cells expansion (FIGS. 15D-15E). EIP1044 and EIP1056 showed a greater level of IFN-Ȗ production than EIP1042, EIP1043, and EIP1057 (FIG. 15F). There was no significant difference in IL-2 induction among CD180-targeting bispecific antibodies (FIG.15G).Attorney Docket No. EVIM-006 / 001WO 339103-2023

[0490] Table 28. CD180 expression on B cell malignancies and AML cell lines

[0491] We measured the antibody binding capacity (ABC) of the CD180 surface expression on various B-cell tumor cell lines and AML cell lines. The average, standard deviation and range of the CD180 ABC were reported in Table 28. Consistently with FIG. 13A, we confirmed the CD180 expression on the surface of B cell malignancies and acute myeloid leukemia.

[0492] Table 29. CD180 expression on AML blast, monocyte, and B cell in AML patients PBMCAttorney Docket No. EVIM-006 / 001WO 339103-2023

[0493] We profiled PBMC (containing AML blast) from multiple AML patients. To characterize the patients, we reported the following information for each patient sample in Table 29: FAB and WHO classification for AML, the clinical treatment for these patients pre- and post-PBMC collection, patients’ clinical responses to the treatment, and standard of care (SoC) response in ex vivo PBMC culture. In Table 29, we summarized the CD180 positive expression frequency and the CD180 antibody binding capacity on AML blast, monocytes, and B cells in AML patients. We observed various CD180 expression frequencies and target expression levels on AML blast within this cohort, and some patients (CTG-2228, CTG-2238, and CTG-2240) showed more than 95+% CD180+ in AML blast. AML blast may show higher CD180 ABC than monocytes and B cells within the same donor, and this indicates the potential of the differential CD180 expression on AML blast than monocytes and B cells.

[0494] Table 30. CD180-targeting bispecific fusion biologics deplete AML blast and

[0495] After we confirmed the CD180 expression on the AML blast, we treated the AML PBMC in ex vivo culture with selected bispecific fusion biologics (EIP0717, EIP0709, EIP0746) and non-targeting control (EIP0614) for 6 days, and we counted the AML blast, T cells, CD4 T cells, and CD8 T cells to understand the biologic-mediated AML blast reduction and T cell expansion. Table 30 shows the results of AML patients CTG-3439: the biologic-mediated AML blast reduction, T cells, CD4 T cells, and CD8 T cells expansion at the indicated biologic concentration. We observed dose-dependent AML blast reduction andAttorney Docket No. EVIM-006 / 001WO 339103-2023 T cell expansion (T cells, CD4 T cells, and CD8 T cells) when treated with EIP0717, EIP0709, and EIP0746, indicating the cytotoxicity of CD180-targeting bispecific fusion biologics in primary AML PBMC samples. OTHER EMBODIMENTS

[0496] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. EVIM-006 / 001WO 339103-2023 CLAIMS What is claimed is:

1. A bispecific antibody comprising a first antigen binding region that binds to CD3 and a second antigen binding region that binds to CD180, wherein the first antigen binding region that binds to CD3 comprises three heavy chain complementarity determining regions (CDRH1, CDRH2, CDRH3) and three light chain complementarity determining regions (CDRL1, CDRL2, CDRL3), wherein a) CDRH1 comprises the amino acid sequence of SEQ ID NO: 30; CDRH2 comprises the amino acid sequence of SEQ ID NO: 34; CDRH3 comprises the amino acid sequence of SEQ ID NO: 37; CDRL1 comprises the amino acid sequence of SEQ ID NO: 42; CDRL2 comprises the amino acid sequence of SEQ ID NO: 43; and CDRL3 comprises the amino acid sequence of SEQ ID NO: 45; and wherein the second antigen binding region that binds to CD180 comprises three heavy chain complementarity determining regions (CDRH1, CDRH2, CDRH3) and three light chain complementarity determining regions (CDRL1, CDRL2, CDRL3): i) CDRH1 comprises the amino acid sequence of SEQ ID NO: 210; CDRH2 comprises the amino acid sequence of SEQ ID NO: 212; CDRH3 comprises the amino acid sequence of SEQ ID NO: 214; CDRL1 comprises the amino acid sequence of SEQ ID NO: 222; CDRL2 comprises the amino acid sequence of SEQ ID NO: 223; and CDRL3 comprises the amino acid sequence of SEQ ID NO:

224.

