Bispecific antibody fusion molecules targeting CD180 and CD3 and their application methods

Abstract

This 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 a portion thereof. Methods for preparing and using the bispecific antibodies to treat cancers expressing CD180 are described herein. This disclosure also relates to methods and kits for detecting susceptibility to CD180-related disorders, determining their risk, and guiding treatment.

Description

[0001] Related applications

[0002] This application claims priority and benefit to U.S. Provisional Application No. 63 / 584166, filed September 20, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] The merging of sequence lists by reference

[0004] The contents of the electronic sequence list (EVIM_006_001WO_SeqList_ST26.xml; size: 378759 bytes; creation date: September 20, 2024) are incorporated herein by reference in their entirety. Technical Field

[0005] This invention relates to pharmaceutical compositions of bispecific antibodies binding to CD180 and CD3 and their uses. The invention also relates to methods and kits for detecting cancer susceptibility, determining cancer risk, and guiding cancer therapy. Background Technology

[0006] CD180 is a type 1 transmembrane toll-like receptor (TLR) with the highest sequence similarity to TLR4. It was initially discovered as a mouse B lymphocyte activation receptor that protects B cells from radiation and dexamethasone-mediated cell death (Miyake et al., 1995, J. Immunol. 154: 3333–3340; Divanovic et al., 2005, Nat. Immunol. 6: 571–578). Both CD180 and MD-1, the interacting proteins required for CD180 surface expression and function, are present on the surface of B cells and monocytes, but not on the surface of T lymphocytes. It is also present on other myeloid cells in humans and mice with similar expression (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 present in hematologic 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) and chronic lymphocytic leukemia (CLL) (Porakishvili et al., 2005, Br. J. Haematol. 131: 313–319; Edwards et al., 2021, Br. J. Haematol. 131: 313–319). Haematol. 195) and acute myeloid leukemia (AML) (Kramer et al., 2022, Blood 140: 1533–1548; Saito et al., 2010, Sci. Transl. Med. 2), suggesting that CD180 / MD-1 could be a novel target for cancer immunotherapy, as an alternative B-cell lineage antigen in relapsed and / or refractory B-cell malignancies where the loss of CD19 and CD20 is due to CD19 / CD20 targeted therapy, and as a differential opportunity in myeloid malignancies of the monocyte lineage (Marshalek et al., 2022, JCO 40: e19537–e19537; Duell et al., 2024, Blood143: 685–696).

[0007] There is a persistent need for the development of agents that target CD180 and are highly effective and / or safe for human use. In this field, there is a need for agents targeting CD180 for the diagnosis and treatment of CD180-related conditions such as cancer. Furthermore, there is a need for methods and kits for identifying whether subjects with CD180-expressing cancers will respond to such antibody-based therapies. This document provides methods and compositions that meet these needs. Summary of the Invention

[0008] This disclosure provides a bispecific antibody comprising a first antigen-binding region for binding CD3 and a second antigen-binding region for binding CD180, wherein the first antigen-binding region for binding 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 for binding 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: 34; CDRH2 comprises the amino acid sequence of SEQ ID NO: 3 ... CDRH2 comprises the amino acid sequence of SEQ ID NO: 34; CDRH3 comprises the amino acid sequence of SEQ ID NO: 35; CDRL1 comprises the amino acid sequence of SEQ ID NO: 3 The amino acid sequence of SEQ ID NO: 212; CDRH3 contains the amino acid sequence of SEQ ID NO: 214; CDRL1 contains the amino acid sequence of SEQ ID NO: 222; CDRL2 contains the amino acid sequence of SEQ ID NO: 223; and CDRL3 contains the amino acid sequence of SEQ ID NO: 224.

[0009] In some embodiments, the first antigen-binding region binding 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 binding 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: 202. The amino acid sequence of NO: 204.

[0010] In some embodiments, the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), the constant region having a constant region 1 domain (CH1). H1 ), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 The first light chain polypeptide (L1) comprises a variable region (VL1) and a constant region (CL1); the second heavy chain polypeptide (H2) comprises a variable region (VH2) and a constant region (CH2), wherein the constant region has a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 ); and the second light chain polypeptide (L2), which comprises a variable region (VL2) and a constant region (CL2), wherein: i） The amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; ii) CH1 H1 The amino acid at position 147 (EU number) is K, and the amino acid at position 131 (EU number) of CL1 is D; iii) CH1 H1The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; iv) The amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; and i） The amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; and ii) The CH2 H1 The amino acid at position 147 (EU number) is D, and the amino acid at position 180 (EU number) of CL2 is R.

[0011] In some embodiments, the bispecific antibody comprises: i) the amino acid at position 87 (Kabat number) of VH1 and / or VH2 is G; and ii) the amino acid at position 45 (Kabat number) of VL1 and / or VL2 is W.

[0012] In some embodiments, the bispecific antibody comprises: i) the CH1 H3 It has a C at position 349, an S at position 366, an A at position 368, and a V (EU number) at position 407; and the CH2 H3 It has a C at position 354 and a W (EU number) at position 366; ii) the CH2 H3 It has a C at bit 349, an S at bit 366, an A at bit 368, and a V (EU number) at bit 407; and CH1 H3 It has a C at position 354 and a W (EU number) at position 366; iii) CH1 H3 It has a C at position 354, an S at position 366, an A at position 368, and a V (EU number) at position 407; and the CH2 H3 It has a C at position 349 and a W at position 366 (EU number); or iv) the CH2 described. H3 It has a C at bit 354, an S at bit 366, an A at bit 368, and a V (EU number) at bit 407; and CH1 H3 It has a C at position 349 and a W (EU number) at position 366.

[0013] In some embodiments, CH1 H3 and / or CH2 H3The amino acid at position 447 (EU number) is missing.

[0014] In some implementations, i) the H1H and / or H2H have an A at bits 234 and 235 (EU number); ii) the H1H and / or H2H have an A at bits 234, 235 and 237 (EU number); or iii) the H1H and / or H2H have an A at bits 234 and 235 and a G (EU number) at bit 329.

[0015] In some implementations, i) the CH1 H3 and / or CH2 H3 A is present at position 297 (EU number); ii) CH1 H3 and / or CH2 H3 It has G at position 297 (EU number); or CH1 as described in iii) H3 and / or CH2 H3 An S is present at position 297 (EU number). In some embodiments, CH1 H3 and / or CH2 H3 It has an S at position 331 (EU number).

[0016] In some embodiments, the polypeptide is fused to the N-terminus or 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.

[0017] 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 CD58 or a fragment thereof. In some embodiments, the CD58 comprises the amino acid sequence of SEQ ID NO: 49.

[0018] In some embodiments, the bispecific antibody is an IgG1 or IgG4 antibody. In some embodiments, the bispecific antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0019] In some embodiments, the first antigen-binding region binding 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 binding 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: 304; The amino acid sequence of SEQ ID NO: 307, and VL contains the amino acid sequence of SEQ ID NO: 306; iii) VH contains the amino acid sequence of SEQ ID NO: 311, and VL contains the amino acid sequence of SEQ ID NO: 310; iv) VH contains the amino acid sequence of SEQ ID NO: 315, and VL contains the amino acid sequence of SEQ ID NO: 314; or v) VH contains the amino acid sequence of SEQ ID NO: 319, and VL contains the amino acid sequence of SEQ ID NO: 318.

[0020] This disclosure provides a polynucleotide comprising a nucleic acid sequence encoding any of the bispecific antibodies of this disclosure. This disclosure provides a vector comprising any of the polynucleotides of this disclosure. This disclosure provides a pharmaceutical composition comprising any of the bispecific antibodies of this disclosure, any of the polynucleotides of this disclosure, or any of the vectors of this disclosure, and a pharmaceutically acceptable vector.

[0021] This disclosure provides a method for treating CD180-expressing cancer in a subject of need, the method comprising administering a therapeutically effective amount of any pharmaceutical composition of this disclosure. This disclosure also provides a method for T-cell retargeting in a subject of need, the method comprising administering a therapeutically effective amount of any pharmaceutical composition of this disclosure.

[0022] This disclosure provides a method for activating T cells in a subject in need, the method comprising administering a therapeutically effective amount of any pharmaceutical composition of this disclosure.

[0023] In some implementations, the cancer is lymphoma or leukemia.

[0024] In some embodiments, the lymphoma or leukemia is Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), B-cell lymphoma, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML).

[0025] In some embodiments, the NHL is small lymphocyte (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, advanced immune cell NHL, advanced lymphocyte NHL, advanced small non-cleaved cell NHL, or massive lesion NHL.

[0026] In some embodiments, the subject in need has cancer. In some embodiments, the subject is concurrently or previously administered a therapeutically effective amount of an additional therapeutic agent. In some embodiments, the additional therapeutic agent is CAR-T cell therapy, an immune checkpoint inhibitor, a co-stimulatory ligand, or a cytokine. Attached Figure Description

[0027] Figure 1A-1B This is a non-reducing SDS-PAGE analysis of the purified (lane 1) human and cynomolgus monkey CD180-MD1 complex against a standard molecular weight marker (lane 2).

[0028] Figure 2 Size exclusion chromatography was used to analyze the purified human and cynomolgus monkey CD180-MD1 complex.

[0029] Figure 3A The structure comparison shows the Fv domain (light color) of the mouse MHR73-11 antibody and the Fv domain (dark color, PDB ID: 4N90) of a human antibody that is structurally similar to the preferred CDR transplantation template. Figure 3B The structure comparison is shown between the Fv domain (light color) of the mouse MHR73-11 antibody and the Fv domain (dark color, PDB ID: 4YE4) of a human antibody that is structurally not very similar to the mouse MHR73-11 antibody and is not preferred as a CDR transplantation template.

[0030] Figures 4A-4E This is a series of schematic diagrams of CD180-targeting bispecific fusion polymers with charged pair mutations, disulfide bond repositioning, and mortising mutations. The gray-shaded domains represent: the first heavy chain polypeptide (H1), which has a heavy chain variable region (VH1) and a constant region 1 domain (CH1). H1), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 The first light chain polypeptide (L1) contains a variable region (VL1) and a constant region (CL1). The white-shaded domains represent the second heavy chain polypeptide (H2), which contains a variable region (VH2) and a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 The constant region (CH2) of the antigen-binding domain; and the second light chain polypeptide (L2), which contains a variable region (VL2) and a constant region (CL2). The + and - symbols between the antigen-binding domains indicate charged pair mutations. CH1 H1 Between the CL1 and CH2 domains H1 The lines between the CL2 domain and the CL2 domain represent disulfide bonds, with solid lines representing intrinsic disulfide bonds and dashed lines representing relocated disulfide bonds. CH1 H3 With CH2 H3 The protrusions and depressions between the domains represent mortising mutations. Charge pair mutations, disulfide bond repositioning, and mortising mutations provide increased heterodimerization of the heavy and light chains, which is beneficial for the production and purification of the bispecific antibodies of this disclosure. Figure 4E The description includes anti-CD3 arm, anti-CD180 arm, and fusion into CH1. H3 A schematic diagram of an exemplary bispecific antibody fusion molecule of the CD58 peptide of the structural domain.

[0031] Figure 5A The prepared chromatography diagram is shown, obtained by tandem purification (protein A elution and size exclusion chromatography) of CD180-targeting bispecific fusion expressed in transient expi293 cells. Figure 5B-5F It is a series of graphs depicting the chromatograms obtained from the analytical size exclusion chromatography of CD180-targeting bispecific fusion antibody. Figure 5B EIP1042 is shown. Figure 5C EIP1043 is shown. Figure 5D EIP1044 is shown. Figure 5E EIP1056 is shown. Figure 5F EIP1057 is shown.

[0032] Figure 6 This is a differential scanning calorimetry analysis of the CD180-targeted bispecific fusion polymer. Samples in PBS were heated from 25°C to 95°C in increments of 1°C / min on a nanoDSC system, and the data were analyzed using a Nanoanalyzer provided by the supplier.

[0033] Figures 7A-7FThis is a series of figures depicting mass spectrometric analysis of the intact and reduced masses of representative CD180-targeted bispecific fusions using Waters' Xevo TQ-XS triple quadrupole mass spectrometer. Figure 7A The intact mass of EIP1042 was determined after PNGAse F deglycosylation under non-reducing conditions and separation using reversed-phase C18 column chromatography. Figure 7B This is a reduction quality analysis of the heavy and light chains of EIP1042, determined after rapid PNGAse F deglycosylation under reducing conditions and separation using reversed-phase C18 column chromatography. Figure 7C The intact mass of EIP1044 was determined after PNGAse F deglycosylation under non-reducing conditions and separation using reversed-phase C18 column chromatography. Figure 7D This is a reduction quality analysis of the heavy and light chains of EIP1044, determined after rapid PNGAse F deglycosylation under reducing conditions and separation using reversed-phase C18 column chromatography. Figure 7E The intact mass of EIP1056 was determined after PNGAse F deglycosylation under non-reducing conditions and separation using reversed-phase C18 column chromatography. Figure 7F This is a reduction quality analysis of the heavy and light chains of EIP1056, determined after rapid PNGAse F deglycosylation under reducing conditions and separation using reversed-phase C18 column chromatography.

[0034] Figures 8A-8B These are a series of graphs obtained by ELISA, depicting the binding of the light chain-paired bispecific fusion antibody to recombinant CD180-MD1. Figure 8A The binding with human CD180-MD1 was demonstrated. Figure 8B The binding with CD180-MD1 of cynomolgus monkeys was shown.

[0035] Figures 9A-9B These are a series of graphs obtained by ELISA, depicting the binding of MHR73-11, humanized bispecific and bispecific fusion antibodies to recombinant CD180-MD1. Figure 9A The binding with human CD180-MD1 was demonstrated. Figure 9B The binding with CD180-MD1 of cynomolgus monkeys was shown.

[0036] Figure 10 The graph, obtained via ELISA, depicts the binding of G28.8 and MHR73-11 antibodies to recombinant human CD180-MD1.

[0037] Figure 11 A-11C is a series of diagrams depicting the binding of CD180-targeted biologics to various B-cell tumor cell lines. Figure 11 A shows JeKo-1, Figure 11 B shows Raji, Figure 11 C shows Ramos. EIP0051 is CD3xG28.8, EIP0133 is CD3xMHR73-11, EIP0546 is CD3xB12 (non-targeted control), EIP0553 is CD3x humanized MHR73-11, and EIP0554 is CD3x humanized MHR73-11-CD58.

[0038] Figure 12 A-12C is a series of line graphs depicting the cytotoxicity mediated by a biologic targeting CD180 in an activated T cell-tumor cell co-culture assay. Figure 12 A shows JeKo-1, Figure 12 B shows Raji, Figure 12 C represents Ramos. EIP0051 is CD3xG28.8, EIP0133 is CD3xCD180 (MHR73-11), and EIP0209 is a non-targeted control. The EC50 table summarizes the EC50 and EC50 ratio of cytotoxicity for each biologic.

[0039] Figure 13 A-13C is a series of diagrams depicting CD180 expression and binding to biologics in cell lines. Figure 13 A shows the CD180 levels on various cell lines (HT, SU-DHL-10, MV-4-11, and JeKo-1) and isotype controls. Figure 13 B illustrates the binding of a biological agent to HT. Figure 13 C shows the binding of the biological agent to MV-4-11.

[0040] Figure 14 A-14G is a series of figures depicting the functional assessment of bispecific fusion antibodies (EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) compared to a control (EIP0614, non-targeted control) in a tumor cell and PBMC co-culture assay. CellTrace Violet-labeled HT tumor cells and PBMCs (at a 15:1 E:T ratio) were treated for 3 days with serially diluted bispecific fusion antibodies. Cell counts or percentages were calculated by measuring cell counts by flow cytometry. Figure 14 A illustrates cell lysis of tumor cells mediated by a bispecific fusion antibody. Figure 14 B indicates B cell depletion. Figure 14 C represents a monocyte. Figure 14 D shows CD4 T cell expansion. Figure 14 E shows CD8 T cell expansion. Figure 14 F shows the release of IFN-γ. Figure 14G shows IL-2 release.

[0041] Figure 15 A-15G is a series of figures depicting the functional assessment of bispecific fusion antibodies (EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) compared to a control (EIP0614, non-targeted control) in a tumor cell and PBMC co-culture assay. GFP-expressing MV-4-11 tumor cells and PBMCs (at a 15:1 E:T ratio) were treated for 3 days with serially diluted bispecific fusion antibodies. Cell counts or percentages were calculated by flow cytometry. Figure 15 A illustrates cell lysis of tumor cells mediated by a bispecific fusion antibody. Figure 15 B indicates B cell depletion. Figure 15 C represents a monocyte. Figure 15 D shows CD4 T cell expansion. Figure 15 E shows CD8 T cell expansion. Figure 15 F shows the release of IFN-γ. Figure 15 G shows IL-2 release. Detailed Implementation

[0042] T-cell retargeting (or T-cell redirection) bispecific antibodies are a novel class of therapeutic agents capable of recruiting T cells to tumor cells and inducing tumor-specific (but MHC-independent) activation of T-cell effector activity. This disclosure relates to 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 the CD180 antigen to T cells. This targeting design promotes T-cell recruitment and brings them into close contact with CD180-expressing cells, leading to the formation of immune synapses, local T-cell activation, and subsequent destruction of target cells (e.g., but not limited to cancer cells) via perforin and granzymes released from T-cell cytotoxic granules into the target cells.

[0043] Since the CD3 binding affinity of bispecific antibodies for T cell retargeting is crucial for T cell recruitment, this invention also relates to the generation of a group of antibodies exhibiting different binding affinities. The affinity of the CD3 arm of the bispecific antibody can significantly modify the functional activity of the bispecific antibody. Therefore, bispecific antibodies with different affinities are ideal and advantageous.

[0044] Furthermore, the bispecific antibodies disclosed herein may contain cytokine or co-stimulatory molecule fusion peptides, which act as antagonists to inhibit or block detrimental interactions or as agonists 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 superior to conventional CD3 bispecific antibodies or tumor-targeting co-stimulatory receptor agonists, which fail to optimally activate T cells and induce (or promote) T cell dysfunction. Therefore, cytokines and / or co-stimulatory fusion peptides are beneficial for enhancing the therapeutic potential of bispecific antibodies. In some embodiments, the co-stimulatory molecule of the anti-CD180 / anti-CD3 bispecific antibody disclosed herein is CD58 or a fragment thereof.

[0045] The bispecific or multispecific T-cell retargeting agents disclosed herein possess drug-like properties of human monoclonal antibodies. Furthermore, T-cell retargeting bispecific and multispecific antibodies are superior to other existing therapies (e.g., CAR-T therapy) because they provide an off-the-shelf product with high safety profiles (e.g., alleviating cytokine release syndrome and reducing stress signaling levels leading to T-cell dysfunction) and the potential for dose titration and escalation.

[0046] Antibody composition and structure

[0047] This disclosure provides an antibody comprising the following structural domains: a) a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), wherein the constant region has a constant region 1 domain (CH1). H1 ), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 a) 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), wherein the constant region has a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 ); and a second light chain polypeptide (L2), which comprises a variable region (VL2) and a constant region (CL2). A schematic diagram of the antibody structure disclosed herein is shown in Figures 4A-4E middle.

[0048] As used herein, the term "antibody" refers to immunoglobulin (Ig) molecules and the immunoactive portion of immunoglobulin molecules, i.e., molecules containing antigen-binding sites that specifically bind to antigens (resulting in an immune response). "Specific binding," "resulting in an immune response," or "targeting" means that the antibody reacts with one or more antigenic determinants of a desired antigen and does not react with other peptides or with much lower affinity (K). d > 10 -6 Antibodies are bound together. Antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, and chimeric antibodies. Antibodies can originate from recombinant sources and / or be produced in transgenic animals.

[0049] The basic structural unit of an antibody is known to be a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, primarily responsible for effector function.

[0050] Generally, antibody molecules obtained from humans involve any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another due to the nature of the heavy chains present in the molecules. Some classes also have subclasses, such as IgG1, IgG2, IgG4, etc. Furthermore, in humans, the light chain may be a κ chain or a λ chain. Therefore, in one embodiment, the antibody disclosed herein is an IgG antibody.

[0051] Antibodies can be purified using known techniques, such as affinity chromatography of protein A or protein G, which primarily provides IgG fractions of the immune serum. Subsequently, or optionally, a specific antigen or epitope that is a target of the desired immunoglobulin can be immobilized on a column to purify the immune-specific antibody by immunoaffinity chromatography. Immunoglobulin purification 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).

[0052] As used herein, the term "antibody fragment" is intended to include, but is not limited to, Fv, Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimers, mini-antibodies, bispecific antibodies and their multimers, 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 bonds, thereby generating Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab', and F(ab')2, scFv, dsFv, ds-scFv, dimers, mini-antibodies, bispecific antibodies, bispecific antibody fragments, and other fragments can also be synthesized using recombinant techniques.

[0053] The techniques can be adapted to produce single-chain antibodies specific to the antigenic proteins of this disclosure (see, for example, U.S. Patent No. 4,946,778). Furthermore, the methods can be adapted to construct Fab expression libraries (see, for example, Huse et al., 1989 Science 246:1275-1281) to allow for rapid and efficient identification of monoclonal Fab fragments with desired specificity to proteins or their derivatives, fragments, analogs, or homologs.

[0054] As used herein, the term "epitope" refers to a site on an antigen that is recognized by the antibodies and fragments disclosed herein. The term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins. Epitope determinants typically consist of chemically active surface clusters of molecules such as amino acid or sugar side chains and generally possess specific three-dimensional structural features and specific charge characteristics. An antibody is said to specifically bind to an antigen when its dissociation constant is <1 micromolar (e.g., <100 nM, preferably <10 nM, more preferably <1 nM).

[0055] A bispecific antibody is an antibody that has binding specificity to at least two different antigens. This 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., a disease-related antigen).

[0056] Antibodies with more than two valentities have also been envisioned. For example, trispecific antibodies could be prepared. (Tutt et al., J. Immunol. 147:60 (1991)).

[0057] Antibody variants

[0058] In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve heavy chain heterodimerization, light chain heterodimerization, binding affinity, and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics (e.g., light chain heterodimerization, heavy chain heterodimerization, antigen binding).

[0059] Amino acids can be grouped according to common side chain properties: (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Alkaline (positively charged): His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatics: Trp, Tyr, Phe.

[0060] Functional variants of the antibody or antigen-binding fragments described herein are also covered in this disclosure. As used herein, the term "functional variant" includes modifications or chemical equivalents of the amino acid and nucleic acid sequences disclosed herein that perform substantially the same function as the polypeptide or nucleic acid molecule disclosed herein in a substantially similar manner. For example, functional variants of the polypeptides disclosed herein include, but are not limited to, conserved amino acid substitutions.

[0061] As used herein, "conserved amino acid substitution" refers to the substitution of one amino acid residue for another, thereby changing an amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and size). Variants of peptides also include additions and deletions of the peptide sequences disclosed herein. Furthermore, variant nucleotide sequences include their analogues and derivatives. Variants of the binding proteins disclosed herein include proteins that bind the same antigen or epitope to said binding protein.

[0062] In some embodiments, the charged amino acid residues are naturally occurring amino acids or non-naturally occurring amino acids. In some embodiments, the naturally occurring charged amino acid residues are arginine, lysine, histidine, glutamic acid, or aspartic acid.

[0063] Light chain and heavy chain replacement variants

[0064] To generate a substantially homogeneous population of bispecific antibodies with the correct pairing of heavy and light chains (i.e., homologous pairing or heterodimerization of the 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) exhibits a strong preference for binding to the first light chain polypeptide (L1) relative to the second light chain polypeptide (L2); and the second heavy chain polypeptide (H2) exhibits a strong preference for binding to the second light chain polypeptide (L2) relative to the first light chain polypeptide (L1). Furthermore, the first heavy chain polypeptide (H1) and the second heavy chain polypeptide (H2) show a stronger preference for heterodimerization (i.e., heavy chain heterodimerization) compared to homodimerization.

