Anti-CD84 antibody and use thereof

Abstract

Provided are an antibody binding to a CD84 antigen, or an antigen-binding fragment thereof, a nucleic acid molecule encoding the specific antibody and the antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a host cell comprising the vector, a chimeric antigen receptor or immunoconjugate comprising the antibody and the antigen-binding fragment thereof, and a use of them in the preparation of a drug for treating or preventing diseases, especially for treating hematologic tumors, and an application of them in detection of products.

Description

Anti-CD84 antibodies and uses thereof Technical Field

[0001] The present invention relates to anti-CD84 antibodies, preparation methods, compositions and uses thereof. The present invention also provides methods for treating CD84-related diseases and / or conditions, such as cancer. Background Art

[0002] CD84 (also known as LY9B or SLAMF5) is a membrane glycoprotein and a member of the signaling lymphocyte activation molecule (SLAM) family, which itself is a subset of the larger CD2 cell surface receptor subset in the Ig superfamily. It self-associates to form homophilic dimers and is primarily expressed by hematopoietic cells.

[0003] CD84 is ubiquitously expressed on most immune cell subsets, acting as a homophilic adhesion molecule. Its signaling can activate or inhibit leukocyte function, depending on the cell type and its stage of activation or differentiation. CD84-mediated signaling regulates multiple immune processes, including T cell cytokine secretion, natural killer cytotoxicity, monocyte activation, autophagy, cognate T:B interactions, and B cell tolerance at the germinal center checkpoint. Recent studies have implicated CD84 in autoimmune diseases and lymphoproliferation. CD84 is widely expressed in various hematologic malignancies, including acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), multiple myeloma (MM), hairy cell leukemia (HCL), and Hodgkin lymphoma (HL). Studies have shown that inhibiting CD84 function has therapeutic potential in hematologic malignancies, particularly AML.

[0004] However, there are currently few studies on antibody therapy targeting CD84 for the treatment of AML. Therefore, the development of drugs targeting CD84 for the treatment of AML has important clinical value.

[0005] SUMMARY OF THE INVENTION

[0006] The present invention provides novel anti-CD84 antibodies. The CD84 antibody molecules of the present invention all have strong affinity for human CD84 and monkey CD84; all specifically recognize CD84 and do not bind to other proteins in the same family; and have high tumor cell killing activity, thereby achieving the purpose of treating hematological malignancies.

[0007] Therefore, in one aspect, the present invention provides an anti-CD84 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region HCDR1 selected from the group consisting of SEQ ID NOs: 1, 8, 13 and 22, and / or a heavy chain variable region HCDR2 selected from the group consisting of SEQ ID NOs: 2, 9, 14, 18 and 23, and / or a heavy chain variable region HCDR3 selected from the group consisting of SEQ ID NOs: 3, 10, 15, 21 and 24, and / or a light chain variable region LCDR1 selected from the group consisting of SEQ ID NOs: 4, 6, 11, 16, 19, 25 and 27, and / or a light chain variable region LCDR2 selected from the group consisting of NAK, FAS, WAS and YAS, and / or a light chain variable region LCDR3 selected from the group consisting of SEQ ID NOs: 5, 7, 12, 17, 20 and 26, or variants of said CDR sequence combinations.

[0008] In some embodiments, the present invention also provides anti-CD84 antibodies or antigen-binding fragments thereof that bind to the same or overlapping epitope as an exemplary antibody of the invention (e.g., an antibody having a combination of antibody VH and VL sequences listed in Table A below) and / or compete for binding to CD84 and / or inhibit (e.g., competitively inhibit) an exemplary antibody of the invention.

[0009] In some embodiments, the antibody is a bispecific antibody, which further comprises an antigen-binding fragment that binds to a second antigen, specifically binds CD3, and the second antigen-binding fragment comprises VH and VL, wherein VH comprises the amino acid sequence of SEQ ID NO: 61, and VL comprises the amino acid sequence of SEQ ID NO: 62, respectively.

[0010] In some embodiments, the present invention provides nucleic acids encoding the antibodies or antigen-binding fragments thereof of the present invention, vectors comprising the nucleic acids, and host cells comprising the vectors.

[0011] In some embodiments, the present invention provides methods for making the antibodies or antigen-binding fragments thereof of the present invention.

[0012] In some embodiments, the present invention provides a chimeric antigen receptor, immunoconjugate, or pharmaceutical combination comprising an antibody of the present invention.

[0013] In some embodiments, the present invention also provides methods and uses of the antibodies or antigen-binding fragments thereof of the present invention for preventing or treating CD84-related diseases, including but not limited to tumors, such as hematological malignancies, further including acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), multiple myeloma (MM), hairy cell leukemia (HCL) or Hodgkin lymphoma (HL).

[0014] In some embodiments, the antibodies of the invention may be the sole active agent, or may be administered in combination with other therapies or therapeutic agents.

[0015] In some embodiments, the present invention also provides methods and kits for detecting CD84 in a sample using the antibodies or antigen-binding fragments thereof of the present invention.

[0016] The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered to limit the scope of the present invention, and changes that are readily apparent to those skilled in the art will be included within the spirit of the present invention and the protection scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the construction of CD84 overexpression cell line

[0018] Figure 2 shows the CDR sequences of the light and heavy chains of chimeric antibodies.

[0019] Figure 3 shows the affinity experiment between chimeric antibodies and 239T-hCD84 cells

[0020] Figure 4 shows the sequence information of humanized antibody VH / VL.