2. The bispecific antibody of claim 1, wherein the first antigen binding region that binds to CD3 comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein: a) VH comprises the amino acid sequence of SEQ ID NO: 17 and VL comprises the amino acid sequence of SEQ ID NO: 22; and wherein the second antigen binding region that binds to CD180 comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein: i) VH comprises the amino acid sequence of SEQ ID NO: 197 andAttorney Docket No. EVIM-006 / 001WO 339103-2023 VL comprises the amino acid sequence of SEQ ID NO: 196; ii) VH comprises the amino acid sequence of SEQ ID NO: 199 and VL comprises the amino acid sequence of SEQ ID NO: 198; iii) VH comprises the amino acid sequence of SEQ ID NO: 201 and VL comprises the amino acid sequence of SEQ ID NO: 200; iv) VH comprises the amino acid sequence of SEQ ID NO: 203 and VL comprises the amino acid sequence of SEQ ID NO: 202; or v) VH comprises the amino acid sequence of SEQ ID NO: 205 and VL comprises the amino acid sequence of SEQ ID NO:

204.

3. The bispecific antibody of any one of claims 1-2, wherein the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1), and a constant region (CH1) having a constant region 1 domain (CH1H1), a hinge region (H1H), a constant region 2 domain (CH1H2) and a constant region 3 domain (CH1H3); and a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1); and a second heavy chain polypeptide (H2) comprising a variable region (VH2), and a constant region (CH2) having a constant region 1 domain (CH2H1), a hinge region (H2H), a constant region 2 domain (CH2H2) and a constant region 3 domain (CH2H3); and second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2), and wherein i) the amino acid at position 39 (Kabat numbering) of the VH1 is a K and the amino acid at position 38 (Kabat numbering) of the VL1 is a D; ii) the amino acid at position 147 (EU numbering) of the CH1H1is a K and the amino acid at position 131 (EU numbering) of the CL1 is a D; iii) the amino acid at position 173 (EU numbering) of the CH1H1is a C and the amino acid at position 162 (EU numbering) of the CL1 is a C; iv) the amino acid at position 220 (EU numbering) in the H1H is a S and the amino acid at position 214 (EU numbering) of the CL1 is a S; and i) the amino acid at position 39 (Kabat numbering) of the VH2 is a D and the amino acid at position 38 (Kabat numbering) of the VL2 is a K; and ii) the amino acid at position 147 (EU numbering) of the CH2H1 is a D and the amino acid at position 180 (EU numbering) of the CL2 is a R.Attorney Docket No. EVIM-006 / 001WO 339103-2023 4. The bispecific antibody of claim 3, wherein i) the amino acid at position 87 (Kabat numbering) of the VH1 and / or VH2 is a G; and ii) the amino acid at position 45 (Kabat numbering) of the VL1 and / or VL2 is a W.

5. The antibody of any one of claims 3-4, wherein: i) the CH1H3 has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3has a C at position 354 and a W at position 366 (EU numbering); ii) the CH2H3has a C at position 349, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3has a C at position 354 and a W at position 366 (EU numbering); iii) the CH1H3has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH2H3 has a C at position 349 and a W at position 366 (EU numbering); or iv) the CH2H3 has a C at position 354, an S at position 366, an A at position 368 and a V at position 407 (EU numbering); and the CH1H3 has a C at position 349 and a W at position 366 (EU numbering).

6. The antibody of any one of claims 3-5, wherein the amino acid at position 447 (EU numbering) of the CH1H3 and / or of the CH2H3 is deleted.

7. The antibody of any one of claims 3-6, wherein: i) the H1H and / or the H2H has an A at positions 234 and 235 (EU numbering); ii) the H1H and / or the H2H has an A at positions 234, 235 and 237 (EU numbering); or iii) the H1H and / or the H2H has an A at positions 234 and 235 and G at position 329 (EU numbering).

8. The antibody of any one of claims 3-7, wherein i) the CH1H3 and / or the CH2H3 has an A at position 297 (EU numbering); ii) the CH1H3 and / or the CH2H3 has a G at position 297 (EU numbering); orAttorney Docket No. EVIM-006 / 001WO 339103-2023 iii) the CH1H3 and / or the CH2H3 has a S at position 297 (EU numbering).

9. The antibody of any one of claims 3-8, wherein the CH1H3 and / or the CH2H3 has an S at position 331 (EU numbering).

10. The bispecific antibody of any one of claims 1-9, wherein a polypeptide is fused to the N-terminus or the C-terminus of the first heavy chain polypeptide or the second heavy chain polypeptide.