[0065] This article provides antibody variants with one or more amino acid substitutions. Exemplary substitution mutation sites include charged substitution pairs shown in Tables 1-6.

[0066] Table 1. κ light and heavy chains – constant domain mutation pairs

[0067] All location information is reported using the EU numbering scheme.

[0068] Wild type (WT) indicates the naturally occurring amino acid at a specified location.

[0069] The charge pairs of negatively and positively charged residues can be reversed between heavy and light chains, where D or E (negatively charged) is replaced by K or R (positively charged), and K or R (positively charged) of the homologous chain is replaced by D or E (negatively charged).

[0070] Table 2. κ light and heavy chains – variable domain mutation pairs

[0071] All location information is reported using the Kabat numbering scheme.

[0072] Wild type (WT) indicates the naturally occurring amino acid at a specified location.

[0073] The charge pairs of negatively and positively charged residues can be reversed between heavy and light chains, where D or E (negatively charged) is replaced by K or R (positively charged), and K or R (positively charged) of the homologous chain is replaced by D or E (negatively charged).

[0074] Table 3. λ light and heavy chains – constant domain mutation pairs

[0075] All location information is reported using the EU numbering scheme.

[0076] The charge pairs of negatively and positively charged residues can be reversed between heavy and light chains, where D or E (negatively charged) is replaced by K or R (positively charged), and K or R (positively charged) of the homologous chain is replaced by D or E (negatively charged).

[0077] Table 4. λ light and heavy chains – variable domain mutation pairs

[0078] All location information is reported using the Kabat numbering scheme.

[0079] The charge pairs of negatively and positively charged residues can be reversed between heavy and light chains, where D or E (negatively charged) is replaced by K or R (positively charged), and K or R (positively charged) of the homologous chain is replaced by D or E (negatively charged).

[0080] Table 5. κ constant chain cysteine ​​mutation pairs

[0081] All location information is reported using the EU numbering scheme.

[0082] Table 6. λ constant chain cysteine ​​mutation pairs

[0083] All location information is reported using the EU numbering scheme.

[0084] In some embodiments, the anti-CD3 / anti-CD180 bispecific antibody of this disclosure comprises an antibody variant comprising substitutions in a variable heavy chain, variable light chain, constant heavy chain, or constant light chain domain of the anti-CD3 or anti-CD180 arm or both, as discussed in PCT application number PCT / US2023 / 064728 and PCT publication number WO2019 / 104075A1, which are incorporated herein by reference in their entirety.

[0085] In some embodiments, the antibody variant comprises a "light chain pairing mutation set D" which includes the following substitutions: a) the heavy and light chains of the anti-CD3 arm comprise the following amino acids: i） The amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; ii) CH1 H1 The amino acid at position 147 (EU number) is K, and the amino acid at position 131 (EU number) of CL1 is D; iii) CH1 H1The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; iv) The amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; and b) the heavy and light chains of the anti-CD180 arm contain the following amino acids: i） The amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; and ii) The CH2 H1 The amino acid at position 147 (EU number) is D, and the amino acid at position 180 (EU number) of CL2 is R.

[0086] In some embodiments, the antibody variant comprises a "light chain pairing mutation set D" which includes the following substitutions: a) the heavy and light chains of the anti-CD180 arm comprise the following amino acids: i） The amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; ii) CH1 H1 The amino acid at position 147 (EU number) is K, and the amino acid at position 131 (EU number) of CL1 is D; iii) CH1 H1 The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; iv) The amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; and b) the heavy and light chains of the anti-CD3 arm contain the following amino acids: i） The amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; and ii) The CH2 H1 The amino acid at position 147 (EU number) is D, and the amino acid at position 180 (EU number) of CL2 is R.

[0087] The antibodies disclosed herein may need to be modified in terms of effector function to enhance, for example, their efficacy in treating diseases and disorders. For instance, cysteine ​​residues may be introduced into the Fc region, allowing interchain disulfide bonds to form therein. The resulting homodimeric antibodies may possess improved internalization capacity and / or enhanced complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). (See Caron et al., J Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922. (1992)). Alternatively, an antibody with a dual Fc region may be designed, thereby enhancing complement cleavage and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0088] Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0089] In some implementations, the antibody variant includes an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbers of residues) of the Fc region.

[0090] In some implementations, changes are made in the Fc region that result in alterations (i.e., improvements or reductions) in C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0091] Antibodies with prolonged half-lives and improved binding to the neonatal Fc receptor (FcRn) (which is responsible for transferring maternal IgG 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.). These antibodies contain one or more replaced Fc regions that improve the binding of the Fc region to the FcRn. Such Fc variants include variants with substitutions at one or more of the following 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, for example, a substitution at residue 434 of the Fc region (US Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); US Patent No. 5,648,260; US Patent No. 5,624,821; and WO 94 / 29351.

[0092] In some embodiments, the antibody may include substitution mutations in the Fc region that reduce effector function. In some embodiments, the substitution mutation is a glycosylation-free site mutation. In some embodiments, the glycosylation-free site mutation is located at amino acid residue 297, and amino acid substitutions at residues 234, 235, 265, and 331 (EU number) to disrupt the Fc receptor binding interface. In some embodiments, the glycosylation-free site mutation reduces the effector function of the antibody.

[0093] In some embodiments, i) the H1H and / or H2H has an A at bits 234 and 235 (EU number); or ii) the H1H and / or H2H has an A at bits 234, 235, and 237 (EU number); iii) the H1H and / or H2H has an A at bits 234 and 235 and a G (EU number) at bit 329. In some embodiments, i) the CH1 H3 and / or CH2 H3 At position 297 (EU number), there is A, ii) CH1 H3 and / or CH2 H3 It has G at position 297 (EU number); or CH1 as described in iii) H3 and / or CH2 H3 An S is present at position 297 (EU number). In some embodiments, CH1 H3 and / or CH2H3 It has an S at position 331 (EU number).

[0094] The use of a pestle and mortar as a method for generating multispecific antibodies is well known in the art. See U.S. Patent No. 5,731,168 to Genentech, March 24, 1998; PCT Publication No. WO2009089004 to Amgen, published July 16, 2009; and U.S. Patent Publication No. 20090182127 to Novo Nordisk A / S, published July 16, 2009. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26(6):649-658 and Kontermann (2005) Acta Pharacol. Sin., 26:1-9.

[0095] A "protrusion" refers to at least one amino acid side chain that protrudes from the interface of the first polypeptide, thus allowing it to be positioned in a compensating cavity at the adjacent interface (i.e., the interface of the second polypeptide) to stabilize the heteropolymeric antibody, thereby favoring heteropolymeric antibody formation, for example, compared to homopolymeric antibody formation. The protrusion can be present in the original interface or introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the first polypeptide is altered to encode the protrusion. To achieve this, the nucleic acid encoding at least one "original" amino acid residue in the interface of the first polypeptide is replaced with at least one "input" amino acid residue encoding a side chain larger in volume than the original amino acid residue. It should be understood that more than one original residue and a corresponding input residue can exist. The upper limit for the number of original residues that can be replaced is the total number of residues in the interface of the first polypeptide.

[0096] The preferred input residues for forming the protrusion are typically naturally occurring amino acid residues, preferably selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Tryptophan and tyrosine are most preferred. In one embodiment, the original residues for forming the protrusion have small side chain volumes, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. CH1 H3 or CH2 H3 Exemplary amino acid substitutions used to form protrusions in the domain include, but are not limited to, the T366W substitution.

[0097] A “cavity” refers to at least one amino acid side chain that recesses from the interface of the second polypeptide, thus accommodating a corresponding protrusion on the adjacent interface of the first polypeptide. The cavity can be present in the original interface or introduced synthetically (e.g., by altering the nucleic acid encoding the interface). Typically, the nucleic acid encoding the interface of the second polypeptide is altered to encode 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 “input” amino acid residue whose side chain volume is smaller than that of the original amino acid residue. It should be understood that more than one original residue and a corresponding input residue can exist. The upper limit for the number of original residues replaced is the total number of residues in the interface of the second polypeptide. The side chain volumes of various amino acid residues are shown in Table 1 above. Preferred input residues for forming the cavity are typically naturally occurring amino acid residues, preferably selected from alanine (A), serine (S), threonine (T), and valine (V). Serine, alanine, or threonine are most preferred. In one embodiment, the original residues for forming the cavity have a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. CH1 H3 or CH2 H3 Exemplary amino acid substitutions used to create cavities in the domain include, but are not limited to, substitutions for T366S, L368A, Y407A, Y407T, and Y407V. In some embodiments, the mortarsal hapten contains a T366W substitution, and the acetabulum hapten contains a T366S / L368A / Y407V substitution.

[0098] In some embodiments, the antibody variant comprises the following substitution: the CH1 H3 It has a C at position 349, an S at position 366, an A at position 368, and a V (EU number) at position 407; and the CH2 H3 It has a C at position 354 and a W (EU number) at position 366.

[0099] In some embodiments, the antibody variant comprises the following substitution: the CH2 H3 It has a C at bit 349, an S at bit 366, an A at bit 368, and a V (EU number) at bit 407; and CH1 H3 It has a C at position 354 and a W (EU number) at position 366.

[0100] In some embodiments, the antibody variant comprises the following substitution: the CH1 H3 It has a C at position 354, an S at position 366, an A at position 368, and a V (EU number) at position 407; and the CH2 H3It has a C at position 349 and a W (EU number) at position 366.

[0101] In some embodiments, the antibody variant comprises the following substitution: the CH2 H3 It has a C at bit 354, an S at bit 366, an A at bit 368, and a V (EU number) at bit 407; and CH1 H3 It has a C at position 349 and a W (EU number) at position 366.

[0102] Differentiation antigen cluster 3 (CD3)

[0103] This disclosure provides a bispecific antibody comprising a first antigen-binding domain that binds to CD3 expressed on T cells and a second antigen-binding domain that binds to the CD180 antigen on the surface of cancer cells. The antibody of this invention can be used, for example, to treat cell proliferation disorders (e.g., cancers expressing CD180) or to slow their progression.

[0104] Unless otherwise stated, the term “differentiation antigen cluster 3” or “CD3” as used herein refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkeys) and rodents (e.g., mice and rats), including, for example, CD3ε, CD3γ, CD3α, and CD3β chains. CD3 is a cell surface complex expressed on T cells and binding to the T cell receptor. The CD3 complex is required for the activation of CD8+ and CD4+ T lymphocytes. It is formed by three distinct but highly related chains: one CD3γ chain, one CD3δ chain, and two CD3ε chains, which bind together to form CD3ε / γ heterodimers and CD3ε / δ heterodimers. These two CD3 heterodimers, together with the T cell receptor (TCR) and the signal transduction ζ chain homodimer, form the T cell receptor complex.

[0105] The term encompasses “full-length” unprocessed CD3 (e.g., unprocessed or unmodified CD3ε or CD3γ), as well as any form of CD3 produced in cells through processing. The term also encompasses naturally occurring CD3 variants, including, for example, splice variants or allelic variants. For example, CD3 includes the human CD3ε protein (NCBI RefSeqNo. NP_000724), which is 207 amino acids in length.

[0106] In some embodiments, the present invention provides isolated antibodies that bind to CD3. In some embodiments, the present invention provides antibodies that bind to CD3ε. In some cases, the anti-CD3ε antibody binds to a human CD3ε peptide or a cynomolgus monkey (cynomolgus monkey) CD3ε peptide. In some cases, the human CD3 peptide or the cynomolgus monkey CD3 peptide is, respectively, the human CD3ε peptide MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID (SEQ ID NO: 419) or cynomolgus monkey CD3ε polypeptide MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIVDICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQQDLYSGLNQRRI (SEQ ID NO: 420). In some cases, the anti-CD3 antibody binds to an epitope within the CD3ε (e.g., human CD3ε) fragment consisting of amino acid residues 1-26 or 1-27 of human CD3ε (SEQ ID NO: 419).

[0107] A useful method for identifying antibodies that can be targeted for mutated residues or regions is called "alanine scanning mutation," as described by Cunningham and Wells (1989). ScienceAs described in 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions may be introduced at the amino acid position to confirm functional sensitivity to the initial substitution. Optionally or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

[0108] In some embodiments, the "SP34" anti-CD3ε antibody is subjected to an alanine scan mutation to produce the affinity-modified anti-CD3ε antibody of the present invention.

[0109] In some embodiments, the first antigen-binding region that binds to CD3 comprises either the VH or VL sequences listed in Table 7. In Table 7, underlined sequences are CDR sequences according to Kabat, and bold sequences are CDR sequences according to Chothia.

[0110] In some embodiments, the first antigen-binding region includes a VH region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the amino acid sequence set forth in any one of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, or 21.

[0111] In some embodiments, the first antigen-binding region includes a VL region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the amino acid sequence set forth in any of SEQ ID NO: 22, 23, 24, 25, 26, 27, and 28.

[0112] In some embodiments, the anti-CD3 antibody of this disclosure comprises: a) a heavy chain variable region (VH) including a VH complementarity-determining region 1 (VH... CDR1 VH complementarity determination region 2 (VH CDR2 ) and VH complementarity determinant 3 (VH CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL CDR1 VL Complementary Determinant Region 2 (VL) CDR2 ) and VL complementarity determinant 3 (VLCDR3 Tables 8 and 9 provide exemplary CDR sequences for the anti-CD3 antibodies presented herein.

[0113] Table 7. Anti-CD3 variable heavy chain and variable light chain domains

[0114] Table 8. Anti-CD3 heavy chain CDR

[0115] Table 9. Anti-CD3 light chain CDR

[0116] This document provides a bispecific antibody comprising a first antigen-binding domain that binds to 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), the constant region having a constant region 1 domain (CH1). H1 ), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 The first light chain polypeptide (L1) comprises a variable region (VL1) and a constant region (CL1); the second heavy chain polypeptide (H2) comprises a variable region (VH2) and a constant region (CH2), wherein the constant region has a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 ); and the second light chain polypeptide (L2), which contains a variable region (VL2) and a constant region (CL2).

[0117] In some embodiments, the bispecific antibody of this disclosure includes a first antigen-binding domain (e.g., CD3-binding) comprising either the VH1 or VL1 sequences listed in Table 7. In Table 7, underlined sequences are CDR sequences according to Kabat, and bold sequences are CDR sequences according to Chothia.

[0118] In some embodiments, the bispecific antibody of this disclosure includes a first antigen-binding domain (e.g., binding to CD3ε), which includes: a) a heavy chain variable region (VH1) containing a VH complementarity-determining region 1 (VH1). CDR1 VH complementarity determinant region 2 (VH1) CDR2 ) and VH complementarity determinant 3 (VH1)CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL1) CDR1 VL Complementary Determinant Region 2 (VL1) CDR2 ) and VL complementarity determinant 3 (VL1) CDR3 Tables 8 and 9 provide exemplary CDR sequences for the anti-CD3 antibodies presented herein.

[0119] In some embodiments, the bispecific antibody comprises any of the anti-CD3 antibodies disclosed herein. Exemplary anti-CD3 antibodies of the present invention include CD3-O1, 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 an "CD3-O1" anti-CD3 antibody. In some embodiments, the bispecific antibody comprises an "CD3-A6" anti-CD3 antibody.

[0120] In some embodiments, the binding affinity (K0) of the first antigen-binding region of the bispecific antibody to CD3 (e.g., human CD3ε (e.g., SEQ ID NO: 419) or cynomolgus monkey CD3ε (e.g., SEQ ID NO: 422)) is [not specified]. D The binding affinity is from about 0.001 nM to about 5000 nM. In some embodiments, the binding affinity with CD3 is from about 0.001 nM to about 0.01 nM, from about 0.01 to about 0.1 nM, or from about 0.1 to about 1 nM. In some embodiments, the binding affinity is from about 1 nM to about 1000 nM, from about 10 nM to about 1000 nM, or from about 100 nM to about 1000 nM.

[0121] 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.

[0122] 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.

[0123] 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, 9 nM, 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.

[0124] CD180 antigen

[0125] This disclosure provides a bispecific antibody comprising a first antigen-binding domain that binds to CD3 expressed on T cells and a second antigen-binding domain that binds to the CD180 antigen on the surface of cancer cells. The antibody of this invention can be used, for example, to treat cell proliferation disorders (e.g., cancers expressing CD180) or to slow their progression.

[0126] CD180 is a type 1 transmembrane toll-like receptor (TLR) with the highest sequence similarity to TLR4. It was initially discovered as a mouse B lymphocyte activation receptor that protects B cells from radiation and dexamethasone-mediated cell death (Miyake et al., 1995, J. Immunol. 154: 3333–3340; Divanovic et al., 2005, Nat. Immunol. 6: 571–578). Both CD180 and MD-1, the interacting proteins required for CD180 surface expression and function, are present on the surface of B cells and monocytes, but not on the surface of T lymphocytes. It is also present on other myeloid cells in humans and mice with similar expression (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 present in hematologic 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) and chronic lymphocytic leukemia (CLL) (Porakishvili et al., 2005, Br. J. Haematol. 131: 313–319; Edwards et al., 2021, Br. J. Haematol. 131: 313–319). Haematol. 195) and acute myeloid leukemia (AML) (Kramer et al., 2022, Blood 140: 1533–1548; Saito et al., 2010, Sci. Transl. Med. 2), suggesting that CD180 / MD-1 could be a novel target for cancer immunotherapy, as an alternative B-cell lineage antigen in relapsed and / or refractory B-cell malignancies where the loss of CD19 and CD20 is due to CD19 / CD20 targeted therapy, and as a differential opportunity in myeloid malignancies of the monocyte lineage (Marshalek et al., 2022, JCO 40: e19537–e19537; Duell et al., 2024, Blood143: 685–696).

[0127] The terms “CD180” and “CD180 antigen” are used interchangeably herein and include any variant, allotype, and species homolog of human CD180, whether naturally expressed in cells or expressed on cells transfected with the CD180 gene. The binding of the antibody of the present invention to the CD180 antigen mediates the killing of CD180-expressing cells (e.g., tumor cells) by inactivating CD180. The killing of said CD180-expressing cells may occur through one or more of the following mechanisms: cell death / apoptosis induction, ADCC, and CDC.

[0128] In some embodiments, the present invention provides isolated antibodies that bind to CD180. In some cases, the anti-CD3180 antibody binds to a human CD180 peptide or a cynomolgus monkey (cyno) CD180 peptide. In some cases, the CD180 peptide is either a human CD180 peptide (SEQ ID NO: 417) or a cynomolgus monkey CD180 peptide.(SEQ ID NO: 418).

[0129] The term "anti-CD180 antibody" according to the present invention refers to an antibody that specifically binds to the CD180 antigen. Depending on the binding characteristics and biological activity of the anti-CD180 antibody to the CD180 antigen, two types of anti-CD180 antibodies (type I and type II anti-CD180 antibodies) can be distinguished according to Cragg, MS et al., Blood 103 (2004) 2738-2743 and Cragg, MS et al., Blood 101 (2003) 1045-1052.

[0130] In some cases, CD180 can be expressed at low copy numbers on target cells (e.g., tumor cells). For example, in some cases, CD180 is expressed or present at fewer than 35,000 copies per target cell. In some embodiments, the low copy number of cell surface CD180 is present at 100 to 35,000 copies per target cell, 100 to 30,000 copies per target cell, 100 to 25,000 copies per target cell, 100 to 20,000 copies per target cell, 100 to 15,000 copies per target cell, 100 to 10,000 copies per target cell, 100 to 5,000 copies per target cell, 100 to 2,000 copies per target cell, 100 to 1,000 copies per target cell, or 100 to 500 copies per target cell. The copy number of cell surface CD180 can be determined, for example, using a standard Scratchcard plot.

[0131] This disclosure provides a bispecific antibody comprising a first antigen-binding region that binds to CD3 and a second binding region that binds to CD180.

[0132] In some embodiments, the bispecific antibody has the following structure: a first heavy chain polypeptide (H1) comprising a variable region (VH1) and a constant region (CH1), the constant region having a constant region 1 domain (CH1). H1 ), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 The first light chain polypeptide (L1) comprises a variable region (VL1) and a constant region (CL1); the second heavy chain polypeptide (H2) comprises a variable region (VH2) and a constant region (CH2), wherein the constant region has a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 ); and the second light chain polypeptide (L2), which contains a variable region (VL2) and a constant region (CL2).

[0133] For example, the second binding region for CD180 may be derived from the binding region of an anti-CD180 antibody. Exemplary anti-CD180 antibodies include, but are not limited to, the MHR73-11 and G28-8 monoclonal antibodies (BD Biosciences, ThermoScientific, Sigma-Aldrich). Anti-CD180 antibody G28.8 (also known as G28-8) is disclosed in U.S. Patent No. 9,260,529, the entire contents of which are incorporated herein by reference.

[0134] In some embodiments, the second antigen-binding region that binds to CD180 comprises either the VH or VL sequences listed in Table 10. In Table 10, the underlined sequences are CDR sequences according to Kabat, and the bold sequences are CDR sequences according to Chothia.

[0135] In some embodiments, the second antigen-binding region binding to CD180 includes a VH region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the 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.

[0136] In some embodiments, the second antigen-binding region binding to CD180 includes a VL region having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the 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.

[0137] In some embodiments, the second binding region associated with CD180 includes: a) a heavy chain variable region (VH) comprising a VH complementarity determination region 1 (VH... CDR1 VH complementarity determination region 2 (VH CDR2 ) and VH complementarity determinant 3 (VH CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL CDR1 VL Complementary Determinant Region 2 (VL) CDR2 ) and VL complementarity determinant 3 (VL CDR3 Tables 11 and 12 provide exemplary CDR sequences for anti-CD180 antibodies.

[0138] Table 10. Variable Heavy Chain and Variable Light Chain Domains of CD180

[0139] Table 11. Anti-CD180 heavy chain CDR

[0140] Table 12. Anti-CD180 light chain CDR

[0141] In some embodiments, the second antigen-binding region comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 210. CDR1 ), ii) the VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 212 CDR2 (iii) contains the VH complementarity-determining region 3 (VH) of the amino acid sequence of SEQ ID NO: 214. CDR3 ).

[0142] In some embodiments, the second antigen-binding region comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 210. CDR1 ), ii) the VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 216 CDR2 (iii) contains the VH complementarity-determining region 3 (VH) of the amino acid sequence of SEQ ID NO: 214. CDR3 ).

[0143] In some embodiments, the second antigen-binding region comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 218. CDR1 ), ii) the VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 220 CDR2 (iii) contains the VH complementarity-determining region 3 (VH) of the amino acid sequence of SEQ ID NO: 221. CDR3 ).

[0144] In some embodiments, the second antigen-binding region comprises: i) a light chain variable region (VL) comprising i) a VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 222. CDR1 ), ii) the VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 223 CDR2 (iii) contains the VL complementarity-determining region 3 (VL) of the amino acid sequence of SEQ ID NO: 224. CDR3 ).

[0145] In some embodiments, the second antigen-binding region comprises: i) a light chain variable region (VL) comprising i) a VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 222. CDR1 ), ii) the VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 225 CDR2 (iii) contains the VL complementarity-determining region 3 (VL) of the amino acid sequence of SEQ ID NO: 224. CDR3 ).

[0146] In some embodiments, the second antigen-binding region comprises: i) a light chain variable region (VL) comprising i) a VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 430. CDR1 ), ii) the VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 431 CDR2 (iii) contains the VL complementarity-determining region 3 (VL) of the amino acid sequence of SEQ ID NO: 432. CDR3 ).