[0021] Figure 5 shows the affinity experiment between humanized antibodies and 293T-hCD84 cells

[0022] Figure 6 shows the affinity experiment between humanized antibodies and HEL cells

[0023] Figure 7: Affinity experiment of humanized antibody and U937 cells

[0024] Figure 8 is a schematic diagram of the CD84XCD3 dual antibody structure

[0025] Figure 9 shows the cytotoxicity of PBMC to U937 cells mediated by CD84xCD3 dual antibody

[0026] Figure 10 shows the affinity experiment between CD84XCD3 dual antibody and CD84 and CD3 proteins

[0027] Figure 11 shows the affinity experiment between CD84XCD3 dual antibody and different cells

[0028] Figure 12 shows the cytotoxicity experiment of CD84XCD3 dual antibody on tumor cells

[0029] Figure 13 shows the in vitro safety evaluation experiment of CD84xCD3 dual antibody

[0030] Figure 14 shows the kinetics experiment of CD84xCD3 dual antibody and CD84 protein

[0031] Figure 15 shows the kinetics experiment of CD84XCD3 dual antibody and CD3 protein

[0032] Figure 16 shows the in vivo anti-tumor experiment of CD84xCD3 dual antibody

[0033] Detailed Description of the Invention

[0034] definition

[0035] The terms used herein are for the purpose of describing the embodiments only and are not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0036] The term "comprising" or "including" means including the stated elements, integers, or steps, but does not exclude any other elements, integers, or steps. In this document, when the term "comprising" or "including" is used, unless otherwise indicated, combinations of the stated elements, integers, or steps are also encompassed. For example, when referring to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of the specific sequence.

[0037] The term "and / or," such as "X and / or Y," should be understood to mean "X and Y" or "X or Y" and should be used to provide clear support for both meanings or either meaning.

[0038] The term "antibody" is used herein in the broadest sense to refer to a protein that contains an antigen binding site and encompasses natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, and antibody fragments.

[0039] The term "whole antibody" (used interchangeably herein with "full-length antibody," "complete antibody," and "intact antibody") comprises at least two heavy chains (H) and two light chains (L). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The variable region is the domain in the heavy or light chain of an antibody that participates in the binding of the antibody to its antigen. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions. The light chain of an antibody can be classified into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. The heavy chain of an antibody can be divided into five main different types depending on the amino acid sequence of its heavy chain constant region: IgA, IgD, IgE, IgG and IgM, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to the different antibody types are called α, δ, ε, γ and μ, respectively. The term "isotype" refers to the antibody type determined by the antibody heavy chain constant region. See, for example, Fundamental Immunology, Ch. 7 (Paul, W. ed., 2nd ed., Raven Press, NY (1989)).

[0040] The term "CD84" refers to an expressed isoform of the CD84 gene. Examples include, but are not limited to, Q9UIB8-1, Q9UIB8-2, Q9UIB8-3, Q9UIB8-4, Q9UIB8-5, Q9UIB8-6, and Q9UIB8-7. An exemplary amino acid sequence of CD84 contemplated by the present invention is shown below:

[0041] The term "CD3" refers to the human CD3 protein multi-subunit complex. The CD3 protein multi-subunit complex is composed of 6 different polypeptide chains. Unless otherwise indicated, the term "CD3" includes any CD3 variant, isoform, and species homolog that is naturally expressed by cells (including T cells) or is capable of being expressed on cells transfected with genes or cDNAs encoding those polypeptides.

[0042] The term "bispecific antibody" in the context of the present invention should be understood as an antibody having two different antigen-binding fragments defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a target. The term "bispecific antibody" as used herein should be understood in its broadest sense, including full-length bispecific antibodies and antigen-binding fragments thereof. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations in glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing the antigenic determinants of antibodies, and immunoglobulin molecules containing any other modifications to the antigen recognition site, as long as these antibodies exhibit the desired biological activity.

[0043] The term "antigen-binding fragment" (used interchangeably herein with "antibody fragment" and "antigen-binding portion") refers to a molecule that is not a complete antibody, which comprises a portion of a complete antibody that binds to the antigen to which the complete antibody binds. As will be appreciated by those skilled in the art, the antigen-binding portion of an antibody typically comprises amino acid residues from a "complementarity determining region" or "CDR." Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab')2, Fab'-SH, Fv, single-chain Fv, diabody, triabody, tetrabody, minibody, and single-domain antibody (sdAb).

[0044] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0045] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, for example, an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0046] The term "humanized antibody" refers to antibodies in which CDR sequences derived from other mammalian species, such as mouse germline, are joined to human framework sequences. Additional framework region modifications can be made within the human framework sequences, and / or additional amino acid modifications can be made in the CDR sequences, for example, to effect affinity maturation of the antibody.

[0047] The term "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman, S. et al., J. Chrom. B 848 (2007) 79-87.

[0048] The term "chimeric antigen receptor" or alternatively "CAR" refers to a group of polypeptides, which are typically two groups in the simplest embodiment. When they are in immune effector cells, the cells are provided with specificity for target cells (usually cancer cells) and have intracellular signal generation. In certain embodiments, CAR includes at least one extracellular binding region, a transmembrane region, and an intracellular signaling region. In some aspects, the polypeptide groups are adjacent to each other.

[0049] The term "epitope" refers to the region of an antigen to which an antibody binds. An epitope can be formed by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.

[0050] The term "Fc region" herein is used to define the C-terminal region of the immunoglobulin heavy chain containing at least a portion of the constant region. The term includes native sequence Fc-regions and variant Fc-regions. In one embodiment, the human IgG heavy chain Fc-region extends from Cys226 of the heavy chain or from Pro230 to the carboxyl terminus. However, the C-terminal lysine (Lys447) in the Fc-region may or may not be present. Unless otherwise noted herein, the numbering of the amino acid residues in the Fc-region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0051] As used herein, the term sequence "identity" refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity. It is known to those skilled in the art that sequence identity can be determined using algorithms such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and ClustalW.

[0052] In the present invention, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. These conservative modifications include conservative substitutions, additions, and deletions of amino acids. Modifications can be introduced into the chimeric antigen receptor of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications may be selected, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the residues involved.

[0053] Various aspects of the invention are described in further detail in the following subsections.

[0054] i. Anti-CD84 Antibodies of the Present Invention

[0055] In one aspect, the present invention provides antibodies or antigen-binding fragments thereof, particularly humanized antibodies or antigen-binding fragments thereof, that specifically bind to CD84, preferably human CD84 protein. In some embodiments, the antigen-binding fragment of an antibody of the present invention is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibodies such as scFv, (Fab')2 fragments, single-domain antibodies, diabodies (dAbs), or linear antibodies.