11. The bispecific antibody of claim 10, wherein the polypeptide is fused to the C- terminus of the first heavy chain polypeptide or the C-terminus of the second heavy chain polypeptide.

12. The bispecific antibody of any one of claims 10-11, wherein the polypeptide is fused via a linker peptide.

13. The bispecific antibody of claim 12, wherein the linker peptide comprises the amino acid sequence of SEQ ID NO:

53.

14. The bispecific antibody of any one of claims 10-13, wherein the polypeptide comprises a CD58 or a fragment thereof.

15. The bispecific antibody of claim 14, wherein the CD58 comprises the amino acid sequence of SEQ ID NO:

49.

16. The bispecific antibody of any one of claims 1-15, wherein the bispecific antibody is an IgG1 or an IgG4 antibody.

17. The bispecific antibody of any one of claims 1-16, wherein the bispecific antibody is a monoclonal antibody, a chimeric antibody or a humanized antibody.Attorney Docket No. EVIM-006 / 001WO 339103-2023 18. The bispecific antibody of any one of claims 1-17, wherein the first antigen binding region that binds to CD3 comprises a heavy chain (HC) and a light chain (LC), wherein: a) HC comprises the amino acid sequence of SEQ ID NO: 305 and LC comprises the amino acid sequence of SEQ ID NO: 304; b) HC comprises the amino acid sequence of SEQ ID NO: 309 and LC comprises the amino acid sequence of SEQ ID NO: 308; c) HC comprises the amino acid sequence of SEQ ID NO: 313 and LC comprises the amino acid sequence of SEQ ID NO: 312; d) HC comprises the amino acid sequence of SEQ ID NO: 317 and LC comprises the amino acid sequence of SEQ ID NO: 316; or e) HC comprises the amino acid sequence of SEQ ID NO: 321 and LC comprises the amino acid sequence of SEQ ID NO: 320; and wherein the second antigen binding region that binds to CD180 comprises a heavy chain (HC) and a light chain (LC), wherein: i) HC comprises the amino acid sequence of SEQ ID NO: 303 and LC comprises the amino acid sequence of SEQ ID NO: 302; ii) VH comprises the amino acid sequence of SEQ ID NO: 307 and VL comprises the amino acid sequence of SEQ ID NO: 306; iii) VH comprises the amino acid sequence of SEQ ID NO: 311 and VL comprises the amino acid sequence of SEQ ID NO: 310; iv) VH comprises the amino acid sequence of SEQ ID NO: 315 and VL comprises the amino acid sequence of SEQ ID NO: 314; or v) VH comprises the amino acid sequence of SEQ ID NO: 319 and VL comprises the amino acid sequence of SEQ ID NO:

318.

19. A polynucleotide comprising a nucleic acid sequence encoding the bispecific antibody according to any one of claims 1-17.

20. A vector comprising the polynucleotide of claim 18.Attorney Docket No. EVIM-006 / 001WO 339103-2023 21. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-18, the polynucleotide of claim 19 or the vector of claim 20 and a pharmaceutically acceptable carrier.

22. A method of treating a cancer expressing CD180 in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21.

23. A method of T-cell re-targeting in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21.

24. A method of T-cell activation in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 21.

25. The method of any one of claims 22-24, wherein the subject in need thereof has cancer.

26. The method of any one of claims 22-25, wherein the cancer is a lymphoma or leukemia.

27. The method of any one of claims 22-26, wherein the lymphoma or leukemia is Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), B-cell lymphoma, mantle cell lymphoma, AIDS-related lymphoma, Waldenstrom's Macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), Hairy cell leukemia, chronic myeloblastic leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML).

28. The method of any one of claims 22-27, wherein the NHL is small lymphocytic (SL) NHL, intermediate grade / follicular NHL, intermediate grade diffuse NHL, high gradeAttorney Docket No. EVIM-006 / 001WO 339103-2023 immunoblastic NHL, high grade lymphoblastic NHL, high grade small non-cleaved cell NHL, or bulky disease NHL.

29. The method of any one of claims 22-28, wherein the subject is simultaneously administered or previously administered with a therapeutically effective amount of an additional therapeutic agent.

30. The method of claim 29, wherein the additional therapeutic agent is a CAR-T cell therapy, an immune checkpoint inhibitor, a co-stimulatory ligand or a cytokine.

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