[0147] In some embodiments, the second antigen-binding region comprises: i) a light chain variable region (VL) comprising i) a VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 433. CDR1 ), ii) the VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 434. CDR2 (iii) contains the VL complementarity-determining region 3 (VL) of the amino acid sequence of SEQ ID NO: 435. CDR3 ).

[0148] In some embodiments, the second antigen-binding region comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 210. CDR1 ), ii) the VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 212 CDR2 (iii) contains the VH complementarity-determining region 3 (VH) of the amino acid sequence of SEQ ID NO: 214. CDR3 ); and b) the light chain variable region (VL), which includes i) the VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 222. CDR1 ), ii) the VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 223 CDR2 (iii) contains the VL complementarity-determining region 3 (VL) of the amino acid sequence of SEQ ID NO: 224.CDR3 ).

[0149] In some embodiments, the second antigen-binding region comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 210. CDR1 ), ii) the VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 216 CDR2 (iii) contains the VH complementarity-determining region 3 (VH) of the amino acid sequence of SEQ ID NO: 214. CDR3 ); and b) the light chain variable region (VL), which includes i) the VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 222. CDR1 ), ii) the VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 225 CDR2 (iii) contains the VL complementarity-determining region 3 (VL) of the amino acid sequence of SEQ ID NO: 224. CDR3 ).

[0150] In some embodiments, the second antigen-binding region comprises: a) a heavy chain variable region (VH) comprising i) a VH complementarity-determining region 1 (VH) containing the amino acid sequence of SEQ ID NO: 210. CDR1 ), ii) the VH complementarity-determining region 2 (VH) containing the amino acid sequence of SEQ ID NO: 216 CDR2 (iii) contains the VH complementarity-determining region 3 (VH) of the amino acid sequence of SEQ ID NO: 214. CDR3 ); and b) the light chain variable region (VL), which includes i) the VL complementarity-determining region 1 (VL) containing the amino acid sequence of SEQ ID NO: 222. CDR1 ), ii) the VL complementarity-determining region 2 (VL) containing the amino acid sequence of SEQ ID NO: 223 CDR2 (iii) contains the VL complementarity-determining region 3 (VL) of the amino acid sequence of SEQ ID NO: 224. CDR3 ).

[0151] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 197 and a VL region containing the amino acid sequence shown in SEQ ID NO: 196.

[0152] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 199 and a VL region containing the amino acid sequence shown in SEQ ID NO: 198.

[0153] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 201 and a VL region containing the amino acid sequence shown in SEQ ID NO: 200.

[0154] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 203 and a VL region containing the amino acid sequence shown in SEQ ID NO: 202.

[0155] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 205 and a VL region containing the amino acid sequence shown in SEQ ID NO: 204.

[0156] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 161 and a VL region containing the amino acid sequence shown in SEQ ID NO: 160.

[0157] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 165 and a VL region containing the amino acid sequence shown in SEQ ID NO: 164.

[0158] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 167 and a VL region containing the amino acid sequence shown in SEQ ID NO: 166.

[0159] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 169 and a VL region containing the amino acid sequence shown in SEQ ID NO: 168.

[0160] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 171 and a VL region containing the amino acid sequence shown in SEQ ID NO: 170.

[0161] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 173 and a VL region containing the amino acid sequence shown in SEQ ID NO: 172.

[0162] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 175 and a VL region containing the amino acid sequence shown in SEQ ID NO: 174.

[0163] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 177 and a VL region containing the amino acid sequence shown in SEQ ID NO: 176.

[0164] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 179 and a VL region containing the amino acid sequence shown in SEQ ID NO: 178.

[0165] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 181 and a VL region containing the amino acid sequence shown in SEQ ID NO: 180.

[0166] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 183 and a VL region containing the amino acid sequence shown in SEQ ID NO: 182.

[0167] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 185 and a VL region containing the amino acid sequence shown in SEQ ID NO: 184.

[0168] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 187 and a VL region containing the amino acid sequence shown in SEQ ID NO: 186.

[0169] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 189 and a VL region containing the amino acid sequence shown in SEQ ID NO: 188.

[0170] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 191 and a VL region containing the amino acid sequence shown in SEQ ID NO: 190.

[0171] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 193 and a VL region containing the amino acid sequence shown in SEQ ID NO: 192.

[0172] In some embodiments, the second antigen-binding region comprises a VH region containing the amino acid sequence shown in SEQ ID NO: 195 and a VL region containing the amino acid sequence shown in SEQ ID NO: 194.

[0173] Exemplary bispecific antibodies that bind to CD3 and CD180

[0174] This document provides a bispecific antibody comprising a first antigen-binding domain that binds to a first antigen (e.g., CD3ε) and a second antigen-binding domain that binds to 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), the constant region having a constant region 1 domain (CH1). H1 ), hinge region (H1H), constant region 2 structural domain (CH1) H2 ) and constant region 3 structural domain (CH1) H3 The first light chain polypeptide (L1) comprises a variable region (VL1) and a constant region (CL1); the second heavy chain polypeptide (H2) comprises a variable region (VH2) and a constant region (CH2), wherein the constant region has a constant region 1 domain (CH2). H1 ), hinge region (H2H), constant region 2 structural domain (CH2) H2 ) and constant region 3 structural domain (CH2 H3 ); and the second light chain polypeptide (L2), which contains a variable region (VL2) and a constant region (CL2).

[0175] In some embodiments, the bispecific antibody of this disclosure includes a first antigen-binding domain (e.g., binding to CD3ε), which includes: a) a heavy chain variable region (VH1) containing a VH complementarity-determining region 1 (VH1). CDR1 VH complementarity determinant region 2 (VH1) CDR2 ) and VH complementarity determinant 3 (VH1) CDR3 ); and b) light chain variable region (VL), which contains VL complementarity determination region 1 (VL1) CDR1 VL Complementary Determinant Region 2 (VL1) CDR2 ) and VL complementarity determinant 3 (VL1) CDR3 ); and a second antigen-binding domain (e.g., binding to CD180), which includes: a) a heavy chain variable region (VH2) containing VH complementarity-determining region 1 (VH2 CDR1VH complementarity determinant region 2 (VH2) CDR2 ) and VH2 complementarity determinant 3 (VH2 CDR3 ); and b) the light chain variable region (VL2), which contains the VL complementarity determination region 1 (VL2). CDR1 VL Complementary Determinant Region 2 (VL2) CDR2 ) and VL complementarity determinant 3 (VL2) CDR3 Tables 8 and 9 provide exemplary CDR sequences for the anti-CD3ε antibody provided herein. Tables 11 and 12 provide exemplary CDR sequences for the anti-CD180 antibody provided herein.

[0176] In some embodiments, the bispecific antibody of this disclosure comprises a first antigen-binding domain (e.g., binding to CD3ε) containing either of the VH1 and VL1 sequences listed in Table 7 and a second antigen-binding domain (e.g., binding to CD180) containing either of the VH2 and VL2 sequences listed in Table 10.

[0177] In some embodiments, the bispecific antibody of this disclosure comprises a first heavy chain polypeptide (H1) and a first light chain polypeptide (L1); and a second heavy chain polypeptide (H2) and a second light chain polypeptide (L2), comprising any of the sequences listed in Tables 13 and 16. Italicized sequences are heavy chain variable regions and light chain variable regions. Underlined sequences are according to Kabat's CDR, and bold sequences are according to Chothia's CDR.

[0178] In some embodiments, the bispecific antibody provided herein comprises an H1 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the amino acid sequence listed in Tables 13 and 16. In some embodiments, the H1 comprises a C-terminal lysine (K) residue.

[0179] In some embodiments, the bispecific antibody provided herein comprises an L1 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the amino acid sequence listed in Tables 13 and 16.

[0180] In some embodiments, the bispecific antibody provided herein comprises H2 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the amino acid sequences listed in Tables 13 and 16. In some embodiments, H1 comprises a C-terminal lysine (K) residue.

[0181] In some embodiments, the bispecific antibody provided herein comprises an L2 having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity with the sequences listed in Tables 13 and 16.

[0182] In some embodiments, the amino acid sequence of H1 is numbered according to SEQ ID NO: 229. In some embodiments, the amino acid sequence of L1 is numbered according to SEQ ID NO: 228. In some embodiments, the amino acid sequence of H2 is numbered according to SEQ ID NO: 227. In some embodiments, the amino acid sequence of L2 is numbered according to SEQ ID NO: 226.

[0183] Table 13. Exemplary bispecific antibodies binding CD3ε and CD180

[0184] The exemplary CD3ε x CD180 bispecific antibodies of the present invention include EIP1870, EIP1871, EIP1872, EIP1873, EIP1874, EIP0696, EIP0706, EIP0710, EIP0698, EIP0708, EIP0745 and EIP0751.

[0185] Bispecific antibodies EIP1870, EIP1871, EIP0872, EIP1873, EIP1874, and EIP0698 contain a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 30. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0186] The bispecific antibody EIP0696 contains a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0187] The bispecific antibody EIP0706 contains a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence of SEQ ID NO: 44 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0188] The bispecific antibody EIP0710 contains a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 39 CDR3; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0189] The bispecific antibody EIP0708 contains a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0190] The bispecific antibody EIP0745 contains a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0191] The bispecific antibody EIP0751 contains a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0192] Bispecific antibodies EIP1870, EIP1871, EIP1872, EIP1873, EIP1874, EIP0696, EIP0706, EIP0710, EIP0698, EIP0708, EIP0745, and EIP0751 contain a second antigen-binding domain that binds to CD180, which contains VH2, said VH2 containing the amino acid sequence having SEQ ID NO: 210. CDR1 VH2 having the amino acid sequence SEQ ID NO: 212 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 214 CDR3 ; and VL2, said VL2 comprising the amino acid sequence having SEQ ID NO: 222. CDR1 VL2 with the amino acid sequence SEQ ID NO: 223 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 224 CDR3 .

[0193] In some embodiments, the bispecific antibody EIP1870 comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 197, and VL2 containing the amino acid sequence of SEQ ID NO: 196.

[0194] In some embodiments, the bispecific antibody EIP1870 comprises H1 containing the amino acid sequence of SEQ ID NO: 229, L1 containing the amino acid sequence of SEQ ID NO: 228, H2 containing the amino acid sequence of SEQ ID NO: 227, and L2 containing the amino acid sequence of SEQ ID NO: 226.

[0195] In some embodiments, the bispecific antibody EIP1871 comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 199, and VL2 containing the amino acid sequence of SEQ ID NO: 198.

[0196] In some embodiments, the bispecific antibody EIP1871 comprises H1 containing the amino acid sequence of SEQ ID NO: 233, L1 containing the amino acid sequence of SEQ ID NO: 232, H2 containing the amino acid sequence of SEQ ID NO: 231, and L2 containing the amino acid sequence of SEQ ID NO: 230.

[0197] In some embodiments, the bispecific antibody EIP1872 comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 201, and VL2 containing the amino acid sequence of SEQ ID NO: 200.

[0198] In some embodiments, the bispecific antibody EIP1872 comprises H1 containing the amino acid sequence of SEQ ID NO: 237, L1 containing the amino acid sequence of SEQ ID NO: 236, H2 containing the amino acid sequence of SEQ ID NO: 235, and L2 containing the amino acid sequence of SEQ ID NO: 234.

[0199] In some embodiments, the bispecific antibody EIP1873 comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0200] In some embodiments, the bispecific antibody EIP1873 comprises H1 containing the amino acid sequence of SEQ ID NO: 241, L1 containing the amino acid sequence of SEQ ID NO: 240, H2 containing the amino acid sequence of SEQ ID NO: 239, and L2 containing the amino acid sequence of SEQ ID NO: 238.

[0201] In some embodiments, the bispecific antibody EIP1874 comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 205, and VL2 containing the amino acid sequence of SEQ ID NO: 204.

[0202] In some embodiments, the bispecific antibody EIP1874 comprises H1 containing the amino acid sequence of SEQ ID NO: 245, L1 containing the amino acid sequence of SEQ ID NO: 244, H2 containing the amino acid sequence of SEQ ID NO: 243, and L2 containing the amino acid sequence of SEQ ID NO: 242.

[0203] In some embodiments, the bispecific antibody EIP0696 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0204] In some embodiments, the bispecific antibody EIP0696 comprises H1 containing the amino acid sequence of SEQ ID NO: 249, L1 containing the amino acid sequence of SEQ ID NO: 248, H2 containing the amino acid sequence of SEQ ID NO: 247, and L2 containing the amino acid sequence of SEQ ID NO: 246.

[0205] In some embodiments, the bispecific antibody EIP0706 comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VL1 containing the amino acid sequence of SEQ ID NO: 27, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0206] In some embodiments, the bispecific antibody EIP0706 comprises H1 containing the amino acid sequence of SEQ ID NO: 257, L1 containing the amino acid sequence of SEQ ID NO: 256, H2 containing the amino acid sequence of SEQ ID NO: 255, and L2 containing the amino acid sequence of SEQ ID NO: 254.

[0207] In some embodiments, the bispecific antibody EIP0710 comprises VH1 containing the amino acid sequence of SEQ ID NO: 16, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0208] In some embodiments, the bispecific antibody EIP0710 comprises H1 containing the amino acid sequence of SEQ ID NO: 265, L1 containing the amino acid sequence of SEQ ID NO: 264, H2 containing the amino acid sequence of SEQ ID NO: 263, and L2 containing the amino acid sequence of SEQ ID NO: 262.

[0209] In some embodiments, the bispecific antibody EIP0698 comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0210] In some embodiments, the bispecific antibody EIP0698 comprises H1 containing the amino acid sequence of SEQ ID NO: 273, L1 containing the amino acid sequence of SEQ ID NO: 272, H2 containing the amino acid sequence of SEQ ID NO: 271, and L2 containing the amino acid sequence of SEQ ID NO: 270.

[0211] In some embodiments, the bispecific antibody EIP0708 comprises VH1 containing the amino acid sequence of SEQ ID NO: 18, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0212] In some embodiments, the bispecific antibody EIP0708 comprises H1 containing the amino acid sequence of SEQ ID NO: 281, L1 containing the amino acid sequence of SEQ ID NO: 280, H2 containing the amino acid sequence of SEQ ID NO: 279, and L2 containing the amino acid sequence of SEQ ID NO: 278.

[0213] In some embodiments, the bispecific antibody EIP0745 comprises VH1 containing the amino acid sequence of SEQ ID NO: 19, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0214] In some embodiments, the bispecific antibody EIP0745 comprises H1 containing the amino acid sequence of SEQ ID NO: 289, L1 containing the amino acid sequence of SEQ ID NO: 288, H2 containing the amino acid sequence of SEQ ID NO: 287, and L2 containing the amino acid sequence of SEQ ID NO: 286.

[0215] In some embodiments, the bispecific antibody EIP0751 comprises VH1 containing the amino acid sequence of SEQ ID NO: 20, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0216] In some embodiments, the bispecific antibody EIP0751 comprises H1 containing the amino acid sequence of SEQ ID NO: 297, L1 containing the amino acid sequence of SEQ ID NO: 296, H2 containing the amino acid sequence of SEQ ID NO: 295, and L2 containing the amino acid sequence of SEQ ID NO: 294.

[0217] Any of the bispecific antibodies shown in Table 13 above can be further modified by replacing any of the anti-CD3ε antigen-binding regions with any of the anti-CD3ε binding regions shown in Tables 7-9. For example, the anti-CD3ε antigen-binding region of the bispecific antibody “EIP1224” can be replaced with any of the anti-CD3ε binding regions shown in Tables 7-9 to produce the bispecific antibody of the present invention. Exemplary antibodies are shown in Table 13.

[0218] Fusion peptide

[0219] This article provides an antibody (e.g., a monospecific antibody or a bispecific antibody) having a fusion peptide fused to the N-terminus or C-terminus of a first heavy chain polypeptide or a second heavy chain polypeptide.

[0220] Key to the initial T cell response is the ability of T cells to detect foreign and mutated proteins via their T cell receptors. This response, commonly referred to as Signal 1 (S1) of T cell activation, occurs when the T cell receptor binds to a cell displaying a foreign or mutated protein fragment or antigen in a specific protein complex called major histocompatibility complex I (MHCI). Activation of the T cell receptor itself is an activation and autoregulation of the T cell. Strong binding of the TCR to the MHCI complex leads to chronic activation of the TCR. This form of signaling is associated with T cells that respond to self-antigens. T cells are programmed to inactivate upon experiencing this form of activation. T cells with a weaker but sufficiently activated TCR undergo acute signal transduction and have the potential to remain active and differentiate into memory T cells. This is becoming an important consideration in the design of T cell therapeutics.

[0221] T cell cytokine activation, commonly referred to as signal 3, is important in the transition of T cells from a non-dividing state to a rapidly dividing state or from one phenotypic state to another. T cell cytokine receptors bind to cytokines produced by both immune and non-immune cells, and depending on the cytokines and the state of the T cell upon receiving cytokine signals, can induce cell proliferation, maintain viability, or induce T cell differentiation into specific cellular states suitable for sustained activation or inactivation after infection.

[0222] One example is the transformation that naive cells undergo through cytokines, which can induce the proliferation of naive T cells and promote their differentiation into memory T cells. Exemplary cytokines include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, and IL-21.

[0223] Activation of co-stimulatory receptors, known as signal 2, provides the background-specific intercellular enhancement for T activation. The most recognized form of co-stimulation occurs when T cells interact with activated antigen-presenting cells via the T cell co-stimulatory receptor CD28 with CD80 and CD86 ligands present on APCs. These interactions can “elicit” specific T cells carrying T cell receptors that are responsive to pathogens or oncoproteins.

[0224] Less understood are the co-stimulatory responses induced at sites of infection and malignancy. This includes co-stimulation via CD2 and NKG2D receptors, which are responsive to ligands such as CD58 and UL16-binding proteins (e.g., ULBP2 / 5 / 6) induced in immune cells and epithelial cells after viral infection. These signals not only provide enhancement of T cell activation but also demonstrate that the lethal effector activity of T cells is targeted at single-cell precision. Although many co-stimulatory receptors have been identified, the specific contextual importance of each receptor and the impact of multiple co-stimulatory receptors signaling simultaneously remain largely unknown. This is an area that greatly advances our understanding of T cell biology and opens up possibilities for the development of novel tumor-targeting T cell therapies.

[0225] Co-stimulatory ligands include, but are not limited to, CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, or ICOSLG.

[0226] CD58 is superior to other costimulatory ligands because it is the primary costimulatory pathway available at the tumor site. This is because tumor-infiltrating T lymphocytes often lose the expression of other costimulatory receptors such as CD28, or because tumor cells do not adequately activate T cells due to their low immunogenicity, thus limiting the potential of inducible costimulatory receptors such as 41BB.

[0227] As previously described, the anti-CD3ε antibody of this disclosure induces varying levels of T cell receptor activation, leading to changes in T cell viability and cytokine production. Therefore, the fusion of the co-stimulatory ligand CD58 with the anti-CD3ε bispecific antibody provides integrated co-stimulatory T cell activation to achieve optimal T cell activation.

[0228] In some embodiments, the bispecific antibody has a peptide fused to the N-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the first heavy chain polypeptide (H1). In some embodiments, the bispecific antibody has a polypeptide fused to the N-terminus of the second heavy chain polypeptide (H2). In some embodiments, the bispecific antibody has a peptide fused to the C-terminus of the second heavy chain polypeptide (H2). Exemplary peptides include, but are not limited to, IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, or portions thereof. Exemplary peptides include, but are not limited to, CD48, CD58, CD86, TNFSF9, OX40L, 4-1BBL, GITL, CD70, CD80, MR1, TNFSF4, ICOSL, ICOSLG, or portions thereof. Exemplary peptide sequences fused to the bispecific antibody include, but are not limited to, those listed in Tables 14.1 and 14.2.

[0229] Table 14.1. Exemplary fusion peptide sequences

[0230] Table 14.2. Exemplary fusion peptide sequences

[0231] In some embodiments, the peptide is directly fused to the bispecific antibody. In some embodiments, the peptide is indirectly fused via a linker. In some embodiments, the bispecific antibody fused to the peptide includes a linker sequence. Exemplary linker sequences include, but are not limited to, those listed in Tables 15.1 and 15.2.

[0232] Table 15.1. Exemplary Connector Sequences

[0233] Table 15.2. Exemplary connector sequences

[0234] In some embodiments, the CD3 x CD180 bispecific antibody of the present invention has a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using linker-1 (SEQ ID NO: 52). In some embodiments, the CD3 x CD180 bispecific antibody of the present invention has a CD58v fusion peptide (SEQ ID NO: 52) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1). Fusion peptide (SEQ ID NO: 50).

[0235] In some embodiments, the CD3 x CD180 bispecific antibody of the present invention has a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1) using linker-2 (SEQ ID NO: 53). In some embodiments, the CD3 x CD180 bispecific antibody of the present invention has a CD58v fusion peptide (SEQ ID NO: 53) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1). Fusion peptide (SEQ ID NO: 50).

[0236] In some embodiments, the CD3 x CD180 bispecific antibody of the present invention has a CD58 fusion peptide (SEQ ID NO: 49) indirectly fused to 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 antibody of the present invention has a CD58v fusion peptide (SEQ ID NO: 54) indirectly fused to the C-terminus of the first heavy chain polypeptide (H1). Fusion peptide (SEQ ID NO: 50).

[0237] Exemplary CD3 x CD180 bispecific antibodies with CD58 fusion are shown in Table 16.

[0238] Table 16. Exemplary bispecific antibodies with C-terminal CD58 fusion peptide binding to CD3ε and CD180

[0239] Exemplary CD3ε x CD180 bispecific antibodies with C-terminal fusion peptides of the present invention include EIP1042, EIP1043, EIP1044, EIP1056, EIP1057, EIP0716, EIP0707, EIP0717, EIP0699, EIP0709, EIP0746 and EIP0752.

[0240] Bispecific antibodies EIP1042, EIP1043, EIP1044, EIP1056, EIP1057, and EIP0699, which contain a CD58 fusion peptide, comprise a first antigen-binding domain that binds to CD3ε, and this domain contains VH1, wherein the VH1 comprises the amino acid sequence having SEQ ID NO: 30. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0241] The bispecific antibody EIP0716, containing a CD58 fusion peptide, comprises a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0242] The bispecific antibody EIP0707, containing a CD58 fusion peptide, comprises a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 comprising the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 37 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence of SEQ ID NO: 44 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 45 CDR3 .

[0243] The bispecific antibody EIP0717, containing a CD58 fusion peptide, comprises a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 39 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0244] The bispecific antibody EIP0709, containing a CD58 fusion peptide, comprises a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 comprising the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 35 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 38 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0245] The bispecific antibody EIP0746, containing a CD58 fusion peptide, comprises a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 containing the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 36 CDR2 and VH1 having the amino acid sequence of SEQ ID NO: 40 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0246] The bispecific antibody EIP0752, containing a CD58 fusion peptide, comprises a first antigen-binding domain that binds to CD3ε, which includes VH1, said VH1 comprising the amino acid sequence having SEQ ID NO: 29. CDR1 VH1 with the amino acid sequence SEQ ID NO: 34 CDR2 and VH1 with the amino acid sequence of SEQ ID NO: 41 CDR3 ; and VL1, said VL1 comprising the amino acid sequence having SEQ ID NO: 42. CDR1 VL1 with the amino acid sequence SEQ ID NO: 43 CDR2 and VL1 with the amino acid sequence of SEQ ID NO: 47 CDR3 .