[0056] Antibody CDR region

[0057] "Complementarity determining regions" or "CDR regions" or "CDRs" (used interchangeably herein with hypervariable regions "HVRs") are the amino acid regions in the variable region of an antibody that are primarily responsible for binding to an antigenic epitope. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The CDRs located within the variable domain of the antibody heavy chain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.

[0058] Table A: VH and VL sequence combinations of exemplary antibodies

[0059] As is well known to those skilled in the art, the CDRs of an antibody can be defined in the art by a variety of methods, such as Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (world wide web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any of the above-mentioned known schemes described herein. Various numbering systems corresponding to CDRs are well known to those skilled in the art, as shown in Table B:

[0060] Table B: Common CDR definition schemes

[0061] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" or "HVR" or "HVR sequence" encompasses an HVR or CDR sequence determined in any of the above ways.

[0062] Unless otherwise specified, the variable region and CDR sequences in the disclosed examples are subject to the "IMGT" numbering convention.

[0063] In a preferred experimental scheme, the CDRs of the antibodies of the present invention are shown in Table C, preferably, the CDR sequences are defined according to the CDR division method expanded by Kabat and IMGT.

[0064] Table C Exemplary CDR sequence combinations of the present invention

[0065] In one embodiment, an antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, wherein the antibody comprises:

[0066] (i) six CDR sequences contained in the VH and VL sequences of any one of the antibodies listed in Table A; or

[0067] (ii) 6 CDR sequences from any combination listed in Table C; or

[0068] (iii) a sequence comprising at least one and no more than 10, or 5, 4, 3, 2, or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the six CDR regions relative to the sequence of (i) or (ii).

[0069] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises three complementarity determining regions (HCDRs) of the heavy chain variable region and three complementarity determining regions (LCDRs) of the light chain variable region, wherein:

[0070] (i) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 4, LCDR2 comprises or consists of NAK, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 5; or

[0071] (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 6, LCDR2 comprises or consists of FAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7; or

[0072] (iii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 12; or

[0073] (iv) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises or consists of YAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17; or

[0074] (v) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 18, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 20; or

[0075] (vi) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 21, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 12; or

[0076] (vii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 22, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 23, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 24, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 25, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 26; or

[0077] (viii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 22, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 23, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 24, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 27, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 26;

[0078] Alternatively, the antibody comprises a variant of one of the CDR sequence combinations of (i) to (viii), wherein the variant comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably amino acid substitution, preferably conservative substitution) in the 6 CDR regions, and preferably the heavy chain CDR3 remains unchanged; wherein the fifth amino acid X in the amino acid sequence of SEQ ID NO: 9 = N, D, Q.

[0079] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises:

[0080] (i) the HCDR1, 2 and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 30, and the LCDR1, 2 and 3 sequences of the light chain variable region as shown in SEQ ID NO: 29, or

[0081] (ii) the HCDR1, 2 and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 43, and the LCDR1, 2 and 3 sequences of the light chain variable region as shown in SEQ ID NO: 38, or

[0082] (iii) the HCDR1, 2 and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 45, and the LCDR1, 2 and 3 sequences of the light chain variable region as shown in SEQ ID NO: 46, or

[0083] (iv) the HCDR1, 2 and 3 sequences of the heavy chain variable region as shown in SEQ ID NO: 53, and the LCDR1, 2 and 3 sequences of the light chain variable region as shown in SEQ ID NO: 54.

[0084] Antibody variable region

[0085] "Variable region" or "variable domain" is the domain of an antibody's heavy or light chain that is involved in binding the antibody to its antigen. The heavy chain variable region (VH) and light chain variable region (VL) can be further subdivided into hypervariable regions (HVRs, also called complementarity determining regions (CDRs)), interspersed with more conserved regions (i.e., framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some cases, a single VH or VL domain is sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated by using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains (see, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887; Clackson, T. et al., Nature 352 (1991) 624-628).

[0086] CDR transplantation is another method of modifying the variable region of an antibody known in the art. Since CDR sequences are responsible for most antibody-antigen interactions, recombinant antibody variants that mimic the properties of known antibodies can be constructed. In this antibody variant, the CDR sequences from a known antibody are transplanted onto the framework regions of a different antibody with different properties. Therefore, in one embodiment, the present invention relates to an anti-CD84 antibody or antigen-binding fragment thereof comprising CDR sequences from the heavy and light chain variable regions of one of the antibodies in Table A, but having different framework region sequences. The framework region sequences for replacement can be obtained from public DNA databases, including germline antibody gene sequences, or from CD84 antibody sequences reported in the open literature. For example, germline DNA encoding human heavy and light chain variable region genes can be obtained from the GenBank database. The antibody protein sequence can be compared with the protein sequences in the database using sequence similarity search tools, such as Gapped BLAST. Preferably, the framework sequence used for replacement has structural similarity with the framework sequence of the antibody of the present invention selected for modification, for example, a framework sequence having a sequence identity of at least 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99%. In some embodiments, humanization of the antibody can be performed in the manner of Example 3.

[0087] Therefore, in one embodiment, the antibody of the present invention comprises the heavy chain variable region VH sequence of any one of the antibodies listed in Table A, or consists of the amino acid sequence. In another embodiment, the antibody of the present invention comprises a variant of the VH sequence.

[0088] In another embodiment, the antibody of the present invention comprises the light chain variable region VL sequence of any one of the antibodies listed in Table A, or consists of the amino acid sequence. In yet another embodiment, the antibody of the present invention comprises a variant of the VL sequence.

[0089] In one embodiment, an antibody of the invention comprises:

[0090] (i) a VH sequence comprising the amino acid sequence shown in SEQ ID NO: 30 or a variant thereof, and / or a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof, or

[0091] (ii) a VH sequence comprising the amino acid sequence shown in SEQ ID NO: 43 or a variant thereof, and / or a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 38 or a variant thereof, or

[0092] (iii) a VH sequence comprising the amino acid sequence shown in SEQ ID NO: 45 or a variant thereof, and / or a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 46 or a variant thereof, or

[0093] (iv) a VH sequence comprising the amino acid sequence shown in SEQ ID NO: 53 or a variant thereof, and / or a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 54 or a variant thereof.

[0094] In one embodiment, the variant of the VH sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in amino acid sequence compared to the reference VH sequence (preferably, over the entire length or in the three regions of CDR1, 2 and 3). In one embodiment, the variant of the VH sequence comprises at least one and no more than 30, 10, or 5, 4, 3, 2, 1, or 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in amino acid sequence compared to the reference VH sequence (preferably, over the entire length or in the three regions of CDR1, 2 and 3). Preferably, the sequence differences do not occur in the CDR regions.

[0095] In a preferred embodiment, the variant of the VL sequence has at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity in amino acid sequence compared to the reference VL sequence (preferably, over the entire length or in the three regions of CDR1, 2 and 3). In a preferred embodiment, the variant of the VL sequence comprises at least one and no more than 30, 10, or 5, 4, 3, 2, 1, 0 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in amino acid sequence compared to the reference VL sequence (preferably, over the entire length or in the three regions of CDR1, 2 and 3). Preferably, the sequence differences do not occur in the CDR regions.

[0096] In a preferred embodiment, the antibody of the present invention comprises a heavy chain variable region and a light chain variable region VH / VL sequence pair of any one of the antibodies listed in Table A, or consists of said amino acid sequence pair. The present invention also provides variants of the antibody, such as variants having at least 95-99% identity in VH, VL, or VH and VL, or comprising no more than 10 amino acid changes.

[0097] In any of the above embodiments, preferably, the heavy chain variable region of the antibody variant comprises no more than 10, preferably no more than 5 (e.g., 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in one or more CDR regions (preferably all three CDRs) relative to the reference antibody.

[0098] In any of the above embodiments, preferably, the light chain variable region VL of the antibody variant comprises no more than 10, preferably no more than 5 (e.g., 3, 2, 1 or 0) amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in one or more CDR (preferably all three CDR) regions relative to the reference antibody.

[0099] Antibody heavy and light chains

[0100] In one embodiment, the antibodies of the present invention comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the antibodies of the present invention comprise a heavy chain Fc region, such as an Fc region of an IgG1, IgG2, IgG3, or IgG4 isotype. In other embodiments, the antibodies of the present invention comprise an IgG1-Fc region, particularly a human IgG1-Fc region. In some embodiments, the antibodies of the present invention comprise a kappa light chain constant region, such as a human kappa light chain constant region.

[0101] In a preferred embodiment, the antibodies of the present invention comprise an IgG1-Fc region variant, wherein the variant comprises an amino acid sequence having at least one, two, or three, but no more than 20, 10, or 5 amino acid changes compared to the corresponding reference sequence, or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or higher identity. Preferably, the amino acid changes do not occur in the CDR region, and more preferably, do not occur in the variable region.

[0102] Bispecific antibody CD84×CD3

[0103] In some embodiments, the anti-CD84 antibody of the invention is a bispecific antibody, which further comprises a second antigen-binding fragment.

[0104] In some embodiments, the first or second antigen-binding fragment of the anti-CD84 bispecific antibody of the present invention is selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibody, scFv, (Fab')2 fragment, single-domain antibody, diabody (dAb) or linear antibody.

[0105] In some embodiments, the second antigen of the anti-CD84 antibody of the present invention is a tumor-associated antigen or an immune cell antigen, preferably a T cell antigen, wherein the T cell antigen is selected from T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) and NKG2D.

[0106] In some embodiments, the anti-CD84 bispecific antibody of the present invention comprises a first antigen-binding fragment that binds to CD84 and a second antigen-binding fragment that binds to CD3, the first antigen-binding fragment comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), the second antigen-binding fragment comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein VL1 and VH1 comprise the CDRs of the antibodies shown in Table A or Table C above, and the VH2 comprises HCDRs 1-3 in the amino acid sequence shown in SEQ ID NO: 61, and the VL2 comprises LCDRs 1-3 having the amino acid sequence shown in SEQ ID NO: 62, respectively.

[0107] In some embodiments, the CD3 antigen-binding fragment of the anti-CD84 bispecific antibody of the present invention comprises a VH domain and a VL domain selected from Table D.

[0108] Table D CD3 terminal VH / VL sequence information

[0109] The bispecific antibodies disclosed herein may comprise an Fc region comprising CH2 and CH3 of an antibody.

[0110] The Fc region can be of any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4, and can contain one or more mutations or modifications. In one embodiment, the Fc region is of the IgG1 isotype or derived therefrom, optionally with one or more mutations or modifications. In another embodiment, the Fc region is of the IgG4 isotype or derived therefrom, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgG1 Fc.

[0111] In a specific embodiment of the present invention, the heavy chain variable region VH and light chain variable region VL in the second antigen-binding fragment adopt crossmab light and heavy chain exchange, that is, VH and VL are replaced with each other, or the constant regions CL and CH1 are replaced with each other.

[0112] Preferably, the constant regions CL and CH1 in the second antigen-binding fragment are replaced with each other, and the heavy chain amino acid sequence containing the constant region CL after replacement is shown in SEQ ID NO: 76, and the light chain amino acid sequence containing CH1 after replacement is shown in SEQ ID NO. 77;

[0113] And / or, the heavy chain amino acid sequence of the first antigen-binding fragment is shown as SEQ ID NO.74, and the light chain amino acid sequence is shown as SEQ ID NO.64.

[0114] In one embodiment, the Fc region has reduced effector function, such as reduced ADCC, ADCP, CDC and / or C1q, FcγRI, FcγRII or FcγRIIIA binding. For example, the Fc region can be an IgG1 isotype, or a non-IgG1 type, such as IgG2, IgG3 or IgG4, which has been mutated so that the ability to mediate effector function is reduced or even eliminated. Such mutations have been described, for example, in Dall'Acqua WF et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). For example, compared to the wild-type sequence, the Fc region may comprise an amino acid sequence having one or more of the following amino acid substitutions: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. In a preferred embodiment of the present invention, the Fc region employs the L234A / L235A / P329G mutation combination.