[0247] Bispecific antibodies EIP1042, EIP1043, EIP1044, EIP1056, EIP1057, EIP0716, EIP0707, EIP0717, EIP0699, EIP0709, EIP0746, and EIP0752, which contain a CD58 fusion peptide, comprise a second antigen-binding domain that binds to CD180, and this domain contains VH2, wherein VH2 comprises the amino acid sequence having SEQ ID NO: 210. CDR1 VH2 having the amino acid sequence SEQ ID NO: 212 CDR2 and VH2 having the amino acid sequence of SEQ ID NO: 214 CDR3 ; and VL2, said VL2 comprising the amino acid sequence having SEQ ID NO: 222. CDR1 VL2 with the amino acid sequence SEQ ID NO: 223 CDR2 and VL2 having the amino acid sequence of SEQ ID NO: 224 CDR3 .

[0248] In some embodiments, the bispecific antibody EIP1042 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 197, and VL2 containing the amino acid sequence of SEQ ID NO: 196.

[0249] In some embodiments, the bispecific antibody EIP1042 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 305, L1 containing the amino acid sequence of SEQ ID NO: 304, H2 containing the amino acid sequence of SEQ ID NO: 303, and L2 containing the amino acid sequence of SEQ ID NO: 302.

[0250] In some embodiments, the bispecific antibody EIP1043 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 199, and VL2 containing the amino acid sequence of SEQ ID NO: 198.

[0251] In some embodiments, the bispecific antibody EIP1043 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 309, L1 containing the amino acid sequence of SEQ ID NO: 308, H2 containing the amino acid sequence of SEQ ID NO: 307, and L2 containing the amino acid sequence of SEQ ID NO: 306.

[0252] In some embodiments, the bispecific antibody EIP1044 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 201, and VL2 containing the amino acid sequence of SEQ ID NO: 200.

[0253] In some embodiments, the bispecific antibody EIP1044 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 313, L1 containing the amino acid sequence of SEQ ID NO: 312, H2 containing the amino acid sequence of SEQ ID NO: 311, and L2 containing the amino acid sequence of SEQ ID NO: 310.

[0254] In some embodiments, the bispecific antibody EIP1056 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0255] In some embodiments, the bispecific antibody EIP1056 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 317, L1 containing the amino acid sequence of SEQ ID NO: 316, H2 containing the amino acid sequence of SEQ ID NO: 315, and L2 containing the amino acid sequence of SEQ ID NO: 314.

[0256] In some embodiments, the bispecific antibody EIP1057 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 205, and VL2 containing the amino acid sequence of SEQ ID NO: 204.

[0257] In some embodiments, the bispecific antibody EIP1057 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 321, L1 containing the amino acid sequence of SEQ ID NO: 320, H2 containing the amino acid sequence of SEQ ID NO: 319, and L2 containing the amino acid sequence of SEQ ID NO: 318.

[0258] In some embodiments, the bispecific antibody EIP0716 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0259] In some embodiments, the bispecific antibody EIP0716 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 329, L1 containing the amino acid sequence of SEQ ID NO: 328, H2 containing the amino acid sequence of SEQ ID NO: 327, and L2 containing the amino acid sequence of SEQ ID NO: 326.

[0260] In some embodiments, the bispecific antibody EIP0707 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 13, VL1 containing the amino acid sequence of SEQ ID NO: 27, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0261] In some embodiments, the bispecific antibody EIP0707 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 337, L1 containing the amino acid sequence of SEQ ID NO: 336, H2 containing the amino acid sequence of SEQ ID NO: 335, and L2 containing the amino acid sequence of SEQ ID NO: 334.

[0262] In some embodiments, the bispecific antibody EIP0717 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 16, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0263] In some embodiments, the bispecific antibody EIP0717 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 345, L1 containing the amino acid sequence of SEQ ID NO: 344, H2 containing the amino acid sequence of SEQ ID NO: 343, and L2 containing the amino acid sequence of SEQ ID NO: 342.

[0264] In some embodiments, the bispecific antibody EIP0699 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 17, VL1 containing the amino acid sequence of SEQ ID NO: 22, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0265] In some embodiments, the bispecific antibody EIP0699 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 354, L1 containing the amino acid sequence of SEQ ID NO: 353, H2 containing the amino acid sequence of SEQ ID NO: 352, and L2 containing the amino acid sequence of SEQ ID NO: 351.

[0266] In some embodiments, the bispecific antibody EIP0709 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 18, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0267] In some embodiments, the bispecific antibody EIP0709 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 363, L1 containing the amino acid sequence of SEQ ID NO: 362, H2 containing the amino acid sequence of SEQ ID NO: 361, and L2 containing the amino acid sequence of SEQ ID NO: 360.

[0268] In some embodiments, the bispecific antibody EIP0746 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 19, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0269] In some embodiments, the bispecific antibody EIP0746 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 372, L1 containing the amino acid sequence of SEQ ID NO: 371, H2 containing the amino acid sequence of SEQ ID NO: 370, and L2 containing the amino acid sequence of SEQ ID NO: 369.

[0270] In some embodiments, the bispecific antibody EIP0752 having the CD58 fusion peptide comprises VH1 containing the amino acid sequence of SEQ ID NO: 20, VL1 containing the amino acid sequence of SEQ ID NO: 26, VH2 containing the amino acid sequence of SEQ ID NO: 203, and VL2 containing the amino acid sequence of SEQ ID NO: 202.

[0271] In some embodiments, the bispecific antibody EIP0752 having the CD58 fusion peptide comprises H1 containing the amino acid sequence of SEQ ID NO: 381, L1 containing the amino acid sequence of SEQ ID NO: 380, H2 containing the amino acid sequence of SEQ ID NO: 379, and L2 containing the amino acid sequence of SEQ ID NO: 368.

[0272] Any of the anti-CD3 / anti-CD180 bispecific antibodies with the CD58 fusion peptide shown in Table 16 above can be further modified by replacing any of the anti-CD3ε antigen-binding regions shown in Tables 7-9 with any of the anti-CD3ε binding regions shown in Tables 7-9. For example, the anti-CD3ε antigen-binding region of the bispecific antibody “EIP1042” can be replaced with any of the anti-CD3ε binding regions shown in Tables 7-9 to produce the bispecific antibody of the present invention. Exemplary antibodies are shown in Tables 13 and 16.

[0273] Production methods

[0274] Various procedures known in the art can be used to produce polyclonal or monoclonal antibodies against a given target such as CD180, a disease-associated antigen, or other targets, or against their derivatives, fragments, analogs, homologs, or orthologs. (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).

[0275] Antibodies are purified using known techniques, such as affinity chromatography of protein A or protein G, which primarily provides IgG fractions of the immune serum. Subsequently, or optionally, a specific antigen or its epitope that is a target of the desired immunoglobulin can be immobilized on a column to purify the immune-specific antibody by immunoaffinity chromatography. Immunoglobulin purification 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).

[0276] In some embodiments, the antibodies of the present invention are monoclonal antibodies. Monoclonal antibodies are generated, for example, using the procedures described in the examples provided herein. For example, antibodies can also be generated by immunizing BALB / c mice, for example, with a combination of cell transfectants expressing high levels of a given target on their surface. The reactivity of hybridomas derived from myeloma / B cell fusions to the selected target is then screened.

[0277] For example, monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to said immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0278] The immunomodulator typically comprises a protein antigen, a fragment thereof, or a fusion protein thereof. Peripheral blood lymphocytes are typically used if human-derived cells are required, or spleen cells or lymph node cells if non-human mammalian-derived cells are required. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent such as polyethylene glycol to form hybridoma cells (Goding, *Monoclonal Antibodies: Principles and Practice*). Monoclonal Antibodies: Principles and Practice (Academic Press, (1986) pp. 59-103). Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells are deficient in hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma will typically include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevent the growth of HGPRT-deficient cells.

[0279] Preferred immortalized cell lines are those that are efficiently fused, support stable and high-level antibody expression in cells producing the selected antibody, and are sensitive to culture media such as HAT medium. More preferred immortalized cell lines are mouse myeloma cell lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Collection of Type Cultures (Manassas, Virginia). Human myeloma and mouse-human heterologous myeloma cell lines have also 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).

[0280] The presence of monoclonal antibodies against the antigen can then be detected in the culture medium in which the hybridoma cells are cultured. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, the binding affinity of a monoclonal antibody can be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity to the target antigen.

[0281] After identifying the desired hybridoma cells, the clone can be subcloned using a limiting dilution procedure and grown using standard methods. (See Goding, *Monoclonal Antibodies: Principles and Practice*) Monoclonal Antibodies: Principles and Practice (Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco modified Eagle medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0282] Monoclonal antibodies secreted by the subclones can be isolated or purified from culture medium or ascites fluid using conventional immunoglobulin purification procedures such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0283] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Hybridoma cells of the present invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into host cells that do not normally produce immunoglobulins, such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example by replacing the homologous mouse sequences with coding sequences for the constant domains of the human heavy and light chains (see U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can replace the constant domain of the antibody of the present invention, or can replace the variable domain of an antigen-binding site of the antibody of the present invention, to produce chimeric bivalent antibodies.

[0284] The monoclonal antibodies of this invention include humanized antibodies or human antibodies. These antibodies are suitable for administration to humans without evoking an immune response in humans against the administered immunoglobulin. Humanized forms of antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding sequence of the antibody) that consist primarily of sequences of human immunoglobulins and contain very few sequences derived from non-human immunoglobulins. For example, humanization is performed by following the methods of Winter et al. (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 replacing the corresponding sequence of a human antibody with one or more rodent CDR sequences. (See also U.S. Patent No. 5,225,539.) In some cases, the Fv framework residues of human immunoglobulins are replaced with corresponding non-human residues. Humanized antibodies also contain residues, for example, that are not present in the receptor antibody or in the input CDR or framework sequence. Generally, humanized antibodies include at least one, typically two, variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the framework regions are framework regions of the human immunoglobulin common sequence. Humanized antibodies preferably also include immunoglobulin constant regions (Fc), typically at least a portion of the human immunoglobulin constant regions (Jones et al., 1986; Riechmann et al., 1988; and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

[0285] Fully human antibodies are antibody molecules in which the entire sequence of both the light and heavy chains (including the CDR) is derived from human genes. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies.” Monoclonal antibodies can be prepared using three-source hybridoma technology, human B-cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4: 72), and EBV hybridoma technology for producing monoclonal antibodies (see Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Monoclonal antibodies can be utilized and can be produced by using human hybridomas (Cote et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein Barr virus (see Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0286] In addition, human antibodies can also be produced using other techniques, including phage display libraries (see Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). The production of human antibodies after irritation is observed to be very similar to that observed in humans in all respects, including gene rearrangement, assembly, and antibody library formation. This method is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, and in Marks et al., Bio / Technology 10, 779-783 (1992); Lonberg et al., Nature 368, 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

[0287] Furthermore, human antibodies can be produced using transgenic nonhuman animals modified to produce fully human antibodies in response to antigenic stimulation, rather than the animal's endogenous antibodies (see PCT Publication WO94 / 02602). The endogenous genes encoding the heavy and light chains of immunoglobulins in the nonhuman host have been deactivated, and active loci encoding human heavy and light chain immunoglobulins have been inserted into the host genome. For example, a yeast artificial chromosome containing the desired human DNA segment is used to incorporate human genes. Animals providing all the desired modifications are then obtained as offspring by hybridizing intermediate transgenic animals containing less than the full set of modifications. An example of such a nonhuman animal is the Xenomouse disclosed in PCT Publications WO 96 / 33735 and WO 96 / 34096. TMMice. These animals produce B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from said animals after immunization with an immunogen of interest, for example as a preparation of polyclonal antibodies, or from immortalized B cells derived from said animals, such as hybridomas that produce monoclonal antibodies. Furthermore, the gene encoding the immunoglobulin having a human variable region can be recovered and expressed to directly obtain said antibody, or it can be further modified to obtain antibody analogs, such as single-chain Fv (scFv) molecules.

[0288] U.S. Patent No. 5,939,598 discloses an example of a method for generating a non-human host (e.g., a mouse) lacking expression of endogenous immunoglobulin heavy chain. This can be achieved through a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and to prevent the formation of transcripts of rearranged immunoglobulin heavy chain loci, said deletion being achieved by a targeting vector containing a gene encoding a selectable marker; and generating transgenic mice from said embryonic stem cells containing somatic and germ cells encoding the gene encoding said selectable marker.

[0289] U.S. Patent No. 5,916,771 discloses a method for producing an antibody of interest (e.g., a human antibody). This method includes introducing an expression vector containing a nucleotide sequence encoding a heavy chain into a cultured mammalian host cell, 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 both the heavy and light chains.

[0290] In a further refinement of this procedure, PCT Publication WO 99 / 53049 discloses a method for identifying clinically relevant epitopes on immunogens and an association method for selecting antibodies that specifically bind to said relevant epitopes with high affinity.

[0291] Antibodies can be expressed by vectors containing DNA segments encoding the aforementioned single-chain antibodies.

[0292] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates having a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid-binding moiety (e.g., polylysine) as described in WO 93 / 64701, viral vectors (e.g., DNA or RNA viral vectors), fusion proteins (e.g., those described in PCT / US 95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specifically targeting a target cell) and a nucleic acid-binding moiety (e.g., protamine), plasmids, bacteriophages, etc. The vectors can be chromosomal, non-chromosomal, or synthetic.

[0293] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include poxvirus vectors such as orthopoxvirus or fowlpoxvirus vectors, herpesvirus vectors such as type I herpes simplex virus (HSV) vectors (see Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F. et al., DNA Cloning: Mammalian Systems, D. Glover ed., (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI et al., Proc Natl. Acad. Sci. USA 87:1149 (1990)), and 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, MG et al., Nat. Genet. 8:148 (1994)).

[0294] Poxvirus vectors introduce genes into the cytoplasm of cells. Fowlpoxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acids into nerve cells. Adenovirus vectors induce expression for a shorter period (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn is shorter than HSV vectors. The specific vector chosen will depend on the target cells and the disease being treated. Introduction can be performed using standard techniques such as infection, transfection, transduction, or transformation. Examples of gene transfer modalities include, for example, naked DNA, Ca2(PO4)3 precipitation, DEAE dextran, electroporation, protoplast fusion, lipid staining, cell microinjection, and viral vectors.

[0295] Vectors can be used to target virtually any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. Additionally, particles can be delivered via intraventricular (ICV) infusion using a small pump infusion system (e.g., the SynchroMed infusion system). A total flow-based approach known as convection has also proven effective in delivering macromolecules to extended areas of the brain and may facilitate vector delivery to target cells. (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 catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known routes of administration.

[0296] A bispecific antibody is an antibody that has binding specificity to at least two different antigens. In the case of this invention, one of the binding specificities is against a first target, such as CD3ε or any fragment thereof. The second binding target is a disease-associated antigen, such as CD180 or any fragment thereof.

[0297] Methods for preparing bispecific antibodies are known in the art. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (tetravalent hybridomas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is typically accomplished by an affinity chromatography step. Similar procedures are disclosed in WO 93 / 08829, published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0298] The bispecific and / or monovalent antibodies of the present invention can be prepared using any of a variety of techniques recognized in the art, including those disclosed in co-pending application WO 2012 / 023053, filed August 16, 2011, the contents of which are incorporated herein by reference in their entirety. The method described in WO 2012 / 023053 produces bispecific antibodies with structures identical to human immunoglobulins. This type of molecule consists of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant κ domain, and a second light chain variable region fused to a constant λ domain. Each binding site exhibits a distinct antigen specificity, which is contributed to by both the heavy and light chains. The light chain variable region can be of the λ or κ family and is preferably fused to the λ and κ constant domains, respectively. This is preferred to avoid the generation of non-natural polypeptide linkages. However, the bispecific antibodies of the present invention can also be obtained by fusing the κ light chain variable domain to the constant λ domain to provide first specificity and fusing the λ light chain variable domain to the constant κ domain to provide second specificity. The bispecific antibody described in WO 2012 / 023053 is called an IgGκλ antibody or "κλ body," which is a novel fully human bispecific IgG form. This κλ body form allows for affinity purification of bispecific antibodies with characteristics indistinguishable from standard monoclonal antibodies and indistinguishable from standard IgG molecules, thus offering advantages over previous forms.

[0299] A necessary step in the method is to identify two antibody Fv regions (each region consisting of a variable light chain and a variable heavy chain domain) that share the same heavy chain variable domain and have different antigen specificities. Numerous methods for generating monoclonal antibodies and their fragments have been described. (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). A fully human antibody is an antibody molecule in which both the light and heavy chains (including CDR1 and 2) are derived from human genes. The CDR3 region may be of human origin or designed synthetically. Such antibodies are referred to herein as “human antibodies” or “fully human antibodies.” Human monoclonal antibodies can be prepared using three-source hybridoma technology, human B-cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4: 72), and EBV hybridoma technology for producing human monoclonal antibodies (see Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized and can be produced by using human hybridomas (see Cole et al., 1983. Proc Natl Acad SciUSA 80: 2026-2030) or by transforming human B cells in vitro with Epstein Barr virus (see Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0300] Monoclonal antibodies are produced by immunizing animals, for example, with a target antigen or an immunogenic fragment, derivative, or variant thereof. Alternatively, animals are immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, thereby expressing the target antigen and binding it to the surface of the transfected cells. Various techniques for producing xenogeneic nonhuman animals are well known in the art. See, for example, U.S. Patents 6,075,181 and 6,150,584, the entirety of which is incorporated herein by reference.

[0301] Alternatively, antibodies can be obtained by screening libraries containing antibody or antigen-binding domain sequences for binding to target antigens. This library can be prepared, for example, in bacteriophages, as a protein or peptide fusion fused with a phage coat protein expressed on the surface of an assembled phage particle, and containing a DNA-coding sequence within the phage particle (i.e., a "phage display library"). Alternatively, the library can be prepared in yeast, as a protein or peptide fusion fused with a cell wall protein on the surface of yeast cells, and containing a DNA-coding sequence within the yeast cell (i.e., a "yeast display library").

[0302] The reactivity of hybridomas derived from myeloma / B-cell fusions to the target antigen is then screened. For example, monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In hybridoma methods, mice, hamsters, or other suitable host animals are typically immunized with an immunogen to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to said immunogen. Alternatively, lymphocytes can be immunized in vitro.

[0303] While not entirely impossible, the chances of identifying different antibodies with the same heavy chain variable domain but targeting different antigens by chance are extremely low. In fact, in most cases, the heavy chain contributes significantly to the antigen-binding surface and is the most sequence-variable. Specifically, CDR3 on the heavy chain is the most diverse CDR in terms of sequence, length, and structure. Therefore, two antibodies specifically targeting different antigens almost always carry different heavy chain variable domains.

[0304] The method disclosed in co-pending application WO 2012 / 023053 overcomes this limitation and greatly facilitates the isolation of antibodies with the same heavy chain variable domain by using antibody libraries in which the heavy chain variable domain is identical for all library members, thus limiting diversity 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 incorporated herein by reference in its entirety. However, since the light chain variable domain is co-expressed with the heavy chain variable domain, both domains contribute to antigen binding. To further facilitate the process, antibody libraries containing the same heavy chain variable domain and multiple λ or κ variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This method enables the identification of two antibodies with a common heavy chain, but one carrying the λ light chain variable domain and the other carrying the κ light chain variable domain, which can be used as building blocks for bispecific antibodies in the form of whole immunoglobulins according to the present invention. The bispecific antibodies of the present invention can have different isotypes, and their Fc moieties can be modified to alter their binding properties to different Fc receptors, thereby modifying the effector function and pharmacokinetic properties of the antibodies. Numerous methods for modifying the Fc moieties have been described, applicable to the antibodies of the present invention. (See, for example, Strohl, WR Curr Opin Biotechnol 2009 (6):685-91; U.S. Patent No. 6,528,624; PCT / US2009 / 0191199, filed January 9, 2009). The methods of the present invention can also be used to generate bispecific antibodies and antibody mixtures in the form of F(ab')2 lacking the Fc moieties.

[0305] The common heavy chain and two different light chains are co-expressed in a single cell to allow assembly of the bispecific antibody of the present invention. If all peptides are expressed at the same level and assembled equally well to form immunoglobulin molecules, the ratio of monospecific (same light chain) to bispecific (two different light chains) should be 50%. However, different light chains may be expressed at different levels and / or not assembled with the same efficiency. Therefore, means of regulating the relative expression of different peptides are used to compensate for their inherent expression characteristics or different propensities to assemble with the common heavy chain. This regulation can be achieved through promoter strength, the use of internal ribosome entry sites (IRES) with different efficiencies, or other types of regulatory elements that can function at the transcriptional or translational level and act on mRNA stability. Different promoters with different strengths can include CMV (immediate early cytomegalovirus promoter), EF1-1α (human elongation factor 1α subunit promoter), Ubc (human ubiquitin C promoter), and SV40 (simian virus 40 promoter). Different IRES from mammalian and viral sources have also been described. (See, for example, Hellen CU and Sarnow P, Genes Dev 2001 15: 1593–612). The length and ribosome recruitment efficiency of these IRES can vary considerably. Furthermore, activity can be further modulated by introducing multiple copies of the IRES (Stephen et al., 2000 Proc Natl Acad Sci USA 97: 1536–1541). Regulation of expression can also be achieved through multiple sequential transfections of cells to increase the copy number of a single gene expressing one or more light chains, thereby altering their relative expression. The examples provided in this article demonstrate that controlling the relative expression of different chains is crucial for maximizing the assembly and overall yield of bispecific antibodies.

[0306] The co-expression of the heavy chain and two light chains produces a mixture of three different antibodies in the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter must be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by using affinity chromatography media such as CaptureSelect Fab κ and CaptureSelect Fab λ affinity matrices (BAC BV, Holland) that specifically interact with the constant domains of the κ or λ light chain. This multi-step affinity chromatography purification method is efficient and generally applicable to the antibodies of this invention. This contrasts sharply with specific purification methods that must be developed and optimized for each bispecific antibody derived from cell lines expressing a mixture of tetravalent somatic tumors or other antibody mixtures. In fact, if the different antibodies in the mixture have similar biochemical properties, separating them using standard chromatography techniques, such as ion exchange chromatography, can be challenging or even impossible.

[0307] Other suitable purification methods include those disclosed in co-pending application PCT / IB2012 / 003028 (published as WO2013 / 088259), filed on October 19, 2012, the contents of which are incorporated herein by reference in their entirety.

[0308] In other embodiments of bispecific antibody production, an antibody variable domain having the desired binding specificity (antibody-antigen binding site) can be fused with an immunoglobulin constant domain sequence. The fusion preferably has an immunoglobulin heavy chain constant domain comprising at least a portion of a hinge, CH2, and CH3 regions. Preferably, at least one fusion contains a first heavy chain constant region (CH1) containing the desired light chain binding site. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0309] According to another method described in WO 96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell cultures. The preferred interface comprises at least a portion of the CH3 region of the antibody's constant structural domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing heterodimer yield compared to other unwanted end products, such as homodimers.

[0310] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical conjugation. The resulting bispecific antibodies can be used as reagents for the selective immobilization of enzymes.

[0311] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell cultures have also been described. For example, bispecific antibodies have been generated using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portion of two different antibodies via gene fusion. The antibody homodimer is reduced at the hinge region to form a monomer, and then re-oxidized to form an antibody heterodimer. This method can also be used to generate antibody homodimers. The “bimeric antibody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for preparing bispecific antibody fragments. The fragments contain a variable domain (V) linked to a light chain via a linker. L Heavy chain variable structural domain (V) H The joint is too short to allow pairing between the two structural domains on the same chain. Therefore, a segment of V... H and V L The structural domain is forced to interact with the complementary V of another segment. L and V H Domain pairing forms two antigen-binding sites. Another strategy for preparing bispecific antibody fragments using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0312] Antibodies with more than two valentities have been envisioned. For example, trispecific antibodies could be prepared. (Tutt et al., J. Immunol. 147:60 (1991)).