[0115] In some embodiments, the mutated Fc region has enhanced binding affinity to the neonatal Fc receptor (FcRn) compared to the wild-type Fc region; for example, the M252Y / S254T / T256E mutations are introduced into the Fc region.

[0116] In some embodiments, the Fc region comprises modifications or mutations that inhibit Fc homodimerization. In some embodiments, the Fc region comprises a variant of the wild-type human IgG1 Fc sequence. Preferably, the amino acid residues on one side of the variant Fc domain are replaced with amino acid residues with larger side chain volumes, i.e., knob mutations, thereby forming a protruding structure; and the amino acid residues on the other side are replaced with amino acid residues with smaller side chain volumes, i.e., hole mutations, thereby forming a cavity; the cavity accommodates the protruding structure, thereby enabling heterodimer formation. Preferably, the Fc domain mutation scheme is selected from: (a) knob mutation to T366W, and hole mutations to T366S, L368A, and Y407V; (b) knob mutations to S354C and T366W, and hole mutations to Y349C, T366S, L368A, and Y407V; the above numbering is based on the EU index.

[0117] In some embodiments, the second antigen-binding fragment of the anti-CD84 bispecific antibody of the present invention is scFv, which comprises the VL2 and the VH2, and the scFv is optionally connected to the N / C terminus of VL1 or VH1 or the N / C terminus of the Fc region via a linker.

[0118] The linker can be any of those described above. For example, the linker can be any flexible linker. In some embodiments, the linker comprises an amino acid sequence selected from (G4S)n and GS(G4S)n, wherein n is an integer selected from 1-5, and preferably the linker comprises an amino acid sequence such as GGGGS.

[0119] In some embodiments, insertions, deletions and / or substitutions may be made in the framework (FR) regions, e.g., FR1, FR2, FR3 and / or FR4; and / or constant regions, e.g., CL, CH1, CH2 and / or CH3.

[0120] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0121] ii. Polynucleotides, vectors, and hosts

[0122] The present invention provides nucleic acids encoding any of the above anti-CD84 antibodies or fragments thereof. Also provided are vectors comprising the nucleic acids. In one embodiment, the vector is an expression vector. Also provided are host cells comprising the nucleic acids or the vectors. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells). In another embodiment, the host cell is prokaryotic.

[0123] The present invention also provides polynucleotides encoding at least one CDR region and generally all three CDR regions from the heavy chain VH or light chain VL sequence of the CD84-binding antibody described above. In some further embodiments, the polynucleotides encode the complete or substantially complete variable region sequence of the heavy chain and / or light chain of the CD84-binding antibody described above.

[0124] As will be apparent to those skilled in the art, because of codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0125] In a preferred embodiment, the nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a heavy chain constant region.

[0126] In one embodiment, one or more vectors comprising a nucleic acid of the present invention are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0127] In one embodiment, a host cell comprising the vector is provided. Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523, also see Charlton, Methods in Molecular Biology, Volume 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes expression of antibody fragments in E. coli. After expression, the antibody can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0128] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For example, fungi and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or completely human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006). Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney line (293HEK or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); and the like. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0129] iii. Chimeric Antigen Receptor

[0130] In one aspect of the invention, the invention relates to an isolated chimeric antigen receptor (CAR) molecule comprising (e.g., sequentially linked) an extracellular binding region that binds CD84, a transmembrane region, and an intracellular signaling region (e.g., comprising a costimulatory domain).

[0131] In one embodiment of the present invention, the transmembrane region used in particular in the present invention may include at least the following transmembrane region: for example, CD28, CD8 (for example, CD8α, CD8β). In some cases, the transmembrane region can be connected to the extracellular region of CAR via a hinge (for example, a hinge from a human protein), such as the antigen binding domain of CAR. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (for example, an IgG4 hinge, an IgD hinge), a GS linker, a KIR2DS2 hinge or a CD8 hinge.

[0132] In one embodiment of the present invention, the intracellular domain of CAR contains the CD3ζ chain, which provides T cell receptor activation signals and proliferation stimulation for genetically modified T cells. In addition to containing the TCRζ chain, the intracellular domain also contains a CD28 or 4-1BB costimulatory signaling domain. The CD28 domain enhances the initial activation and proliferation of CAR-T cells and improves effector function, while the 4-1BB domain mainly improves the expansion and long-term persistence of CAR-T cells.

[0133] In addition, the intracellular domain co-expresses some small molecules (such as the pro-inflammatory cytokine IL-12), which can trigger cytokine-induced signals or block some signaling pathways that affect the function of CAR-T cells.

[0134] iv. Immunoconjugates

[0135] In another aspect, the present invention provides immunoconjugates produced by conjugating an antibody of the present invention, or an antigen-binding fragment thereof, to a heterologous molecule. In one embodiment, in the immunoconjugate, an antibody of the present invention, or an antigen-binding fragment thereof, is conjugated to a therapeutic or diagnostic agent. In some embodiments, the antibody of the present invention can be conjugated to the heterologous molecule in the form of a full-length antibody or antibody fragment. For example, conjugation can be performed in the form of a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a single-chain scFab antibody, a single-chain scFv, a VHH, or other fragment.

[0136] Linkers can be used to covalently link the different entities of the conjugate. Suitable linkers include chemical linkers or peptide linkers. Advantageously, the linker is a "cleavable linker" that is beneficial for releasing the polypeptide after delivery to the target site. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker or a disulfide-containing linker can be used.

[0137] Therapeutic agents suitable for use in conjugates include, but are not limited to, cytotoxins (e.g., cytostatic or cell-killing agents), drugs, or radioisotopes. Examples of cytotoxic agents (e.g., chemotherapeutic agents) suitable for forming immunoconjugates are known in the art, see, for example, WO 05 / 103081. For example, cytotoxic agents include, but are not limited to, radioisotopes; growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof; and various known anti-tumor or anti-cancer agents.

[0138] In another aspect, the antibodies of the present invention can be conjugated to diagnostic or detectable agents. Such conjugates can be used as part of clinical testing methods (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnosis and detection can be achieved by coupling the antibody to a detectable agent, including but not limited to a variety of enzymes, such as, but not limited to, horseradish peroxidase; prosthetic groups, such as, but not limited to, streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metals and non-radioactive paramagnetic metal ions used in various positron emission tomography techniques.

[0139] v. Pharmaceutical compositions and pharmaceutical preparations

[0140] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-CD84 antibodies or immunoconjugates thereof or chimeric antigen receptors thereof, and compositions comprising polynucleotides encoding anti-CD84 antibodies or immunoconjugates thereof or chimeric antigen receptors. These compositions may also optionally contain suitable pharmaceutical excipients, such as pharmaceutical carriers and excipients known in the art, including buffers.

[0141] Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those with petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When intravenously administering pharmaceutical compositions, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. Oral formulations can include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin.

[0142] Pharmaceutical formulations comprising the present invention can be prepared by mixing an anti-CD84 antibody, immunoconjugate or chimeric antigen receptor of the present invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or aqueous solution.

[0143] In the pharmaceutical compositions and pharmaceutical preparations of the present invention, the antibodies of the present invention may be the sole active agent, or may be combined with other therapeutic agents. Therapeutic agents that may be combined with the antibodies of the present invention include, but are not limited to, therapeutic agents that have beneficial therapeutic efficacy for the disease and / or condition to be treated. For example, the active ingredients may be those required for the specific indication being treated, preferably having complementary activities that do not adversely affect each other. For example, other pharmaceutical ingredients may provide anti-cancer activity. The antibodies of the present invention are suitably combined with the active ingredients in pharmaceutical compositions and pharmaceutical preparations in an amount effective for the intended use.

[0144] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0145] vi. Combination products

[0146] In yet another aspect, the present invention also provides a combination product comprising an antibody or antigen-binding fragment thereof, an immunoconjugate or a chimeric antigen receptor of the present invention, and one or more other therapeutic agents (e.g., chemotherapeutic agents, other antibodies, cytotoxic agents, anti-tumor drugs, etc.). The combination product of the present invention can be used in the method of treatment of the present invention. In some embodiments, the present invention provides a combination product, wherein the other therapeutic agent is, for example, an effective stimulating immune response to further enhance, stimulate, or upregulate the immune response of the subject, such as an antibody.

[0147] In some embodiments, the combination product is used to prevent or treat a tumor. In some embodiments, the tumor is a hematological malignancy, such as acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), multiple myeloma (MM), hairy cell leukemia (HCL) or Hodgkin lymphoma (HL).

[0148] vii. Therapeutic / preventive / diagnostic uses

[0149] In another aspect, the present invention also provides a method for treating and / or preventing and / or diagnosing a disease associated with CD84 expression, comprising administering the humanized antibody, chimeric antigen receptor, immunoconjugate or pharmaceutical composition described above to a subject.

[0150] In one embodiment, diseases associated with CD84 expression include, but are not limited to, CD84-positive acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), multiple myeloma (MM), hairy cell leukemia (HCL), or Hodgkin lymphoma (HL).

[0151] viii. Detection Application

[0152] The present invention also provides methods and kits for detecting CD84 in a sample, wherein the method comprises: (a) contacting the sample with an antibody, antigen-binding fragment thereof, or immunoconjugate thereof of the present invention; and (b) detecting the formation of a complex between the antibody, antigen-binding fragment thereof, or immunoconjugate, or chimeric antigen receptor, and CD84 protein. In some embodiments, the sample is from a cancer patient, such as a leukemia patient. The detection can be in vitro or in vivo.

[0153] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads complexed with antibody molecules, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from a hyperproliferative or cancerous lesion. In certain embodiments, the CD84 to be detected is human CD84.

[0154] In one embodiment, an anti-CD84 antibody is used to select a subject suitable for treatment with an anti-CD84 antibody, e.g., wherein CD84 is a biomarker for selecting the subject. In one embodiment, the antibodies of the invention can be used to diagnose cancer or tumors, e.g., to evaluate (e.g., monitor) the treatment or progression of a disease described herein (e.g., a hyperproliferative or cancerous disease), diagnose, and / or stage the disease in a subject.

[0155] In certain embodiments, labeled anti-CD84 antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes 32P, 14C, 125I, 3H, and 131I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferase, e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HR), alkaline phosphatase, β-galactosidase, glucoamylase, lytic enzymes, carbohydrate oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, and enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HR, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, and the like.

[0156] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention. DETAILED DESCRIPTION

[0157] Example 1 Construction of CD84 overexpressing cell line

[0158] 1.1 Construction of HEK293T-hCD84 and HEK293T-Cyno-CD84 Overexpressing Cells

[0159] Human CD84 and monkey CD84 sequences were obtained from the NCBI database, with sequence numbers NP_001171808.1 and XM_005541244.3, respectively. First, CD84 sequences from different species were cloned into the lentiviral overexpression vector pCDH-CMV.MCS-EF1.CopGFP.T2A.Puro by PCR. The constructed overexpression plasmid and viral packaging plasmids (psPAX2 and pMD2.G) were then transferred into HEK293T cells via lipofectae2000 at a specific ratio for viral packaging. The supernatant containing the virus was collected approximately 48 hours later. HEK293T cells were infected with the collected viral supernatant to construct HEK293T-human-CD84 and HEK293T-cyno-CD84 cell lines. All overexpression cell lines were cultured in DMEM + 10% FBS + 1% P / S + 5 μg / mL puromycin. The constructed overexpression cell lines were evaluated using CD84.1.21 (Biolegend; Catalog No. 326002) as a positive control molecule (BMK). The construction results are shown in Figure 1: Both HEK293T-hCD84 and HEK293T-cyno-CD84 overexpression cell lines were successfully constructed.