[0313] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which is derived from the protein antigen of the present invention. Alternatively, the anti-antigen arm of an immunoglobulin molecule can be combined with an arm that binds to triggering molecules on leukocytes (e.g., T-cell receptor molecules (e.g., CD2, CD3, CD28, or B7) or Fc receptors (FcγRs) of IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16)) to focus cellular defense mechanisms on cells expressing a specific antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells expressing a specific antigen. These antibodies have an antigen-binding arm and an arm that binds to cytotoxic agents or radionuclide chelators such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to the protein antigen described herein and further binds to tissue factor (TF).

[0314] Heterovalent antibodies are also within the scope of this invention. Heterovalent antibodies consist of two covalently linked antibodies. For example, such antibodies have been proposed to target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980) and for the treatment of HIV infection (see WO 91 / 00360, WO 92 / 200373, EP 03089). It is contemplated that said antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including methods involving cross-linking agents. For example, immunotoxins can be constructed via disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiols and methyl-4-mercaptobutyrylimide esters, as well as reagents disclosed, for example, in U.S. Patent No. 4,676,980.

[0315] The antibodies of the present invention may need to be modified in terms of effector function to enhance, for example, their efficacy in treating cancer and / or other diseases and disorders associated with aberrant CD180 expression and / or activity. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in that region. The resulting homodimeric antibodies may have improved internalization capacity and / or enhanced complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, the antibodies may be engineered to have a double Fc region, thereby having enhanced complement cleavage and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)).

[0316] The present invention also relates to immunoconjugates comprising antibodies conjugated to cytotoxic agents such as toxins (e.g., bacterial, fungal, plant or animal-derived enzyme-active toxins or fragments thereof) or radioisotopes (i.e., radioconjugates).

[0317] Available enzymatically active toxins and their fragments include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from *Pseudomonas aeruginosa*), ricin A chain, abrin A chain, *Saccharitoxin* A chain, α-Dacococcus toxin, *Aleurites fordii* protein, carnation toxin protein, *Phytolaca americana* protein (PAPI, PAPII, and PAP-S), *Momordica charantia* inhibitor, *Jatropha curcas* toxin protein, croton toxin protein, *Sapaonaria officinalis* inhibitor, *Alternaria leucocephala* toxin protein, mitocytoxin, aspergillus oryzae, phenolmycin, enoxacin, and trichothecenes. Various radionuclides can be used to produce radioconjugated antibodies. Examples include... 212 Bi、 131 I, 131 In、 90 Y and 186 Re.

[0318] Antibody-cytotoxic agent conjugates are prepared using various bifunctional protein conjugates, such as N-succinimide-3-(2-pyridyldithiool)propionate (SPDP), iminothiacyclopentane (IT), bifunctional derivatives of imine esters (e.g., dimethyl diimide adipate HCl), active esters (e.g., disuccinimide octanoate), aldehydes (e.g., glutaraldehyde), diazid compounds (e.g., bis(p-azidobenzoyl)hexamethylenediamine), diazido derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and difluorinated compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanate-benzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. (See WO94 / 11026).

[0319] Those skilled in the art will recognize that a variety of possible components can be conjugated to the antibodies derived in this invention. (See, for example, “Conjugate Vaccines,” Contributions to Microbiology and Immunology, eds. J.M. Cruse and RE. Lewis, Jr., Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0320] As long as the antibody and the other moiety retain their respective activities, conjugation can be achieved through any chemical reaction that binds the two molecules. Such conjugation can include many chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordination binding, and complexation. However, covalent binding is preferred. Covalent binding can be achieved through the direct condensation of existing side chains or by incorporating an external bridging molecule. Many divalent or multivalent conjugates can be used to conjugate protein molecules (such as the antibodies of this invention) to other molecules. For example, representative conjugates can include organic compounds such as thioesters, carbodiimides, succinimidyl esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be an exhaustive list of all types of conjugates known in the art, but rather an example of more common conjugates. (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)).

[0321] Preferred linkers are described in the literature. See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimide benzoyl-N-hydroxysuccinimide ester). See also U.S. Patent No. 5030719, which describes the use of haloacetylhydrazide derivatives conjugated to antibodies via oligopeptide linkers. Particularly preferred connectors include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride); (ii) SMPT (4-succinimide-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene) (Pierce Chem. Co., catalog 21558G); (iii) SPDP (succinimide-6-[3-(2-pyridyl-dithio)propamido]hexanoate) (Pierce Chem. Co., catalog 21651G); (iv) sulfonyl-LC-SPDP (sulfosuccinimide-6-[3-(2-pyridyl-dithio)propamido]hexanoate) (Pierce Chem. Co., catalog 2165-G); and (v) sulfonyl-NHS (N-hydroxysulfonyl-succinimide) coupled to EDC (Pierce Chem. Co., catalog 24510).

[0322] The aforementioned connectors contain components with different properties, resulting in conjugates with varying physicochemical properties. For example, sulfonated NHS esters of alkyl carboxylic acids are more stable than sulfonated NHS esters of aromatic carboxylic acids. Connectors containing NHS esters have lower solubility than sulfonated NHS esters. Furthermore, the SMPT connector contains sterically hindered disulfide bonds, which can form conjugates with even greater stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer usable conjugates. In particular, sulfonated NHS can enhance the stability of carbodiimide conjugates. When carbodiimide conjugates (e.g., EDC) are used in combination with sulfonated NHS, the resulting esters are more resistant to hydrolysis than carbodiimide conjugates alone.

[0323] The antibodies disclosed herein can also be formulated into immunoliposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77: 4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced cycle times are disclosed in U.S. Patent No. 5,013,556.

[0324] Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a specified pore size to produce liposomes with the desired diameter. The Fab' fragment of the antibody of the present invention can be coupled to the liposome via a disulfide exchange reaction, as described by Martin et al., J. Biol. Chem., 257:286-288 (1982).

[0325] How to use

[0326] Any bispecific antibody disclosed herein (e.g., anti-CD3ε and anti-CD180 antibodies; anti-CD3ε and anti-CD180 antibodies having a CD58 fusion peptide) can be used in therapeutic methods. In one aspect, the bispecific antibodies of this disclosure can be used as pharmaceuticals. In a further aspect, the bispecific antibodies of this disclosure can be used to treat cell proliferation disorders (e.g., cancer) or to delay their progression. In some embodiments, a bispecific antibody for therapeutic purposes is provided. In some embodiments, the present invention provides a bispecific antibody for a method of treating an individual with a cell proliferation disorder, the method comprising administering an effective amount of the bispecific antibody to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, such as the additional therapeutic agent described below. In other embodiments, the present invention provides a bispecific antibody for enhancing immune function in an individual with a cell proliferation disorder. In some embodiments, the present invention provides a bispecific antibody for enhancing immune function in an individual with a cell proliferation disorder, the method comprising administering an effective amount of the bispecific antibody to the individual to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) the effector cell population, reduce the target cell population, and / or kill target cells (e.g., target tumor cells). The “individual” according to any of the above embodiments can be a human being.

[0327] In another aspect, the present invention provides the use of the bispecific antibodies disclosed herein (e.g., anti-CD3ε and anti-CD180 antibodies; anti-CD3ε and anti-CD180 antibodies having a CD58 fusion peptide) in the manufacture or preparation of a pharmaceutical. In one embodiment, the pharmaceutical is used to treat a cell proliferation disorder (e.g., cancer, such as esophageal cancer or adenocarcinoma). In another embodiment, the pharmaceutical is used as a method of treating a cell proliferation disorder or an autoimmune disorder, the method comprising administering an effective amount of the pharmaceutical to an individual suffering from a cell proliferation disorder or an autoimmune disorder. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent, such as the additional therapeutic agent described below, to the individual. In another embodiment, the pharmaceutical is used to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) effector cell populations, reduce target cell populations (e.g., cells expressing CD180), and / or kill target cells (e.g., target tumor cells) in the individual. In another embodiment, the drug is used in a method of enhancing immune function in an individual suffering from a cell proliferation disorder or an autoimmune disorder, the method comprising administering an effective amount of the drug to the individual to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) the effector cell population, reduce the target cell population (e.g., cells expressing CD180), and / or kill target cells (e.g., target tumor cells). The “individual” according to any of the above embodiments can be a human being.

[0328] In another aspect, the present invention provides a method for treating a cell proliferation disorder (e.g., cancer). In one embodiment, the method includes administering an effective amount of a bispecific antibody to an individual suffering from such a cell proliferation disorder. In one such embodiment, the method further includes administering an effective amount of at least one additional therapeutic agent to the individual, such as the additional therapeutic agent described below. An "individual" according to any of the above embodiments can be a human being. In another aspect, the present invention provides a method for enhancing immune function in an individual suffering from a cell proliferation disorder or an autoimmune disorder. In one embodiment, the method includes administering an effective amount of a bispecific antibody to the individual to activate effector cells (e.g., T cells, such as CD8+ and / or CD4+ T cells), expand (increase) the effector cell population, reduce the target cell population (e.g., cells expressing CD180), and / or kill target cells (e.g., target tumor cells).

[0329] In some embodiments, the present invention provides a method for treating cancers expressing CD180. In some embodiments, the method of treating cancer includes administering an effective amount of a bispecific antibody of the present disclosure (e.g., anti-CD3ε and anti-CD180 antibodies; anti-CD3ε and anti-CD180 antibodies having a CD58 fusion peptide).

[0330] In some embodiments, the cancer expresses CD180 at high levels. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer is at least one type of lymphoma, including Hodgkin's lymphoma, non-Hodgkin's lymphoma, and B-cell lymphoma, including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, giant lesion NHL, mantle cell lymphoma, and AIDS-related lymphoma. In some embodiments, the cancer is Waldenström macroglobulinemia. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML). In some implementations, any type of cancer described herein can be treated with CD180 antibodies.

[0331] In some embodiments, CD180 expression is induced by adding 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 an FLT-3 inhibitor. Exemplary FLT-3 inhibitors include, but are not limited to, Rydapt / Midottolin, Xospata / Giratetinib, Vanflyta / Quizatinib, and Crenolanib. In some embodiments, the additional therapeutic agent is an IDH1 / 2 inhibitor. Exemplary IDH1 / 2 inhibitors include, but are not limited to, Tibsovo / Evonib, Idhifa / Entidipine, and Rezlidia / Olutenib. In some embodiments, the additional therapeutic agent is chemotherapy and / or a hypomethylating agent. Exemplary chemotherapy and / or hypomethylating agents include, but are not limited to, Vyxeos / daunorubicin + cytarabine liposomes, Onureg / CC-486 / oral azacitidine, and Inqovi / Inaqovi / ASTX727. In some embodiments, the additional therapeutic agent is a hedgehog pathway inhibitor. Exemplary hedgehog pathway inhibitors include, but are not limited to, Daurismo / grajib. In some embodiments, the additional therapeutic agent is an antibody-drug conjugate. Exemplary antibody-drug conjugates include, but are not limited to, Mylotarg / geutuzumab / orzomicin. In some embodiments, the additional therapeutic agent is a radioimmunoassay conjugate. Exemplary radioimmunoassay conjugates include, but are not limited to, Iomab-B / 131 I. Actimab-A and Lintuzumab-Ac 225 .

[0332] In some embodiments, the additional therapeutic agent is a 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 bispecific antibody that binds to T cells. Exemplary bispecific antibodies that bind to T cells include, but are not limited to, Lunsumio / mucuzumab, Epkinly / Tepkinly / icoretuzumab, Columvi / glucantuzumab, and Ordspono / ornituzumab. 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 / obituzumab, Monjuvi / Minjuvi / tancituzumab, and molotovimab. In some embodiments, the additional therapeutic agent is a BTK inhibitor. Exemplary BTI inhibitors include, but are not limited to, Brukinsa / zanubrutinib, Calquence / acabrutinib, Imbruvica / ibrutinib, Jaypirca / pitobrutinib, neltabrutinib, NX-5948, NX-2127, and BGB-16673. In some embodiments, the additional therapeutic agent is a treatment combination. Exemplary treatment combinations include, but are not limited to, R-CHOP (rituximab, cyclophosphamide, doxorubicin / daunorubicin hydrochloride, vincristine sulfate / vincristine, and prednisone), R-EPOCH (rituximab, etoposide phosphate, prednisone, vincristine sulfate / vincristine, cyclophosphamide, and doxorubicin / daunorubicin hydrochloride), 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.

[0333] In some embodiments, the additional therapeutic agent is a BCL-2 inhibitor. Exemplary BCL-2 inhibitors include, but are not limited to, Venclexta / Venclyxto, Sonrotoclax / BGB-11417, and lisatoclax. In some embodiments, the additional therapeutic agent is a CAR-T cell therapy. Exemplary CAR-T cell therapies include, but are not limited to, MB-106 and Breyanzi / lisocabtagene maraleucel.

[0334] In some embodiments, a method is provided for identifying a subject receiving CD180 therapy. The method includes testing for high levels of CD180 expression in a cancer sample. In some embodiments, the cancer is one or more lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, as well as B-cell lymphomas, including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaved cell NHL, giant cell lesion NHL, mantle cell lymphoma, and AIDS-related lymphoma. In some embodiments, the cancer is Waldenström macroglobulinemia. In some embodiments, the leukemia is chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML).

[0335] The method further includes comparing CD180 expression levels in cancer samples with CD180 expression levels in healthy / control / reference tissues to determine whether CD180 expression levels are higher in the cancer samples. If CD180 expression levels are higher in the cancer samples, the subject receives a CD180 targeting agent. In some embodiments, the CD180 targeting agent is one or more antibodies provided herein. In some embodiments, any method for measuring CD180 levels can be used. In some embodiments, this is any amount greater than a negative amount in a staining assay. In some embodiments, this is any amount higher than the level present in surrounding healthy / control / reference tissues or in the corresponding tissue from a healthy / control / reference subject. In some embodiments, a high expression level of CD180 is defined compared to CD180 expression levels in non-cancer samples.

[0336] In some embodiments, the CD180 expression level 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 CD180 expression level in the non-cancer sample. In some embodiments, the CD180 expression level in the cancer sample is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher than the CD180 expression level in the non-cancer sample. In some embodiments, the CD180 expression level in the cancer samples is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, 425%, 450%, 475%, or 500% higher than the CD180 expression level in the non-cancer samples. In some embodiments, the CD180 expression level in the cancer samples is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than the CD180 expression level in the non-cancer samples.

[0337] In some embodiments, the healthy / control / reference sample is a sample from normal tissue. In some embodiments, the normal tissue is tissue adjacent to the cancer in the subject. For example, when expression is determined by staining, the protein level is higher than a level that would be considered “negative” by a particular assay. Exemplary assays include, but are not limited to, RNA-based assays, flow cytometry, FACS, and IHC, each with its appropriate levels of positive and negative results. Any method for detecting protein levels in a sample is contemplated. A person skilled in the art can select an appropriate method based on the type of sample being analyzed and the identity and quantity of the protein being detected. Non-limiting exemplary such methods include immunohistochemistry, ELISA, Western blotting, multiplex analyte detection (using, for example, Luminex technology), mass spectrometry, etc. Similarly, any method for detecting mRNA levels in a sample is contemplated. A person skilled in the art can select an appropriate method based on the type of sample being analyzed and the identity and quantity of the mRNA being detected. Non-limiting exemplary such methods include RT-PCR, quantitative RT-PCR, and microarray-based methods, etc.

[0338] CD180-positive cancers according to any of the above embodiments can be, for example, CD180-positive B-cell lymphoma, CD180-positive MDS / AML, and CD180-positive CLL. In some embodiments, CD180-positive cancers are cancers with an anti-CD180 immunohistochemical (IHC) or in situ hybridization (ISH) score greater than "0" (which corresponds to very weak or no staining in >90% of tumor cells). In another embodiment, CD180-positive cancers express CD180 at 1+, 2+, or 3+ levels. In some embodiments, CD180-positive cancers are cancers that express CD180 according to a reverse transcriptase PCR (RT-PCR) assay detecting CD180 mRNA. In some embodiments, the RT-PCR is quantitative RT-PCR. In some embodiments, the RT-PCR is quantitative RT-PCR. In some embodiments, CD180-positive cancers are cancers that express CD180 according to a flow cytometry assay detecting CD180 expression on the surface of tumor cells.

[0339] In another aspect, the present invention provides pharmaceutical formulations comprising any bispecific antibody provided herein (e.g., bispecific antibodies for any of the above-described treatment methods). In one embodiment, the pharmaceutical formulation comprises any bispecific antibody provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any bispecific antibody provided herein and at least one additional therapeutic agent, such as the additional therapeutic agent described herein.

[0340] The antibodies of the present invention can be used alone or in combination with other agents for therapeutic purposes. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent, an agent used in radiotherapy, an anti-angiogenic agent, an apoptotic agent, an anti-microtubule agent, or other agents, such as epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (Tarceva™), platelet-derived growth factor inhibitors (e.g., Gleevec™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferon, cytokines, antibodies other than the anti-CD3 antibody of the present invention (e.g., antibodies that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, GPRC5D, CD20, CD19, BCMA). VEGF or VEGF receptor, TRAIL / Apo2, PD-1 (e.g., nivolumab, pembrolizumab, cimipril), PD-L1 (atezolizumab, avelumab, durvalumab), PD-L2), or another biologically active or organic chemical agent. In some embodiments, the invention provides a method in which the additional therapeutic agent is a glucocorticoid. In one embodiment, the glucocorticoid is dexamethasone.

[0341] 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, the entirety of which is incorporated herein by reference.

[0342] In some respects, the additional therapeutic agent is a checkpoint inhibitor. The term “inhibition” or “inhibitor” includes a reduction in a parameter (e.g., activity) of a given molecule, such as an immune checkpoint inhibitor. For example, the term includes inhibiting the activity of, for example, PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFRβ by at least 5%, 10%, 20%, 30%, 40%, or more. The level of inhibition is not necessarily 100%.

[0343] In some aspects, the checkpoint inhibitor is a PD-1 inhibitor. In some aspects, the PD-1 inhibitor is an anti-PD1 antibody. In some aspects, the PD-1 inhibitor is an anti-PD-1 monoclonal antibody. Exemplary anti-PD-1 monoclonal antibodies include, but are not limited to, cimipril (Libtayo), nivolumab (Opdivo), and pembrolizumab (Keytruda). In some aspects, the checkpoint inhibitor is a PD-L1 inhibitor. Exemplary PD-L1 inhibitors include, but are not limited to, avelumab (Bavencio), durvalumab (Imfinzi), and atezolizumab (Tecentriq).

[0344] The aforementioned combination therapy encompasses both combined administration (where two or more therapeutic agents are contained in the same or separate formulation) and single administration, in which case the antibody of the present invention may be administered before, simultaneously with, and / or after the administration of the one or more additional therapeutic agents. In one embodiment, the administration of the bispecific antibody and the administration of the additional therapeutic agent occur within approximately one month, or approximately one week, two weeks, or three weeks, or approximately one day, two days, three days, four days, five days, or six days. The bispecific antibody of this disclosure may also be used in combination with radiotherapy.

[0345] In some aspects, the additional therapeutic agent is a chimeric antigen receptor (CAR) T-cell therapy. In some embodiments, the CAR-T cell therapy specifically binds to CD19. Exemplary CAR-T cell therapies that specifically bind to CD19 include, but are not limited to, BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™ (axicabtagene ciloleucel), ABECMA® (idecabtagene vicleucel), or CARVYKTI™ (ciltacabtagene autoleucel).

[0346] The antibodies (and / or any additional therapeutic agents) of the present invention can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and may also include intralesional administration if local treatment is required. Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, the antibodies are administered subcutaneously. In some embodiments, anti-CD3ε antibodies administered subcutaneously result in less toxic responses in patients compared to the same anti-CD3ε antibodies administered intravenously. Administration can be carried out via any suitable route, such as by injection, for example, intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations at different time points, bolus administration, and pulsatile infusion.

[0347] The antibodies of this invention will be formulated, dosed, and administered in accordance with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to a medical practitioner. The antibody is not necessarily, but optionally, formulated with one or more agents currently used for the prevention or treatment of the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. They are generally used at the same dose and route of administration as described herein (or approximately 1% to 99% of the dose described herein), or at any dose and route determined empirically / clinically as appropriate.

[0348] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (whether used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibody is appropriately administered to the patient once or as part of a series of treatments.

[0349] Generally, whether administered once or multiple times, the therapeutically effective dose of bispecific anti-CD3ε and anti-CD180 antibodies administered to humans will be in the range of about 0.01 to about 100 mg / kg of patient body weight. In some embodiments, the antibodies used are, for example, administered daily at doses of about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg. In one embodiment, on day 1 of a 21-day cycle, the bispecific antibody described herein is administered to a person at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg. The dose may be administered as a single dose or multiple doses (e.g., 2 or 3 doses), such as by infusion. For repeated administration over several days or longer, treatment typically continues until the desired suppression of disease symptoms is achieved, depending on the condition. An exemplary dose of the antibody is in the range of about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses can be administered intermittently, for example, once weekly or every three weeks (e.g., giving the patient about 2 to about 20 doses, or for example about 6 doses of the bispecific antibody). An initial higher loading dose can be administered, followed by one or more lower doses. Progression of this therapy can be easily monitored using conventional techniques and assays.

[0350] In some embodiments, the method may further include additional therapies. These additional therapies may be radiotherapy, surgery, chemotherapy, gene therapy, DMA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the above therapies. The additional therapies may take the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzyme inhibitors or anti-transfer agents. In some embodiments, the additional therapy is the administration of side effect limiters (e.g., agents designed to reduce the occurrence and / or severity of treatment side effects, such as antinausea agents). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be the separate administration of one or more of the above therapeutic agents.

[0351] It should be understood that the application of the therapeutic entity according to the invention will be carried out in conjunction with suitable carriers, excipients, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. Many suitable formulations can be found in all formularies known to medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) vesicles (e.g., Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbon waxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbon waxes. Any of the above mixtures may be used for the treatments and therapies according to the invention, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible with and tolerable to the route of administration. For additional information relating to formulations, excipients, and carriers known to medicinal chemists, see also Baldrick P., “Pharmaceutical excipient development: the need for preclinical guidance,” Regul. Toxicol Pharmacol. 32(2):210-8 (2000); Wang W., “Lyophilization and development of solid protein pharmaceuticals,” Int. J. Pharm. 203(1-2):1-60 (2000); Charman WN, “Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts,” J Pharm Sci. 89(8):967-78 (2000); Powell et al., “Compendium of excipients for parenteral formulations,” PDA J Pharm SciTechnol. 52:238-311 (1998), and quotations therein.

[0352] Therapeutic formulations of the present invention, containing antibodies of the present invention, are used to treat or alleviate symptoms associated with cancer, such as, but not limited to, leukemia, lymphoma, breast cancer, colon cancer, ovarian cancer, bladder cancer, prostate cancer, glioma, lung and bronchial cancer, colorectal cancer, pancreatic cancer, esophageal cancer, liver cancer, bladder cancer, kidney and renal pelvis cancer, oral and pharyngeal cancer, endometrial cancer, and / or melanoma. The present invention also provides methods for treating or alleviating symptoms associated with cancer. Treatment regimens are implemented by identifying subjects (e.g., human patients with cancer (or at risk of developing cancer)) using standard methods.

[0353] The efficacy of the treatment is determined in conjunction with any known methods for diagnosing or treating a specific immune-related disorder. Relief of one or more symptoms of the immune-related disorder indicates that the antibody provides clinical benefit.

[0354] Methods for screening antibodies with desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immune-mediated techniques known in the art.

[0355] Antibodies (or fragments thereof) targeting targets such as CD3ε, CD180, or combinations thereof can be used in methods known in the art related to the localization and / or quantification of these targets, such as for measuring the levels of these targets in appropriate physiological samples, for diagnostic methods, for protein imaging, etc. In one given embodiment, an antibody containing an antibody-derived antigen-binding domain specifically targeting any of these targets or their derivatives, fragments, analogs, or homologs is used as a pharmacologically active compound (hereinafter referred to as a "therapeutic agent").

[0356] The antibodies of the present invention can be used to separate specific targets using standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. The antibodies (or fragments thereof) of the present invention can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, such as to determine the efficacy of a given treatment regimen. Detection can be facilitated by conjugating (i.e., physically linking) the antibodies to a detectable substance. Examples of detectable substances include various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and jellyfish luminescent protein; examples of suitable radioactive materials include… 125 I, 131 I, 35 S or3 H.

[0357] The antibodies of this invention (including polyclonal, monoclonal, humanized, and fully human antibodies) can be used as therapeutic agents. Such agents are commonly used to treat or prevent diseases or pathologies associated with the aberrant expression or activation of a given target in a subject. The antibody preparation (preferably an antibody preparation with high specificity and high affinity for its target antigen) is administered to the subject and is generally effective due to its binding to the target. The administration of the antibody may abolish, inhibit, or interfere with the signal transduction function of the target. The administration of the antibody may abolish, inhibit, or interfere with the binding of the target to its naturally bound endogenous ligands.

[0358] The therapeutically effective dose of the antibody of the present invention generally refers to the amount required to achieve the therapeutic target. As mentioned above, this may be due to the binding interaction between the antibody and its target antigen, which in some cases interferes with the function of said target. Furthermore, the amount required to be administered will depend on the binding affinity of the antibody to its specific antigen, and will also depend on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. As a non-limiting example, a common range of therapeutically effective doses of the antibody or antibody fragment of the present invention may be from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Common dosing frequencies may be, for example, in the range of twice daily to once weekly.

[0359] The antibodies or fragments thereof of the present invention can be administered in the form of pharmaceutical compositions for the treatment of a variety of diseases and disorders. The principles and considerations involved in the preparation of such compositions, as well as guidance on the selection of components, are provided in, for example, the following literature: *Remington: The Science and Practice of Pharmacy*, 19th edition (edited by Alfonso R. Gennaro et al.), Mack Pub. Co., Easton, Pa.: 1995; *Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends*, Harwood Academic Publishers, Langhorne, Pa., 1994; "Peptide and Protein Drug Delivery," *Advances in Parenteral Sciences*, Vol. 4, 1991, M. Dekker, New York.

[0360] When using antibody fragments, minimally inhibitory fragments that specifically bind to the binding domain of the target protein are preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation may also contain more than one active compound as required for a particular indication being treated, preferably those compounds having complementary activities that do not adversely affect each other. Optionally or additionally, the composition may contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are suitably combined in amounts that effectively achieve the intended purpose.

[0361] The active ingredient can also be encapsulated in microcapsules, for example, prepared by coagulation technology or interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in crude emulsions.

[0362] Formulations intended for internal administration must be sterile. This can be easily achieved through filtration using sterile membrane filters.

[0363] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semi-permeable matrices of solid hydrophobic polymers containing antibodies, said matrices being in the form of molded articles such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactide (US Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, and degradable lactic-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic-glycolic acid copolymer and leuprolide acetate) and poly(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for a shorter time.

[0364] The antibodies according to the invention can be used as reagents to detect the presence of a given target (or a protein fragment thereof) in a sample. In some embodiments, the antibody contains a detectable marker. The antibody is polyclonal, or more preferably monoclonal. A complete antibody or a fragment thereof (e.g., Fab, scFv, or F(ab)2) is used. With respect to probes or antibodies, the term "labeled" is intended to cover both direct labeling of the probe or antibody by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of the probe or antibody by reactivity with another directly labeled reagent. Examples of indirect labeling include detecting a first antibody using a fluorescently labeled second antibody, and end-labeling a DNA probe with biotin so that it can be detected by fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within the subject. Thus, the use of the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analytes mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described in, for example, the following literature: “ELISA: Theory and Practice,” Methods in Molecular Biology, Vol. 42, JR Crowther (ed.), Human Press, Totowa, NJ, 1995; “Immunoassay,” E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and “Practice and Theory of Enzyme Immunoassays,” P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detecting analyte proteins include introducing labeled anti-analyte protein antibodies into a subject. For example, the antibody may be labeled with a radioactive marker, and its presence and location in the subject can be detected using standard imaging techniques.

[0365] Pharmaceutical Composition

[0366] The antibodies of the present invention (also referred to herein as “active compounds”) and their derivatives, fragments, analogs, and homologs may be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody and a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents compatible with drug administration. Suitable carriers are described in the latest edition of the standard reference in the art, Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as non-volatile oils, may also be used. Such media and reagents for the use of pharmaceutically active substances are well known in the art. Their use in the compositions is conceivable unless any conventional media or reagent is incompatible with the active compound. Additional active compounds may also be incorporated into the compositions.

[0367] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous application may include the following components: a sterile diluent, such as water for injection, saline solution, non-volatile oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as acetate, citrate, or phosphate; and a reagent for adjusting osmolality, such as sodium chloride or glucose. pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral formulation may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0368] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (in the water-soluble case) or dispersions, as well as sterile powders for immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL... TM(BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be an easily injectable fluid. It must be stable under manufacturing and storage conditions and must be preserved to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, maintaining the desired particle size in the case of a dispersion, and using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyols such as mannitol, sorbitol, and sodium chloride, are preferably included in the composition. The absorption of the injectable composition can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0369] Sterile injectable solutions can be prepared by incorporating the desired amount of the active compound with one or a combination of the ingredients listed above into a suitable solvent as required, followed by sterile filtration. Typically, dispersions are prepared by incorporating the active compound into a sterile carrier containing a base dispersion medium and the other desired ingredients listed above. In the case of sterile powders used to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0370] Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound may be combined with excipients and administered in tablet, lozenge, or capsule form. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally, rinsed, and spat out or swallowed. Pharmaceutically compatible binders and / or excipients may be included as part of the composition. Tablets, pills, capsules, lozenges, etc., may contain any of the following components or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginate, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring.

[0371] For inhalation administration, the compound is delivered as an aerosol spray from a pressure vessel or dispenser or sprayer containing a suitable propellant, such as a gas like carbon dioxide.

[0372] Systemic administration can also be performed via transmucosal or transdermal routes. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are well known in the art and, for example, include detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated as an ointment, cream, gel, or cream, as is known in the art.

[0373] The compound can also be prepared in the form of suppositories (e.g., using conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0374] In one embodiment, the active compound is prepared using a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulation delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are readily apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes containing monoclonal antibodies against viral antigens that target infected cells) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0375] For ease of administration and dosage uniformity, it is particularly advantageous to formulate oral or parenteral compositions in dosage units. As used herein, dosage units refer to physically discrete units suitable as unit doses for use on a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect when combined with the desired drug carrier. The specifications of the dosage unit forms of this invention are determined by and directly depend on the unique characteristics of the active compound, the specific therapeutic effect to be achieved, and the inherent limitations in the technology of formulating such active compounds for individual treatment.

[0376] The pharmaceutical composition may be included in a container, package, or applicator along with the instructions for use.

[0377] The present invention will be further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0378] definition

[0379] Unless otherwise defined, scientific and technical terms used in connection with this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terms and techniques described herein that are associated with cell and tissue culture, molecular biology, protein and oligonucleotide or polynucleotide chemistry and hybridization are well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid staining). Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications or as commonly performed in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods known in the art, as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., *Molecular Cloning: A Laboratory Manual* (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The terms, laboratory procedures and techniques described herein that are associated with analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry are well-known and commonly used in the art. Standard technologies are used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0380] When used in accordance with this disclosure, unless otherwise stated, the following terms shall be understood to have the following meanings: The term "CD180-positive cancer" refers to cancer comprised of cells that express CD180 on their surface. In some implementations, the expression of CD180 on the cell surface is determined using methods such as immunohistochemistry and FACS, for example. Alternatively, the expression of CD180 mRNA is considered to be associated with the expression of CD180 on the cell surface and can be determined by methods selected from in situ hybridization and RT-PCR (including quantitative RT-PCR).

[0381] The term "CD180 positive cell" refers to a cell that expresses CD180 on its surface.

[0382] The terms “cancer” and “cancerous” refer to or describe a physiological condition in mammals typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer (including triple-negative (ER- / PR- / Her2-) breast cancer), colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancers.

[0383] The phrases “reference sample,” “reference cell,” or “reference tissue” refer to a sample having at least one known characteristic that can be used for comparison with a sample having at least one unknown characteristic. In some embodiments, the reference sample can be used as a positive or negative indicator. In contrast to the levels of proteins and / or mRNA present in a sample with unknown characteristics, the reference sample can be used to establish the levels of proteins and / or mRNA present, for example, in healthy tissue. In some embodiments, the reference sample is from the same subject being tested, but from a different site within the subject. In some embodiments, the reference sample is from a tissue region surrounding or adjacent to cancer. In some embodiments, the reference sample is not from the subject being tested, but from a sample from a subject known to have or not have the discussed impairment (e.g., a specific cancer or CD180-related impairment). In some embodiments, the reference sample is from the same subject, but from a time point prior to the development of cancer in the subject. In some embodiments, the reference sample is from a benign cancer sample (e.g., a benign breast cancer sample) from the same or different subjects. When a negative reference sample is used for comparison, the expression level or amount of the molecule in question in the negative reference sample will indicate a level that a person skilled in the art would understand, given this disclosure, that the molecule is absent and / or present at a low level. When a positive reference sample is used for comparison, the expression level or amount of the molecule in question in the positive reference sample will indicate a level that a person skilled in the art would understand, given this disclosure, that the molecule is present at a certain level.

[0384] As used in this article, “disease” or “disorder” refers to a condition that requires and / or is expected to be treated.

[0385] Unless otherwise specified, the terms “tumor cell,” “cancer cell,” “cancer,” “tumor,” and / or “proliferation” are used interchangeably herein and refer to cells (or cells) exhibiting uncontrolled growth and / or abnormally increased cell survival and / or suppressed apoptosis that interfere with the normal function of organs and systems of the body. This definition includes benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases. The terms “cancer” and “tumor” cover solid cancers and hematologic / lymphomas, and also encompass malignant, pre-malignant, and benign growths, such as dysplasia. Furthermore, this definition also includes cells with abnormal proliferation that is not hindered by the immune system (e.g., immune evasion and immune escape mechanisms) (e.g., virus-infected cells). Exemplary tumor cells include, but are not limited to: basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and central nervous system cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancers, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, liver cancer, hepatocellular carcinoma, intraepithelial neoplasia, kidney or renal cell carcinoma; laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system cancers, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer. Cancers of the urinary system, vulvar cancer; lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, and B-cell lymphomas, including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, advanced immunoblastic NHL, advanced lymphoblastic NHL, advanced small non-cleaved cell NHL, giant lesion NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenström macroglobulinemia; chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, and chronic myeloid leukemia; other cancers and sarcomas; post-transplant lymphoproliferative disorder (PTLD), and abnormal angiogenesis associated with nevus hamartomatosis, edema (e.g., edema associated with brain tumors), and Meigs syndrome.

[0386] In some implementations, the term cancer may encompass lung cancer, breast cancer, head and neck cancer, ovarian cancer, and / or endometrial cancer. Where necessary, the distinction between "cancer" and "cancer cells" can be made by explicitly using the phrase "cancer cells"; however, the term "cancer" will encompass concepts such as a subject with cancer, multicellular tumors, and single cancer cells.

[0387] "Increase or decrease" refers to a statistically significant increase or decrease. Those skilled in the art will appreciate that "modulation" can also involve achieving changes (which may be increases or decreases) in the affinity, specificity, and / or selectivity of the target or antigen for one or more ligands, binding couplers, couplers bound to homomeric or heteromeric forms, or substrates; achieving changes (which may be increases or decreases) in the sensitivity of the target or antigen under one or more conditions in the presence of the target or antigen (e.g., pH, ionic strength, presence of cofactors, etc.) compared to the same conditions in the absence of antibodies, bispecific or multispecific peptide reagents. This can be determined, depending on the target involved, in any suitable manner and / or using any suitable assay known per se or described herein.

[0388] When used in this article, “immune response” is intended to encompass cellular and / or humoral immune responses sufficient to suppress or prevent the onset of a disease (such as cancer or cancer metastasis) or to improve the symptoms of a disease. “Immune response” can encompass aspects of both the innate and adaptive immune systems.

[0389] When used herein, “treatment” is a method of achieving a beneficial or desired clinical outcome. As used herein, “treatment” encompasses any administration or application of a therapeutic agent for a disease in mammals, including humans. For the purposes of this disclosure, a beneficial or desired clinical outcome includes, but is not limited to, any or more of the following: relief of one or more symptoms, reduction of the severity of the disease, prevention or delay of disease spread (e.g., metastasis, such as to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or slow disease progression, improvement of the disease state, inhibition of the disease or disease progression, inhibition or slowing of the disease or its progression, prevention of its development, and remission (whether partial or complete). “Treatment” also encompasses reducing the pathological consequences of proliferative diseases. The methods provided herein take into account any or more of these aspects of treatment. Consistent with the foregoing, the term treatment does not require the complete elimination of all aspects of the obstruction.

[0390] "Improvement" refers to the reduction or improvement of one or more symptoms compared to not administering CD180 antibodies. "Improvement" also includes shortening or reducing the duration of symptoms.

[0391] The term "biological sample" refers to a quantity of material derived from or formerly from a living organism. Such material includes, but is not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0392] The term "control" refers to a composition known to contain no analyte ("negative control") or a composition containing analyte ("positive control"). A positive control may contain a known concentration of analyte. "Control," "positive control," and "calibrator" are used interchangeably herein and refer to a composition containing a known concentration of analyte. A "positive control" can be used to establish the performance characteristics of an assay and is a useful indicator of the integrity of reagents (e.g., analytes).

[0393] "Predetermined cutoff value" and "predetermined level" generally refer to assay cutoff values ​​used to assess diagnostic / prognostic / treatment efficacy outcomes by comparing assay results to predetermined cutoff values / levels, where the predetermined cutoff values / levels have been associated with or correlated with various clinical parameters, such as disease severity, progression / non-progression / improvement, etc. While this disclosure may provide exemplary predetermined levels, it is well known that cutoff values ​​can vary depending on the nature of the immunoassay (e.g., the antibodies used). Those skilled in the art are also fully capable of adapting the disclosure herein to other immunoassays to obtain assay-specific cutoff values ​​for those other immunoassays based on this disclosure. Although the precise values ​​of predetermined cutoff values / levels may vary from assay to assay, the relevance described herein, if any, is likely to apply generally.

[0394] The term "inhibition" refers to a reduction or cessation of any phenotypic trait, or a reduction or cessation of the incidence, extent, or likelihood of that trait. "Reduction" or "inhibition" means a decrease, reduction, or cessation of activity, function, and / or amount compared to a reference. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 20% or more. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 50% or more. In some embodiments, "reduction" or "inhibition" means the ability to result in an overall reduction of 75%, 85%, 90%, 95%, or more. In some embodiments, the aforementioned amounts are inhibited or reduced over the same time period relative to a control (e.g., placebo) over a period of time.

[0395] When used herein, “delay disease progression” means to postpone, hinder, slow, delay, stabilize, inhibit, and / or slow the development of a disease (such as cancer). Such delay can have varying durations, depending on the individual’s medical history and / or the individual being treated. As will be apparent to those skilled in the art, adequate or significant delay can effectively encompass prevention, as the individual does not develop the disease. For example, the development of advanced cancer, such as metastasis, can be delayed.

[0396] When used herein, “prevention” includes providing prevention of the onset or recurrence of the disease in subjects who may be susceptible to the disease but have not yet been diagnosed with it. Unless otherwise specified, the terms “reduction,” “suppression,” or “prevention” do not imply or require complete prevention at all times.

[0397] When used herein, "inhibitory" function or activity means a reduction in said function or activity compared to the same conditions other than the condition or parameter of interest, or compared to another condition. For example, an antibody that inhibits tumor growth reduces the growth rate of said tumor (compared to the tumor growth rate in the absence of said antibody).

[0398] The term "antibody-binding region" refers to a region of an antigen containing the epitope to which the antibody binds. Antibody-binding regions can be determined using epitope binning with biolayer interferometry, alanine scanning, or domain mixing assays (using antigen constructs in which a region of an antigen is exchanged with an antigenic region of another species, and determining whether the antibody still binds to the antigen). The amino acids within the antibody-binding region that interact with the antibody can be determined by hydrogen / deuterium exchange mass spectrometry and / or by crystallography of the antibody bound to the antigen.

[0399] As used herein, the term "antibody" refers to both immunoglobulin molecules (i.e., molecules containing antigen-binding sites that specifically bind to antigens (responding to an immune response to an antigen)) and the immunologically active portion of immunoglobulin (Ig) molecules. "Specific binding," "immune response to," or "immunospecific binding" means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other peptides or with much lower affinity (K+). d > 10 -6 ) binding. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, F ab F ab’ and F (ab')2 Fragments, scFv and Fab Expression Library.

[0400] The basic structural unit of an antibody is known to be a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having a "light" chain (approximately 25 kDa) and a "heavy" chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids, primarily responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region, primarily responsible for effector function. Generally, antibody molecules derived from humans involve any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another due to the nature of the heavy chain present in the molecule. Some classes also have subclasses, such as IgG1, IgG2, IgG4, etc. Furthermore, in humans, the light chain may be either a κ chain or a λ chain.

[0401] As used herein, the term "monoclonal antibody" (MAb) or "monoclonal antibody composition" refers to a population of antibody molecules containing only one type of antibody molecule, said antibody molecule being composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining region (CDR) of a monoclonal antibody is identical in all molecules of said population. MAbs contain antigen-binding sites capable of inducing an immune response to a specific epitope of an antigen, characterized by a unique binding affinity to said epitope.

[0402] The terms "antigen-binding region," "antigen-binding site," or "binding portion" refer to the portion of an immunoglobulin molecule involved in antigen binding. An antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") region of the heavy ("H") and light ("L") chains. Three highly divergent segments (referred to as "hypervariates") within the V regions of the heavy and light chains are interspersed between more conserved flanking segments referred to as "framework regions" or "FRs." Therefore, the term "FR" refers to the naturally occurring amino acid sequence between and near the hypervariates in an immunoglobulin. In an antibody molecule, the three hypervariates of the light chain and the three hypervariates of the heavy chain are arranged opposite each other in three-dimensional space to form an antigen-binding surface. This antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariates of each heavy and light chain are referred to as "complementarity-determining regions" or "CDRs." Various methods for numbering the amino acid sequence of an antibody and identifying the complementarity-determining regions are known in the art. For example, the Kabat numbering system (see Kabat, EA et al., Sequences of Protein of immunological interest, 5th edition, US Department of Health and Human Services, US Government Printing Office (1991)) or the IMGT numbering system (see IMGT ®, the internationalImMunoGeneTics information system ® The IMGT exon numbering system is available online at http: / / www.imgt.org / . It is routinely used and recognized in the art as a reliable and accurate system for determining amino acid positions in coding sequences, allele alignment, and facilitating comparison of sequences in immunoglobulins (IG) and T-cell receptors (TR) from all vertebrate species. The accuracy and consistency of IMGT data are based on IMGT-NTOLOGY, the first and, to date, only ontology for immunogenetics and immunoinformatics (see Lefranc. MP et al., Biomolecules, 2014 Dec; 4(4), 1102-1139). IMGT tools and databases operate against the IMGT reference catalog built from large sequence libraries. In the IMGT system, IG V-DOMAIN and IG C-DOMAIN are delimited with exon delimitation taken into account where appropriate. Therefore, with the greater availability of sequences in the IMGT database, those skilled in the art can and "use" the IMGT exon numbering system to reliably determine amino acid positions in coding sequences and perform allele alignment. In addition, the correspondence between the IMGT unique number and other numbers (i.e., Kabat) can be obtained from the IMGT scientific chart (see Lefranc. MP et al., Biomolecules, 2014 Dec; 4(4), 1102-1139).

[0403] The terms "hypervariant region" or "variable region" refer to the amino acid residues of an antibody that are typically responsible for antigen binding. The hypervariant region usually contains amino acid residues from the "complementarity-determining region" or "CDR" (e.g., when numbered according to the Kabat numbering system, V...). L Residues approximately at positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the V... H Residues approximately 31–35 (H1), 50–65 (H2), and 95–102 (H3) in the sequence; Kabat et al., Sequences of Protein of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and / or those residues from the “hypervariant ring” (e.g., when numbered according to the Chothia numbering system, V). LResidues at positions 24-34 (L1), 50-56 (L2), and 89-97 (L3), as well as V H Residues at positions 26-32 (H1), 52-56 (H2), and 95-101 (H3) in the VCDR; Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987); and / or those residues from the “hypervariant ring” VCDR (e.g., when numbered according to the IMGT numbering system, V L Residues at positions 27-38 (L1), 56-65 (L2), and 105-120 (L3), and V H Residues at positions 27-38 (H1), 56-65 (H2), and 105-120 (H3) in the antibody; Lefranc, MP, et al., Nucl. Acids Res. 27:209-212 (1999); Ruiz, M., et al., Nucl. Acids Res. 28:219-221 (2000)). Optionally, when numbered according to AHo, the antibody has symmetrical insertions at one or more of the following sites: V L The 28th, 36th (L1), 63rd, 74-75th (L2) and 123rd (L3) bits, and V H Positions 28, 36 (H1), 63, 74-75 (H2) and 123 (H3) in; Honneger, A. and Plunkthun, A., J. Mol. Biol. 309:657-670 (2001)).

[0404] As used herein, the term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins, scFv, or T-cell receptors. Epitope determinants typically consist of chemically active surface clusters of molecules such as amino acid or sugar side chains and generally possess specific three-dimensional structural features and specific charge characteristics. An antibody is considered to specifically bind to an antigen when its dissociation constant is ≤ 1 µM (e.g., ≤ 100 nM, preferably ≤ 10 nM, more preferably ≤ 1 nM).

[0405] As used herein, the terms "immunobinding" and "immunobinding properties" refer to a class of non-covalent interactions that occur between immunoglobulin molecules and antigens specifically targeted by those immunoglobulins. The strength or affinity of an immune-binding interaction can be determined based on the dissociation constant (K0) of the interaction. d Let K be the smaller one. dThis indicates a high affinity. The immunobinding properties of the selected peptide can be quantified using methods known in the art. One such method requires measuring the rates of antigen-binding site / antigen complex formation and dissociation, where these rates depend on the concentration of the complex's partner, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Therefore, by calculating the concentration and the actual association and dissociation rates, the "association rate constant" (K0) can be determined. on ) and "dissociation rate constant" (K off (See Nature 361:186-87 (1993)). K off / K on The ratio can eliminate all parameters unrelated to affinity and is equal to the dissociation constant K. d (Generally speaking, see Davies et al., (1990) Annual Rev Biochem 59:439-473). When the equilibrium binding constant (K... d When the concentration is ≤ 1 µM (e.g. ≤ 100 nM, preferably ≤ 10 nM, more preferably ≤ 1 nM), the antibody of the present invention is said to specifically bind to its target, as measured by assays such as radioligand binding assays or similar assays known to those skilled in the art.

[0406] As used herein, the term "isolated polynucleotide" means a polynucleotide of genomic, cDNA, or synthetic origin, or certain combinations thereof, in which, due to its origin, "isolated polynucleotide" (1) is not associated with all or part of the polynucleotide that is present in nature, (2) is operatively linked with a polynucleotide that is not linked to in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides according to the invention include nucleic acid molecules encoding heavy chain immunoglobulin molecules and nucleic acid molecules encoding light chain immunoglobulin molecules as described herein.