[0160] Example 2: Screening and preparation of hybridoma cells and monoclonal antibodies

[0161] 2.1 Preparation, screening and identification of hybridoma cells

[0162] Spleens from immunized mice were harvested under sterile conditions. Gently crushed, ground, and passed through a 40-mesh sieve to separate the cells into sterile PBS. The cells were washed twice by centrifugation at 1500 rpm for 5 minutes, resuspended in a volume of electroporation buffer, and allowed to stand for 5 minutes. Mouse myeloma cells were collected in PBS and, following the same procedure as for spleen cells, washed twice by centrifugation at 1500 rpm for 5 minutes, resuspended in a volume of electroporation buffer, and allowed to stand for 5 minutes. The mouse spleen cells and myeloma cells were then thoroughly mixed at a ratio of 1.5:1. Electroporation was performed on an electroporator according to pre-set conditions to allow fusion. After fusion, the appropriate amount of culture medium (DMEM + 10% FBS + 1% HT + 1% P / S) was added and the cells were transferred to 96-well flat-bottom plates at a density of 2E4 cells / well. After approximately 7-10 days, after the cells had grown to a certain number, hybridoma supernatants were identified according to a pre-set screening protocol to identify clones producing CD84-specific antibodies. Hybridoma screening first uses ELISA to screen hybridoma supernatants using human and monkey CD84 proteins, then further combines ELISA-positive samples and performs species cross-detection through flow cytometry experiments, and finally selects ELISA- and FCM-positive clones for verification through ligand blocking tests to determine the final positive clone molecules.

[0163] 2.2 Preparation of chimeric antibody molecules and determination of binding activity to 239T-hCD84 cells

[0164] Chimeric antibody expression

[0165] The heavy chain sequences of the positive clones obtained from the screening were synthesized by a conventional service provider and cloned into the pTT5-hIgG1.CH vector containing the amino acid sequence of the IgG1 heavy chain constant region via homologous recombination to generate chimeric antibody heavy chain expression plasmids. The light chain sequences of the positive clones obtained from the screening were synthesized by a conventional service provider and cloned into the pTT5-hKappa.CL vector via homologous recombination. Well-growing 293F cells in the logarithmic phase were seeded into a 250 mL cell culture flask and cultured in 50 mL of culture medium. Polyethylene glycol (PEI) was then co-transfected with 25 μg of each of the light and heavy chain expression plasmids. Cell supernatants were collected on day 7 post-transfection, centrifuged, and filtered through a 0.45 μM filter. Antibodies were purified using Protein A media and exchanged by dialysis into PBS (pH 7.2). Antibody concentration and purity were determined by absorbance measurement using a Nanodrop analyzer, and purity was verified by sodium dodecyl sulfate gel electrophoresis and Coomassie staining. Finally, 8 chimeric antibodies (1A6E9A1, 1A6G12A13, 14A3A13, 5H6A13, 3F11A13, 39E1A13, 27F7E9A13, 27F7B11A1) were obtained. The amino acid sequences of the CDR regions of the above chimeric antibodies are listed in Figure 2.

[0166] Flow cytometry screening

[0167] The pre-cultured cells were digested and resuspended in FACS buffer (PBS + 1% BSA). After counting the cells, 2E5 cells / 100 μL were plated in a 96-well V-bottom plate. After the cells were treated, 100 μL of antibody was added to each well at the pre-set concentration, mixed with the cells, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and the cell pellet was retained. The cells were resuspended in 200 μL of FACS buffer and centrifuged and washed twice under the same conditions. Then, 100 μL of secondary antibody was added and incubated for another 0.5 hour. After centrifugation and washing twice, the cells were resuspended in 100 μL of FACS buffer and analyzed. The number of cells in each sample should not be less than 10,000. The experimental results are shown in Table 1 and Figure 3. Compared with the control antibody BMK, the chimeric antibodies 1A6G12A13, 14A3A13, 5H6A13, and 39E1A13 have better binding ability to HEK293T-hCD84 and HEK293T-Cyno-CD84 cells.

[0168] Table 1 Affinity test results of chimeric antibodies

[0169] Example 3 Humanization of the anti-CD84 antibody of the present invention

[0170] The resulting chimeric antibodies 1A6G12A13 (abbreviated as 1A6 in the examples below), 14A313 (abbreviated as 14A3), 39E1A13 (abbreviated as 39E1), and 5H6A13 (abbreviated as 5H6) were humanized. The steps are as follows:

[0171] ① Determine the CDR sequence according to the Kabat and IMGT extended CDR division method;

[0172] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the human germline sequence database; ③ Select the human germline with the best match for the heavy and light chains and the lowest number of back mutations;

[0173] ④ Constructing the CDR region of the chimeric antibody onto the human framework region;

[0174] ⑤ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0175] ⑥ Perform back mutation at the sequence position determined to be important;

[0176] ⑦Optimize amino acids at risk sites.

[0177] For the specific sequence information of the light chain variable region and heavy chain variable region of the obtained humanized antibody, see Figure 4.

[0178] Example 4 Purification and expression of the humanized anti-CD84 antibody of the present invention

[0179] 293F cells were used to transiently express the CD84 humanized antibody constructed in Example 3, and the supernatant was collected and purified with Protein A to obtain the antibody. The heavy chain and light chain humanized variable region sequences were synthesized by Boshang Biotechnology (Shanghai) Co., Ltd. and cloned into the pTT5-hIgG1.CH vector and pTT5-hKappa.CL vector containing the amino acid sequence of the IgG1 heavy chain constant region to obtain the antibody expression plasmid. 293F cells in a logarithmic growth phase with good growth status were inoculated into a 250mL cell culture flask and cultured in 50mL of culture medium. The vectors carrying the heavy and light chains of the antibody molecules were transferred into the 293F cells using a chemical transfection method. The chemical transfection reagent used was PEI (purchased from Polysciences), and the cultured 293F cells were transiently transfected according to the protocol provided by the manufacturer. The cell supernatant on the 5th day after transfection was collected and centrifuged at 4000 rpm for 20 min. The supernatant was purified with Protein A medium (purchased from Bogelon), and then the antibody was replaced into PBS pH 7.2 buffer by dialysis.