[0407] The term “isolated protein” as used in this article means a protein of cDNA, recombinant RNA or synthetic origin or some combination thereof, which, due to its origin or derivative, is (1) not associated with a protein present in nature, (2) does not contain other proteins from the same source, such as marine proteins, (3) is expressed by cells from a different species, or (4) does not exist in nature.

[0408] The term "peptide" is used herein as a general term to refer to a native protein, fragment, or analogue of a polypeptide sequence. Thus, native protein fragments and analogues are types of polypeptides. Peptides according to the invention include heavy chain immunoglobulin molecules and light chain immunoglobulin molecules as described herein, as well as antibody molecules (and vice versa) comprising combinations of heavy chain immunoglobulin molecules and light chain immunoglobulin molecules (e.g., κ light chain immunoglobulin molecules), and their fragments and analogues.

[0409] As used in this article, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that exists in organisms (including viruses) that can be isolated from natural sources and has not been intentionally modified by humans in a laboratory or elsewhere is naturally occurring.

[0410] As used herein, the term "operably linked" refers to the position of the described components in a relationship that allows them to function in the intended manner. A control sequence "operably linked" to a coding sequence is connected in a way that enables the representation of the coding sequence to be realized under conditions compatible with the control sequence.

[0411] As used herein, the term "control sequence" refers to a polynucleotide sequence essential to the expression and processing of the coding sequence to which it is linked. The nature of such control sequences varies depending on the host organism; in prokaryotes, they typically include promoters, ribosome binding sites, and transcription termination sequences, while in eukaryotes, they typically include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include other components whose presence is favorable, such as leader sequences and fusion partner sequences. The term "polynucleotide" as used herein refers to a polymer of nucleotides at least 10 bases in length, which may be ribonucleotides or deoxyribonucleotides, or a modified form of any type of nucleotide. The term includes both single-stranded and double-stranded DNA.

[0412] When used herein, the twenty common amino acids and their abbreviations follow conventional usage. See *Immunology - A Synthesis* (2nd ed., E.S. Golub and DR. Gren, Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the twenty common amino acids (e.g., D-amino acids), non-natural amino acids such as α-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components of the peptides of this invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-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 peptide notation used herein, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[0413] When applied to peptides, the term "substantial identity" means that two peptide sequences share at least 80% sequence identity when optimally aligned (e.g., using the procedures GAP or BESTFIT with default vacancy weights), preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity.

[0414] Preferably, the different residue positions are due to conserved amino acid substitutions.

[0415] Conservative amino acid substitution refers to the interchangeability of residues with similar side chains. For example, a group of amino acids with aliphatic side chains includes glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains includes serine and threonine; a group of amino acids with amide-containing side chains includes asparagine and glutamine; a group of amino acids with aromatic side chains includes phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains includes lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains includes cysteine ​​and methionine. Preferred conserved amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0416] As discussed herein, the present invention envisions minute changes in the amino acid sequence of antibody or immunoglobulin molecules, provided that such changes retain at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99% of the activity. Specifically, conserved amino acid substitutions are envisioned. Conserved substitutions are substitutions occurring within side-chain-related amino acid families. Genetically encoded amino acids are generally classified into the following families: (1) acidic amino acids are aspartic acid and glutamic acid; (2) basic amino acids are lysine, arginine, and histidine; (3) nonpolar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. Other amino acid families include (i) serine and threonine, which are aliphatic hydroxyl families; (ii) asparagine and glutamine, which are amide-containing families; (iii) alanine, valine, leucine, and isoleucine, which are aliphatic families; and (iv) phenylalanine, tryptophan, and tyrosine, which are aromatic families. For example, it is reasonable to expect that individually replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or similarly replacing amino acids with structure-related amino acids, will not have a significant impact on the binding or properties of the resulting molecule, especially if the replacement does not involve amino acids within the framework site. Whether an amino acid change results in a functional peptide can be readily determined by measuring the specific activity of the polypeptide derivative. The assay is described in detail herein. Fragments or analogues of antibody or immunoglobulin molecules can be readily prepared by those skilled in the art. Preferred amino and carboxyl terms of fragments or the like appear near the boundaries of functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with public or proprietary sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformations that appear in other proteins with known structures and / or functions. Methods for identifying protein sequences folded into known three-dimensional structures are known. (Bowie et al., Science 253:164 (1991)). Therefore, the above examples demonstrate that those skilled in the art can identify sequence motifs and structural conformations that can be used to define the structural and functional domains according to the present invention.

[0417] Preferred amino acid substitutions are those that: (1) reduce sensitivity to proteolysis; (2) reduce sensitivity to oxidation; (3) alter the binding affinity for forming protein complexes; (4) alter the binding affinity; and (5) impart or alter other physicochemical or functional properties to such analogs. Analogs may include various mutant proteins with sequences other than naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conserved amino acid substitutions) may be made in the naturally occurring sequence (preferably in portions outside the domains forming intermolecular contacts of the polypeptide). Conserved amino acid substitutions should not substantially alter the structural characteristics of the parental sequence (e.g., the substituted amino acid should not tend to break helices present in the parental sequence or disrupt other types of secondary structures characteristic of the parental sequence). Examples of recognized peptide secondary and tertiary structures in this field are described in the following literature: Proteins, Structures and Molecular Principles (edited by Creighton, WH Freeman and Company, New York (1984)); Introduction to Protein Structure (edited by C. Branden and J. Tooze, Garland Publishing, New York, NY (1991)); Thornton et al., Nature 354:105 (1991).

[0418] As used herein, the terms "labeled" or "labeled" refer to the incorporation of a detectable label (e.g., by incorporation of a radiolabeled amino acid) or the attachment of a biotinylated moiety detectable by a labeled avidin (e.g., streptavidin containing a fluorescent label or enzyme activity detectable by optical or calorimetric methods) to a polypeptide. In some cases, the label or label can also be a therapeutic agent. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., radioactive isotopes or radionuclides). 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 1311) Fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotin groups, and predetermined polypeptide epitopes recognized by a second reporter (e.g., leucine zipper pairs, binding sites of second antibodies, metal-binding domains, epitope tags). In some embodiments, the labels are attached via spacer arms of various lengths to reduce potential steric hindrance. As used herein, the term "pharmaceutical agent or drug" refers to a compound or composition that, when properly administered to a patient, can induce a desired therapeutic effect.

[0419] Other chemical terms used in this article are used according to their conventional usage in the field, as exemplified in The McGraw-Hill Dictionary of Chemical Terms (edited by Parker, S., McGraw-Hill, San Francisco (1985)).

[0420] When used herein, “substantially pure” means that the target substance is the dominant substance present (i.e., more abundant in moles than any other individual substance in the composition), and preferably, the substantially purified fraction is a composition in which the target substance constitutes at least about 50% (in moles) of all macromolecules present.

[0421] Typically, a substantially pure composition will comprise more than 80% of all macromolecules present in the composition, more preferably more than 85%, 90%, 95%, and 99%. Most preferably, the target substance is purified to a substantially homogeneous state (in which contaminants cannot be detected by conventional detection methods), wherein the composition consists substantially of a single macromolecule.

[0422] The term "patient" includes both human and veterinary subjects.

[0423] Example

[0424] Example 1: Design and analysis of anti-CD3 / CD180 bispecific antibody fusion molecules

[0425] A schematic diagram of the anti-CD3 / anti-CD180 targeting bispecific antibody disclosed herein is depicted in Figure 4 and comprises the following parts: (1) a first antigen-binding region that binds to CD3, which contains the SP34 antibody variant (“anti-CD3” or “anti-CD3 arm”); (2) a second antigen-binding region that binds to CD180 (“anti-CD180” or “anti-CD180 arm”); (3) a mortising mutation in the heavy chain; (4) a mutation that forces the light chain to heterodimerize; (5) a protein linker; and (6) a full-length CD58 peptide fused to the C-terminus of the heavy chain of the CD3-binding arm.

[0426] Antibody sequencing

[0427] Antibody sequencing of the mouse monoclonal MHR73-11 antibody clone was performed at Bioinformatics Solutions Inc. (Waterloo, ON, Canada). Briefly, the antibody was reduced with DTT, alkylated with iodoacetamide, and then digested into concatenated peptides using Asp N, chymotrypsin, elastase, trypsin, and pepsin, followed by reversed-phase chromatography purification. Full 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, the peptides were analyzed on an Orbitrap analyzer (Orbitrap Fusion Lumos, Thermo Fisher Scientific). Both full MS scans and MS2 scans were acquired on a 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 differential analysis, using PEAKS AB 2.0 software (Bioinformatics Solutions Inc.).

[0428] Mouse antibody structure modeling

[0429] Homology models of the variable regions of the mouse anti-CD180 antibody MHR73-11 were generated using the Antibody Structure Prediction tool in BioLuminate software (Schrödinger, LLC, New York, NY, 2021). Based on the mouse VH and VL sequences of MHR73-11, the following templates were selected from the curated antibody database: (1) PDB structure 6J5D was used as the light chain template, (2) PDB structure 1XIW was used as the heavy chain template, and (3) 6J5D was also used as a general template for aligning the heavy and light chain architectures. Templates for the loops were automatically determined based on the sequence similarity of the loop clusters. The generated models were visualized and refined to address any structural issues, such as spatial conflicts, missing atoms, etc. This was achieved by using the Protein Preparation Workflow in Bioluminate, with constraint minimization using the OPLS_2005 force field.

[0430] Antibody humanization

[0431] The mouse MHR73-11 antibody was humanized using BioLuminate (Schrodinger). In short, a mouse MHR73-11 homology model was compared with a human antibody structure database to identify human antibody frameworks with the highest level of sequence and structural similarity to the mouse antibody. The CDR loop was then transplanted onto the human framework. Structure-directed mutagenesis was introduced to eliminate spatial conflicts and maximize the original geometry of the stem in the CDR region.

[0432] A total of 16 proposed humanized antibodies were obtained. These 16 proposed humanized antibodies are derived from combinations of 7 heavy chain and 7 light chain sequences.

[0433] Small-scale antibody expression and purification

[0434] The obtained heavy and light chain DNA sequences were synthesized in the pDT5 vector (ATUM, Newark CA). All proposed humanized variants were expressed on a small scale by transfecting Expi293 cells with plasmid DNA (1 μg / ml) (ThermoFisher) according to the manufacturer's protocol. Cells were grown in culture flasks at 37°C and 8% CO2, rotated (125 rpm). Five days after transfection, cells were centrifuged (3000 xg) for 30 minutes and discharged with 0.45... Conditioned medium was harvested from cells using an M filter. Antibodies were then purified using protein A magnetic agarose beads (from Pierce, catalog 78610). The beads-bound antibodies were eluted with 0.1 M citrate buffer (pH 3.0) and neutralized with 1 M Tris (pH 8.0). The antibody buffer was replaced with PBS (pH 7.4) using a Zeba rotary desalting column (Thermofisher, catalog 89892) and stored at 4°C.

[0435] Enzyme-linked immunosorbent assay (ELISA)

[0436] The binding of the humanized variant to human and cynomolgus monkey CD180-MD1 was analyzed by ELISA. Maxisorp plates (Nunc) were coated with protein at a concentration of 4 µg / ml in PBS (pH 7.2) at 4 °C for 16 hours. 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 with PBS (pH 7.2) and 0.1% Tween. The humanized variant was diluted in blocking buffer at a 1:4 ratio starting from 100 nM and incubated with the blocked wells for 1 hour. The plates were then washed and incubated with HRP-conjugated goat anti-human IgG Fcγ fragment-specific antibody (Jackson Immune Research Labs, code: 109-035-098) diluted 1 / 800. After the final wash, the luminescent substrate was added according to the manufacturer's instructions (SeraCare). The luminescence was measured using an Ensight plate reader (Perkin Elmer).

[0437] Surface plasmon resonance (SPR) Biacore 8K dynamics combined analysis

[0438] The binding of the exemplary bispecific antibody to the recombinant human CD180-MD1 heterodimer was further analyzed by surface plasmon resonance (SPR). The analysis was performed using a Biacore 8K system (Cytiva). Briefly, the bispecific antibody was diluted to 10 μg / mL in 10 mM sodium acetate at pH 5.0 and immobilized on a CM5 sensor chip via standard amine coupling recommended by Cytiva. Multiple-cycle kinetic analysis was then performed by injecting different concentrations of CD180-MD1 samples diluted in running buffer (10 mM HEPES, 150 mM NaCl, 0.05% v / v surfactant P20, pH 7.4) into each channel at a flow rate of 30 μL / min. Binding kinetics were performed at 25 °C. The association and dissociation times were 300 s and 600 s, respectively. The chip surface was regenerated by injecting 10 mM glycine (pH 2.0) at a flow rate of 30 μL / min for 30 s. The kinetic and affinity constants were determined by fitting the data to a 1:1 combined model using Biacore Insight evaluation software (Cytiva).

[0439] Development of stable cell lines: CD180 protein expression and purification Stable cell lines were prepared using the manufacturer's protocol (Lonza Group AG). To achieve co-expression of human CD180 and MD1, a dual-gene vector (DGV) was created, which incorporated single-gene vectors (SGV) containing CD180 and MD1, respectively.

[0440] For human CD180, the extracellular domain sequence (Uniprot accession number Q99467; residues 24-626 (SEQ ID NO: 418)) was used, and a C-terminal double Strep tag was added for protein purification. For human MD1, residues 21-162 of Uniprotocol accession number O95711 (SEQ ID NO: 417) were taken, and a C-terminal (His)6 tag was added.

[0441] Similarly, a dual-gene vector for co-expressing the cynomolgus CD180-MD1 complex was constructed. For this purpose, the extracellular domain sequence of cynomolgus CD180 with a C-terminal double Strep tag (Uniprot accession number A0A2K5VMS6, residues 24-626) (SEQ ID NO: 416) and the cynomolgus MD1 sequence with a C-terminal (His)6 tag (Uniprot accession number A0A2K5UVX3, residues 21-162) (SEQ ID NO: 415) were used.

[0442] The prepared plasmid was then transfected into the CHOK1SV GS-KO host cell line (part of Lonza's GS Xceed system) via electroporation. After cell recovery, the cells were scaled up for fed-batch shake-flask culture according to the manufacturer's protocol. At the end of 12 days of fed-batch culture, the culture medium was centrifuged and filtered through a 0.2 μm filter for purification.

[0443] Protein expression and purification

[0444] The filtered supernatant was incubated with Biolock (IBA life sciences, 2-0205-050) for 1 hour to chelate any biotin that may be present in the culture medium. The CD180-MD1 complex was purified by elution using a Strep-Tactin® XT 4Flow® high-capacity FPLC column (IBA life sciences, 2-5027-001) and eluted with elution buffer (0.1 M Tris-Cl, 0.15 M NaCl, 1 mM EDTA, 0.25 mM dethiobiotin, pH 8). The molecular weight and integrity of the CD180-MD1 complex were confirmed by SDS-PAGE and size exclusion chromatography, respectively. Figure 1A and 1B as well as Figure 2 ).

[0445] Large-scale antibody expression and purification

[0446] The obtained heavy and light chain DNA sequences were synthesized in the pDT5 vector (ATUM, Newark CA). Following the manufacturer's protocol, the plasmid DNA (1 μg / ml) was transfected into Expi293 cells (ThermoFisher). Cells were grown in culture flasks at 37°C and 8% CO2, rotated (125 rpm). Five days post-transfection, cells were centrifuged (3000 xg) for 30 minutes and cultured with 0.45... Conditioned medium was harvested from the cells using an m-filter. The antibody was then purified using a tandem purification method with an AKTA Avant chromatography system (Cytiva) and a HiTrap Mabselect Protein A and HiLoad Superdex 200 pg column (Cytiva). After purification and before analysis, the antibody was stored in PBS at 4°C at pH 7.4.

[0447] Differential scanning calorimetry

[0448] Differential scanning calorimetry (DSC) was performed using a Nano DSC instrument from TA Instruments – Waters LLC. Protein samples were prepared in PBS at a concentration of 1 mg / mL, loaded into the sample cell, and scanned from 25 to 95 °C at an increase rate of 1 °C / min. Data were analyzed using the NanoAnalyzer program, with PBS buffer background subtracted from each individual scan.

[0449] Mass spectrometry

[0450] The integrity of the purified fusion protein and antibody chain was confirmed using a Xevo G2-XS QTof quadrupole time-of-flight mass spectrometer coupled to an Acquity UHPLC system (Waters) equipped with a Protein BEH C4 (300 Å 1.7 µm) column. First, the antibody sample was deglycosylated using a rapid PNGase F enzyme (New England Biolabs) under both reducing and non-reducing conditions, following the supplier's protocol. The reaction mixture was diluted to 1:10 in 50% acetonitrile containing 0.1% formic acid, and 2 μL was injected into LC-MS. Total ion chromatograms and m / z data of the protein were obtained using gradient runs of 10 to 70 mL HPLC-grade acetonitrile over 12 min. The mass of the protein sample was deconvoluted from the total ion chromatogram using BYOS software from Protein Metrics.

[0451] Hematologic cancer cell line

[0452] Hematologic cancer cell lines ordered from ATCC: HT (DLBCL), SU-DHL-10 (DLBCL), MV-4-11-GFP-luc (AML), JeKo-1 (mantle cell lymphoma), Raji (Burkitt lymphoma), and Ramos (Burkitt 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 for internal use.

[0453] Target expression assessment and biobinding with tumor cell lines

[0454] The BD Biosciences FACSymphony A3 instrument was used for analytical flow cytometry. For CD180 surface expression assessment, cell lines were stained with the Zombie NIR™ Immobilizable Activity Kit (BioLegend). Cells were then incubated with human TruStain FcX Fc blocking solution (BioLegend) and stained with an anti-CD180 antibody (MHR73-11 clone) or an isotype antibody conjugated with APC (MOPC-21) (BioLegend) to assess target expression on the cell surface.

[0455] The binding of antibodies (EIP0051, EIP0133, EIP0209, EIP0553, EIP0554, EIP0546, EIP1042, EIP1043, EIP0614, EIP1056, EIP1057) to tumor cell lines was determined by flow cytometry using a BD Biosciences FACSymphony A3. Cell lines were stained with the Zombie NIR™ Immobilizable Viability Kit (BioLegend) or by scattering-based staining to facilitate gating analysis of live cells, followed by incubation with serially diluted biologics. Surface-bound bispecific antibodies were detected by incubating cells with R-phycoerythrin (PE) of an Alexa Fluor 488-labeled anti-human IgG Fcγ antibody (Jackson ImmunoResearch). Cells were then analyzed by flow cytometry, or fixed in 2% PFA in PBS prior to flow cytometry analysis. Data were processed using FlowJo software (BD) and geometric mean fluorescence (MFI) of stained live cells was obtained.

[0456] Assay for activated T cell-mediated cytotoxicity

[0457] The efficacy of bispecific antibody-mediated tumor cell lysis was evaluated in activated T cell-tumor cell line co-culture assays (JeKo-1-GFP, Raji, Ramos). Pan-CD3 T cells were enriched from healthy human donor PBMCs using the EasySep Human T Cell Isolation Kit (Stemcell Technologies). To activate T cells, Dynabead coated with αCD3 and αCD28 antibodies (Invitrogen) was added to T cells at a ratio of 25 µL beads per million cells and incubated at 37°C in 5% CO2 for 48–72 hours. After removing the Dynabead, the T cells were incubated for another 7 days with IL-7 (10 ng / ml) in supplemental medium. The supplemental medium was replaced with freshly thawed cytokines every 48–72 hours. In the presence of serially diluted antibody, 100,000 expanded T cells and 20,000 GFP-expressing JeKo-1 cells or CellTrace Violet-labeled Raji or Ramos cells (E:T=5:1) were seeded per well and incubated at 37°C for 2 days. Cells were stained with the ZombieNIR™ Immobilizable Viability Kit (BioLegend) and then analyzed by flow cytometry. Live tumor cells were analyzed in FlowJo software. Cell lysis curves of tumor cells were generated using Prism software (GraphPad) by comparing normalized cell counts with bispecific antibody concentrations. Four-parameter nonlinear regression analysis was performed using Prism software (GraphPad).

[0458] Pan-CD3 T cell-mediated cytotoxicity assay

[0459] The efficacy of bispecific antibody-mediated tumor cell lysis was evaluated in a pan-CD3 T cell-JeKo-1 co-culture assay. Pan-CD3 T cells were enriched from healthy human donor PBMCs using the EasySep Human T Cell Isolation Kit (Stemcell Technologies) and seeded at 75,000 T cells per well with 10,000 GFP-expressing JeKo-1 cells (E:T = 7.5:1) in the presence of serially diluted CD180-targeting bispecific antibody, and incubated at 37°C for 3 days. Cells were stained with the ZombieNIR™ Immobilizable Viability Kit (BioLegend) and analyzed by flow cytometry. Viable tumor cells were analyzed in FlowJo software. Cell lysis curves of tumor cells were generated using Prism software (GraphPad) by comparing normalized cell counts with bispecific antibody concentrations. Four-parameter nonlinear regression analysis was performed using Prism software (GraphPad) to obtain EC50 and maximum killing.

[0460] PBMC-mediated cytotoxicity assay

[0461] The efficacy of bispecific antibodies in mediating tumor cell lysis was evaluated in a PBMC-tumor cell co-culture assay. PBMCs from healthy human donors were added to 96-well cell rejection plates (Greiner Bio-One) at a density of 150,000–200,000 cells per well. CellTrace Violet-labeled or GFP-luc-expressing human tumor cells were seeded at an E:T ratio of 8,000–15,000 cells per well in the presence or absence of antibody serial dilutions and incubated at 37°C. Acrylonitrile research-grade biosimilar (ProSci, catalog number 10-978) was purchased from ProSci Incorporated. After 3 days, the plates were centrifuged, and the supernatant was collected and frozen for further cytokine analysis. Cell deposits were stained with the Zombie NIR™ Immobilizable Activity Kit (BioLegend) followed by Fc receptor blocking and surface marker staining. Flow cytometry antibodies targeting surface cell markers (CD3, CD4, CD8, CD19, CD14, CD33) were purchased from BioLegend and BD Bioscience, and stained cells were analyzed by flow cytometry. The numbers of tumor cells, CD4 T cells, CD8 T cells, B cells, and monocytes were analyzed using FlowJo software. Cell counts or normalized cell counts were plotted against bispecific antibody concentrations using Prism software (GraphPad) to generate curves for tumor cell lysis, B cell and monocyte exhaustion, and CD4 T cell and CD8 T cell proliferation. Four-parameter nonlinear regression analysis was performed using Prism software (GraphPad) to obtain EC50 and maximum killing power.

[0462] Quantitative analysis of cytokine release using enzyme-linked immunosorbent assay (ELISA)

[0463] Cytokines (IFN-γ, IL-2, IL-6) were measured using the LEGEND MAX ELISA kit (BioLegend) and the LumiGlo peroxidase chemiluminescent substrate kit (SeraCare), following the manufacturer's protocol. Luminescence was measured using an Ensight plate reader (Perkin Elmer). Cytokine values ​​were interpolated using Excel. Four-parameter nonlinear regression analysis was performed using Prism software (GraphPad) to obtain EC50 and maximum cytokine release.

[0464] Measurement of CD180 antibody binding capacity of tumor cell lines

[0465] The BD Biosciences FACSymphony A3 instrument was used for analytical flow cytometry. For CD180 surface expression assessment, cell lines were stained with the Zombie Violet™ Immobilizable Activity Kit (BioLegend). Cells were then incubated with human TruStain FcX™ Fc Blocking Solution (BioLegend) and stained with an anti-CD180 antibody (MHR73-11 clone) or an isotype antibody conjugated with APC (MOPC-21) (BioLegend) to assess target expression on the cell surface. Quantum™ Simply Cellular was used according to the manufacturer's protocol. ® Anti-mouse IgG beads (Bangs Laboratories, Inc.) were used to determine the binding capacity of CD180 antibodies to tumor cell lines. Where applicable, the mean and standard deviation of antibody binding capacity were used.

[0466] Measurement of CD180 antibody binding capacity of AML embryonic cells in AML PBMCs

[0467] Thaw frozen AML PBMCs and stain them with BD Horizon™ fixable viable staining agent 780. Then stain with a flow cytometry antibody (Biolegend or BD) labeled with fluorescent dyes against human surface proteins, including CD45, CD3, CD4, CD8, CD14, CD33, CD20, CD25, CD69, CD123, and CD180. In a live single-cell population, AML embryonic cells were defined as CD45+CD33+ cells, B cells as CD45+CD33-CD20+ cells, and monocytes as CD45+CD33-CD14+ cells. Use Quantum™ Simply Cellular according to the manufacturer's protocol. ® Anti-mouse IgG beads (Bangs Laboratories, Inc.) were used to determine the binding capacity of CD180 antibodies on AML embryonic cells, B cells, and monocytes.

[0468] In vitro culture of AML PBMCs to evaluate the cytotoxicity of the bispecific fusion protein to AML embryonic cells.

[0469] Frozen AML PBMCs were thawed and cultured in 12-well plates, treated for 6 days with a specified concentration of the bispecific fusion protein or a control biologic. Cells were harvested on day 6 and then stained with a mixture of BD Horizon™ immobilizable active staining agent 780 and flow cytometry antibodies targeting human surface proteins (including CD45, CD3, CD4, CD8, CD14, CD33, CD20, CD25, CD69, CD123, and CD180) (Biolegend or BD). In a live single-cell population, AML embryonic cells were defined as CD45+CD33+ cells, T cells as CD45+CD33-CD3+, CD4 T cells as CD45+CD33-CD3+CD4+, and CD8 T cells as CD45+CD33-CD3+CD8+. The percentage of remaining AML embryonic cells in wells compared to the non-targeted control was normalized: AML embryonic cell reduction = 100% - remaining AML embryonic cells (%) compared to the non-targeted control. T cell fold expansion (%), CD4 T cell expansion, and CD8 T cell expansion were normalized for the corresponding non-targeted control wells.

[0470] Results and discussion: MHR73-11 is a mouse monoclonal antibody that recognizes human CD180 or RP105 (radioactive protein 105) (Miura et al., Blood. 1998 Oct 15;92(8):2815-22). The complete sequences of the variable heavy chain (SEQ ID NO: 161) and variable light chain (SEQ ID NO: 160) of the mouse monoclonal anti-human CD180 antibody MHR73-11 were determined by de novo protein sequencing using mass spectrometry. After elucidating the antibody sequence, MHR73-11 was humanized using a CDR transplantation method. In the absence of known crystal structures available for this antibody, a homology-based structural model of the Fv region of MHR73-11 (Figure 3) was queried in human antibody architecture databases to understand structural homology and sequence similarity. Figure 3A It emphasizes selecting templates based on higher structural and sequence similarity to successfully perform CDR transplantation, thereby maintaining binding to the recombinant CD180-MD1 complex, rather than templates with lower structural and sequence similarity. Figure 3BThe mouse CDR region, including Vernier region residues, canonical structure residues, and interface residues, was transplanted onto the candidate human scaffold. Structure-guided reversion mutations were introduced to eliminate spatial conflicts and maximize the stem geometry of the CDR region. Any spatial conflicts in the resulting humanized structures were examined and resolved via reversion mutations. Another set of humanized variants was prepared by selecting the closest human germline sequence as the scaffold template. In this case, the mouse MHR73-11 structure was structurally mutated to the selected template sequence, and any insertions or deletions were reconstructed.

[0471] In summary, we obtained a list of humanized MHR73-11 variant sequences for both the heavy and light chains. These humanized variants were expressed and purified on a small scale, as described in the Materials and Methods section. The binding of the purified humanized variants and mouse MHR73-11 antibody to recombinant human and cynomolgus monkey CD180-MD1 protein expressed and purified through stable cell lines was tested by sandwich ELISA (as described in the Materials and Methods section) (Table 17).

[0472] Table 17. Binding of humanized MHR73-11 variants and mouse MHR73-11 (EIP0464) obtained by ELISA to recombinant human CD180-MD1 protein.

[0473]

[0474] Variants that then exhibited similar or better binding to human CD180-MD1 as the mouse MHR73-11 antibody were expressed at larger scale and purified by SEC purification to further characterize their biophysical properties. These variants were then used to generate bispecific fusion molecules targeting CD180.

[0475] A schematic diagram of the CD180-targeting bispecific fusion described herein is depicted in Figure 4, which consists of the following components: (1) a CD180-targeting antibody, (2) a CD3-binding antibody consisting of previously described SP34 antibody variants (Table 18), (3) a mortis mutation in the heavy chain as previously described in WO2023178357A1 (the contents of which are incorporated herein in their entirety), and (4) a mutation corresponding to group D that forces heterodimerization of the light chain as previously described (refer to the platform patent) (Table 19), a protein linker, and a full-length CD58.

[0476] Table 18. Nomenclature and mutations associated with CD3 variants

[0477] Table 19. Light chain mutant group D

[0478] Bispecific and bispecific fusion antibodies targeting CD180 were generated in a transient Expi293 system. Figure 5 shows preparative size exclusion chromatography (ASC) images of several bispecific fusion proteins after protein A purification. The integrity and purity of the purified variants were also verified by analytical size exclusion chromatography (aSEC). Figure 5B-5F .

[0479] The thermal stability of the CD180-targeting bispecific fusion polymer was evaluated using differential scanning calorimetry (DSC). Figure 6 As shown in Table 20, all proteins exhibited a first major melt transition between 69 and 70 degrees Celsius, corresponding to the CH2 and CH3 domains with the mortising mutation. Following the first transition, a second major melt transition corresponding to the Fab domain was observed.

[0480] Table 20: Differential scanning calorimetry analysis of CD180-targeting bispecific fusion variants. The Tm values ​​for each variant are listed in the table.

[0481]

[0482] The degree of peptide chain mismatch for each bispecific fusion compound was assessed using mass spectrometry. This was achieved using EIP1042 (…). Figure 7A EIP1044 Figure 7C ) and EIP1056 ( Figure 7E Non-reduced intact antibody quality analysis was performed to observe the expected quality of correctly paired bispecific fusions. In EIP1042 ( Figure 7B EIP1044 Figure 7D ) and EIP1056 ( Figure 7E After reduction, four different polypeptide chains were observed that corresponded to the expected mass of two light chains and two heavy chains.

[0483] Table 21 and Figure 8A and 8B The study summarized the binding of a bispecific fusion targeting CD180 to human and cynomolgus monkey CD180-MD1.

[0484] Table 21. EC5 assays of the humanized CD180-targeting bispecific fusion polymer against the recombinant human and cynomolgus CD180-MD1 complex obtained by ELISA 50 value

[0485] The binding of the exemplary bispecific antibody to the recombinant human CD180-MD1 heterodimer was further analyzed using surface plasmon resonance (SPR). The kinetics and affinity constant (k) of the human CD180-MD1 heterodimer were also analyzed. a kd and K D (This is shown in Table 22.)

[0486] Table 22: Biacore surface plasmon resonance values ​​of the binding of CD180-targeted bispecific fusion complex to human CD180-MD1 heterodimer complex.

[0487]

[0488] The mouse antibody MHR73-11 (EIP0464) was compared with humanized bispecific MHR73-11 antibodies EIP0698 and EIP0699 to determine their binding to the human and cynomolgus monkey CD180-MD1 protein complex. Figure 9A and 9B As shown in Table 23, compared with the mouse MHR73-11 antibody (EIP0464), the humanized bispecific (EIP0698) and bispecific fusion molecule (EIP0699) exhibited comparable binding affinity to the human and cynomolgus CD180-MD1 protein complex. This indicates that the humanized molecules in bispecific fusion form are similar to the mouse CD180 antibody MHR73-11 and retain affinity for the antigen.

[0489] Table 23. Comparison of EC5 activity of mouse MHR73-11 antibody (EIP0464) and humanized CD180-targeting bispecific antibody (EIP0698) and bispecific fusion (EIP0699) against recombinant human and cynomolgus monkey CD180-MD1 complexes obtained by ELISA. 50 value.

[0490]

[0491] To conduct a proof-of-concept study, we compared the MHR73-11 antibody (EIP0464) with the G28.8 antibody (EIP0100) (US Patent No. 9260529). Figure 10 As shown, MHR73-11 has a higher binding affinity for recombinant CD180-MD1 compared to G28.8.

[0492] Table 24. EC50 of MHR73-11 (EIP0464) antibody versus G28.8 (EIP0100) antibody binding to recombinant human CD180-MD1 obtained by ELISA.

[0493]

[0494] In addition, we used G28.8 or MHR73-11, in a bispecific form with and without CD58 fusion, to generate chimeric antibodies by inserting mouse-derived mouse sequences or humanized MHR73-11 as tumor-targeting arms into the human IgG backbone: EIP0051 is CD3xG28.8, EIP0133 is CD3xMHR73-11, EIP0546 is CD3xB12 (non-targeted control), EIP0553 is CD3x humanized MHR73-11, and EIP0554 is CD3x humanized MHR73-11-CD58. We evaluated the binding of these biologics to CD180-expressing cell lines: JeKo-1 ( Figure 11 A), Raji ( Figure 11 B) and Ramos Figure 11 C), and we consistently observed that antibodies derived from mouse MHR73-11 (EIP0133) and humanized MHR73-11 (EIP0553, EIP0664) showed similar binding to tumor cells, while the mouse G28.8-derived bispecific antibody (EIP0051) showed weaker binding. We then further tested the bispecific antibodies in an activated T cell-tumor cell co-culture assay to assess cell lysis activity (EIP0051, EIP0133), with EIP0133 (based on MHR73-11) showing greater potency than EIP0051 (based on G28.8): JeKo-1 ( Figure 12 A), Raji ( Figure 12 B) and Ramos Figure 12 C). EIP0133 showed an approximately 5 to 10-fold increase in tumor-killing efficacy compared to EIP0051, which is reflected in the EC50 ratio.

[0495] We selected a subset of B-cell malignancies (HT, SU-DHL-10, JeKo-1) and acute myeloid leukemia (MV-4-11) based on CD180 expression levels determined by RNA-seq (CCLE database), and confirmed surface CD180 expression (MFI) by flow cytometry (Table 25). Figure 13 A).

[0496] Table 25. Quantification of CD180 expression at the transcriptional level (RNA-seq, CCLE database) and the protein level (flow cytometry).

[0497]

[0498] To evaluate the binding of bispecific fusion biologics to CD180-expressing cell lines, we evaluated a series of bispecific fusion antibodies (EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) to HT ( Figure 13 B) and MV-4-11-GFP-luc ( Figure 13 C) Binding to cell lines. We observed that all antibodies showed comparable binding curves with cells, but in both cell line models, EIP1042, EIP1044, and EIP1056 may have shown relatively stronger binding compared to EIP1043 and EIP1057, which is reflected in higher maximum MFI of CD180.

[0499] To determine the effects of CD3 affinity regulation and CD58 fusion on the activity of bispecific antibodies, we generated a series of biologics targeting CD180 (with the same tumor antigen targeting arm but varying CD3 affinities) and evaluated their function in pan-CD3 T cell-JeKo-1 co-culture assays (Table 26) and PBMC-JeKo-1 co-culture assays (Table 27). The bispecific fusion biologics consistently outperformed their respective bispecific antibodies: most tested biologics achieved 90%+ tumor killing, except for EIP0751 (a very weak CD3 affinity bispecific antibody) which reached 87.2% (pan-CD3 T cells) and 75% (PBMCs); the CD58 fusion bispecific antibody showed greater cytolytic activity, as indicated by EC50. Cytokine measurements from PBMC-JeKo-1 co-cultures also showed that, compared to the bispecific design, the CD58 fusion bispecific antibody tended to induce comparable (IL-6) or higher (IFN-γ, IL-2) maximal cytokine production at lower concentrations. The therapeutic window of the bispecific antibody can be modulated by altering CD3 affinity, as defined by cytotoxicity and cytokine release.

[0500] Table 26. EC50, maximum killing and maximum cytokine release in vitro during naive T cell-JeKo-1 co-culture assays.

[0501]

[0502] Table 27. EC50, maximum killing and maximum cytokine release in vitro during PBMC-JeKo-1 co-culture assays.

[0503]

[0504] Two model cell lines, HT (B-cell malignant tumor), were used. Figure 14A-14G) and MV-4-11 (AML, Figure 15 A-15G), bispecific fusion antibodies (identical CD3 arm, different tumor antigen targeting arms, with CD58 fusion; EIP1042, EIP1043, EIP1044, EIP1056, EIP1057) were evaluated in a PBMC-tumor cell line co-culture assay. In the PBMC-HT co-culture assay, the activity of all five antibodies was assessed in tumor cell depletion (…). Figure 14 A) Decrease in B cells and monocytes ( Figure 14 Both showed comparable performance in terms of B-14C. When compared to the bispecific icoretuzumab research-grade biosimilar targeting CD20 (ProSci, catalog number 10-978), the CD180-targeting biologic showed higher potency in exhausting tumor cells and lower potency in reducing B cells. No monocyte exhaustion was observed under icoretuzumab research-grade biosimilar treatment (ProSci, catalog number 10-978).

[0505] We observed that T cell expansion was similar across all five variants with varying concentrations of the bispecific fusion antibody. Figure 14 D-14E), and EIP1043-induced T cell expansion was relatively low compared to other bispecific antibodies targeting CD180, but still higher than that of the research-grade biosimilar of icoretuzumab (ProSci, catalog number 10-978). The levels of cytokine release (IFN-γ and IL-2) induced by bispecific antibodies targeting CD180 were also lower. Figure 14 F-14G was superior to the research-grade biosimilar of Ectoretinib (ProSci, catalog number 10-978), and EIP1044 and EIP1056 induced more IL-2 at higher biologic concentrations. Figure 15 G). In the PBMC-MV-4-11 co-culture experiment, all five variants were exhausted in tumor cells (G). Figure 15 A) Decrease in B cells and monocytes ( Figure 15 B-15C), CD4 and CD8 T cell expansion ( Figure 15 EIP1044 and EIP1056 showed similar potency in terms of D-15E. Compared to EIP1042, EIP1043, and EIP1057, EIP1044 and EIP1056 showed higher levels of IFN-γ production. Figure 15 F). Among bispecific antibodies targeting CD180, there was no significant difference in IL-2 induction (F). Figure 15 G).

[0506] Table 28. CD180 expression in B-cell malignancies and AML cell lines

[0507] We measured the antibody binding capacity (ABC) of CD180 expressed on the surface of various B-cell tumor cell lines and AML cell lines. The mean, standard deviation, and range of CD180 ABC are reported in Table 28. Figure 13 Consistent with A, we confirmed the expression of CD180 on the surface of B-cell malignancies and acute myeloid leukemia cells.

[0508] Table 29. CD180 expression on AML germ cells, monocytes, and B cells in PBMCs of AML patients

[0509] We analyzed PBMCs (containing AML germ cells) from multiple AML patients. To characterize the patients, we report the following information for each patient sample in Table 29: AML FAB and WHO classification, clinical treatment of these patients before and after PBMC collection, clinical response to treatment, and Standard of Care (SoC) response in ex vivo PBMC culture. In Table 29, we summarize the frequency of CD180 positive expression and CD180 antibody binding capacity on AML germ cells, monocytes, and B cells in AML patients. We observed a variety of CD180 expression frequencies and target expression levels on AML germ cells in this cohort, with some patients (CTG-2228, CTG-2238, and CTG-2240) exhibiting more than 95+% CD180+ in AML germ cells. Within the same donor, AML germ cells may show higher CD180 ABCs than monocytes and B cells, indicating the potential for differential expression of CD180 on AML germ cells and monocytes or B cells.

[0510] Table 30. A bispecific fusion biologic targeting CD180 depletes AML embryonic cells and induces target-dependent T cell expansion in AML PBMC ex vivo cultures (CTG-3439).

[0511] After confirming CD180 expression in AML embryonic cells, we treated AML PBMCs in in vitro cultures for 6 days with selected bispecific fusion biologics (EIP0717, EIP0709, EIP0746) and a non-targeted control (EIP0614), and counted AML embryonic cells, T cells, CD4 T cells, and CD8 T cells to understand the biologic-mediated reduction and expansion of AML embryonic cells. Table 30 shows the results for AML patient CTG-3439: biologic-mediated reduction and expansion of AML embryonic cells, T cells, CD4 T cells, and CD8 T cells at the specified biologic concentrations. We observed dose-dependent reduction and expansion of AML embryonic cells (T cells, CD4 T cells, and CD8 T cells) when treated with EIP0717, EIP0709, and EIP0746, indicating the cytotoxicity of the CD180-targeting bispecific fusion biologics in primary AML PBMC samples.

[0512] Other implementation methods

[0513] Although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A bispecific antibody comprising a first antigen-binding region binding to CD3 and a second antigen-binding region binding to CD180. 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 contains the amino acid sequence of SEQ ID NO: 30; CDRH2 contains the amino acid sequence of SEQ ID NO: 34; CDRH3 contains the amino acid sequence of SEQ ID NO: 37; CDRL1 contains the amino acid sequence of SEQ ID NO: 42; CDRL2 contains the amino acid sequence of SEQ ID NO: 43; and CDRL3 contains the amino acid sequence of SEQ ID NO: 45; and 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 contains the amino acid sequence of SEQ ID NO: 210; CDRH2 contains the amino acid sequence of SEQ ID NO: 212; CDRH3 contains the amino acid sequence of SEQ ID NO: 214; CDRL1 contains the amino acid sequence of SEQ ID NO: 222; CDRL2 contains the amino acid sequence of SEQ ID NO: 223; and CDRL3 contains the amino acid sequence of SEQ ID NO:

224.

2. The bispecific antibody according to claim 1, wherein the first antigen-binding region binding to CD3 comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein: a) VH contains the amino acid sequence of SEQ ID NO: 17, and VL contains the amino acid sequence of SEQ ID NO: 22; and 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 contains the amino acid sequence of SEQ ID NO: 197, and VL contains the amino acid sequence of SEQ ID NO: 196; ii) VH contains the amino acid sequence of SEQ ID NO: 199, and VL contains the amino acid sequence of SEQ ID NO: 198; iii) VH contains the amino acid sequence of SEQ ID NO: 201, and VL contains the amino acid sequence of SEQ ID NO: 200; iv) VH contains the amino acid sequence of SEQ ID NO: 203, and VL contains the amino acid sequence of SEQ ID NO: 202; or v) VH contains the amino acid sequence of SEQ ID NO: 205, and VL contains 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 (CH1 H1 ), a hinge region (H1H), a constant region 2 domain (CH1 H2 ), and a constant region 3 domain (CH1 H3 ); a first light chain polypeptide (L1) comprising a variable region (VL1) and a constant region (CL1); a second heavy chain polypeptide (H2) comprising a variable region (VH2) and a constant region (CH2) having a constant region 1 domain (CH2 H1 ), a hinge region (H2H), a constant region 2 domain (CH2 H2 ), and a constant region 3 domain (CH2 H3 ); and a second light chain polypeptide (L2) comprising a variable region (VL2) and a constant region (CL2), and wherein the first and second heavy chain polypeptides are linked by a disulfide bond between the hinge regions (H1H and H2H), and the first and second light chain polypeptides are linked by a disulfide bond between the constant region 3 domains (CH1 H3 and CH2 H3 ).​​​​​​​​​​​​ i） The amino acid at position 39 (Kabat number) of VH1 is K, and the amino acid at position 38 (Kabat number) of VL1 is D; ii) The CH1 H1 The amino acid at position 147 (EU number) is K, and the amino acid at position 131 (EU number) of CL1 is D; iii) The CH1 H1 The amino acid at position 173 (EU number) is C, and the amino acid at position 162 (EU number) of CL1 is C; iv) The amino acid at position 220 (EU number) of H1H is S, and the amino acid at position 214 (EU number) of CL1 is S; and i） The amino acid at position 39 (Kabat number) of VH2 is D, and the amino acid at position 38 (Kabat number) of VL2 is K; and ii) The CH2 H1 The amino acid at position 147 (EU number) is D, and the amino acid at position 180 (EU number) of CL2 is R.

4. The bispecific antibody according to claim 3, wherein... i) The amino acid at position 87 (Kabat number) of VH1 and / or VH2 is G; and ii) The amino acid at position 45 (Kabat number) of VL1 and / or VL2 is W.

5. The antibody according to any one of claims 3-4, wherein i) CH1 H3 It has a C at position 349, an S at position 366, an A at position 368, and a V (EU number) at position 407; and the CH2 H3 It has a C at position 354 and a W (EU number) at position 366. ii) The CH2 H3 It has a C at bit 349, an S at bit 366, an A at bit 368, and a V (EU number) at bit 407; and CH1 H3 It has a C at position 354 and a W (EU number) at position 366. iii) CH1 H3 It has a C at position 354, an S at position 366, an A at position 368, and a V (EU number) at position 407; and the CH2 H3 It has a C at position 349 and a W (EU number) at position 366; or iv) The CH2 H3 It has a C at bit 354, an S at bit 366, an A at bit 368, and a V (EU number) at bit 407; and CH1 H3 It has a C at position 349 and a W (EU number) at position 366.

6. The antibody according to any one of claims 3-5, wherein the CH1 H3 and / or CH2 H3 The amino acid at position 447 (EU number) is missing.

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

8. The antibody according to any one of claims 3-7, wherein i) CH1 H3 and / or CH2 H3 It has an A at position 297 (EU number); ii) CH1 H3 and / or CH2 H3 It has a G at position 297 (EU number); or iii) CH1 H3 and / or CH2 H3 It has an S at position 297 (EU number).

9. The antibody according to any one of claims 3-8, wherein the CH1 H3 and / or CH2 H3 It has an S at position 331 (EU number).

10. The bispecific antibody according to any one of claims 1-9, wherein the polypeptide is fused to the N-terminus or 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 according to any one of claims 10-11, wherein the polypeptide is fused via a linker peptide.

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

53.

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

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

49.

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

17. The bispecific antibody according to any one of claims 1-16, wherein the bispecific antibody is a monoclonal antibody, a chimeric antibody, or a humanized antibody.

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

318.

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

20. A vector comprising the polynucleotide according to claim 18.

21. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1-18, a polynucleotide according to claim 19, or a carrier according to claim 20, and a pharmaceutically acceptable carrier.

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

23. A method for retargeting T cells in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 21.

24. A method for activating T cells in a subject in need, the method comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 21.

25. The method according to any one of claims 22-24, wherein the subject in need suffers from cancer.

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

27. The method according to any one of claims 22-26, wherein the lymphoma or leukemia is Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), B-cell lymphoma, mantle cell lymphoma, AIDS-related lymphoma, Waldenström macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloid leukemia, myelodysplastic syndrome (MDS), or acute myeloid leukemia (AML).

28. The method according to any one of claims 22-27, wherein the NHL is small lymphocyte (SL) NHL, intermediate / follicular NHL, intermediate diffuse NHL, advanced immune cell NHL, advanced lymphocyte NHL, advanced small non-cleaved cell NHL, or massive lesion NHL.

29. The method according to any one of claims 22-28, wherein the subject is simultaneously or previously administered 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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