[0180] Example 5 Detection of Binding of Humanized Antibodies to Overexpressing Cells

[0181] 5.1 Affinity determination of humanized antibodies to 293T-hCD84, HEL, and U937 cells

[0182] The pre-cultured cells were digested and resuspended in FACS Buffer (PBS + 1% BSA). After cell counting, 2E5 cells / 100 μL were seeded per well in a 96-well V-bottom plate. After cell treatment, 100 μL of antibody was added to each well at the pre-set concentration, mixed with the cells, and incubated at 4°C for 1 hour. After incubation, the cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and the cell pellet was retained. The cells were resuspended in 200 μL of FACS buffer and centrifuged and washed twice under the same conditions. Then, 100 μL of secondary antibody was added and incubated for another 0.5 hour, followed by centrifugation and washing twice. Finally, the cells were resuspended in 100 μL of FACS Buffer and analyzed on the analyzer. The number of cells in each sample should not be less than 10,000 during the analysis. The experimental results are shown in Figures 5, 6 and 7. After the humanization of the chimeric antibody molecule 5H6A13, the antibody molecules 5H6Z01A1-5H6Z06A1 all showed a strong ability to bind to human CD84 at the cellular level; after the humanization of the chimeric antibody molecule 14A3A13, the antibody molecules 14A3Z01A1-14A3Z07A1 all showed a strong ability to bind to human CD84 at the cellular level; after the humanization of the chimeric antibody molecule 1A6E9A1, except for 1A6Z05A1 and 1A6Z06A1, the other antibody molecules all showed a strong ability to bind to human CD84 at the cellular level; after the humanization of the chimeric antibody molecule 39E1A13, the antibody molecules 39E1Z01A1-39E1Z08A1 all showed a strong ability to bind to human CD84 at the cellular level.

[0183] Example 6 Preparation of CD84×CD3 Dual Antibody

[0184] According to the combinations listed in Table 2, the anti-CD84 clone 39E1Z01A1 and the anti-CD3 clone (VH: SEQ ID NO 61; VL: SEQ ID NO 62) were combined to construct a total of nine bispecific antibodies. The bispecific antibody structures are shown in Figure 8 . The peptide chains corresponding to the above bispecific antibodies were cloned into vectors to obtain antibody expression plasmids. 293F cells in the logarithmic growth phase of healthy growth were seeded into 250 mL cell culture flasks and cultured in 50 mL of culture medium. The vectors carrying the heavy and light chains of the antibody molecules were then transfected into the 293F cells using chemical transfection. The chemical transfection reagent used was PEI (purchased from Polysciences), and the cultured 293F cells were transiently transfected according to the manufacturer's protocol. Cell supernatants were collected on day 5 of culture and centrifuged at 4000 rpm for 20 minutes. The supernatant was purified using Protein A medium (purchased from Boglund). The antibodies were then exchanged into PBS pH 7.2 buffer by dialysis, and further purified to obtain the corresponding bispecific antibodies.

[0185] Table 2: Bispecific antibody CD84×CD3 sequence information

[0186] Example 7 In vitro T cell-dependent cytotoxicity of bispecific antibodies

[0187] U937 is a human histiocytic lymphoma cell, a type of acute myeloid leukemia cell. It serves as the target cell, and human PBMC serves as the effector cell. The efficacy of the bispecific antibody CD84×CD3 in Example 6 on T-cell-mediated cytotoxicity was determined and analyzed. The specific operation is as follows: RPMI-1640 culture medium containing 1% FBS was prepared as a killing detection medium. The frozen PBMC effector cells were revived, the PBMC cells were transferred to a 15 mL centrifuge tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were washed twice with culture medium, resuspended, and counted using a cell counter. The effector cell density was adjusted to 1.25×10 6 / mL and place in a 37°C, 5% CO2 incubator until ready to use. Add 5mL of culture medium to a 15mL centrifuge tube, thaw the frozen target cells in a 37°C water bath, and transfer them to 5mL of culture medium. Centrifuge at 1500rpm for 5min. Discard the supernatant, wash the cells twice with culture medium, resuspend the cells, and count them using a cell counter. Adjust the target cell density to 1.88×10 5 / mL, and placed in a 37°C, 5% CO2 incubator for later use.

[0188] Antibody dilution: The initial concentrations of the dual antibody CD84×CD3 of Example 6 were set at 20 nM and 10 nM, and then diluted 5-fold in sequence, for a total of 10 different concentrations.

[0189] Remove the reserved PBMC effector and target cells, and aliquot 60 μL / well of each into a 96-well plate for culture. Using a dispenser, dispense 60 μL of the diluted antibody into the 96-well plate and incubate. Observe the cells for uniformity under a microscope. Add 60 μL of culture medium to the control wells. Incubate the cells at 37°C, 5% CO₂ for 24 hours. Forty-five minutes before the end of the co-culture, add 20 μL of 10x lysis buffer to the wells showing maximum LDH release from the target cells. Mix thoroughly and incubate the cells at 37°C, 5% CO₂ for 45 minutes. Centrifuge the co-cultured cells at 3000 rpm for 2 minutes. Transfer 10 μL of the supernatant to a new 384-well microtiter plate. Add 10 μL of CytoTox 96 reagent to each well, centrifuge at 1000 rpm for 1 minute, and shake for 1 minute. Incubate at room temperature, protected from light, for 30 minutes, maintaining a maximum reading between 1 and 2. Add 10 μL of stop solution to each well and shake for 1 minute. The results are shown in Figure 9. The experimental results showed that the bispecific antibody molecules BIS02, BIS03, BIS04 and BIS05 all showed good mediating PBMC killing effect on U937 cells, and BIS07, BIS08 and BIS09 showed certain activity.

[0190] Example 8 Detection of Binding of Bispecific Antibodies to CD84 and CD3 Proteins

[0191] This example is an affinity experiment of a bispecific antibody to CD84 and CD3 proteins, demonstrating whether the construction of a bispecific antibody will affect its affinity for binding to CD84 and CD3 proteins.

[0192] The specific procedure is as follows: 100 μL of 1 μg / mL CD84 or CD3 protein solution was added to each well of a 96-well microtiter plate and incubated overnight in a refrigerator at 2-8°C. The wells were aspirated and washed twice with wash buffer to remove excess antigen solution. After washing, the plate was inverted to remove residual liquid. 100 μL of 2% BSA (prepared in PBS buffer) was added to each well and blocked for 1 hour at room temperature. The wells were aspirated and washed twice with wash buffer to remove excess antigen solution. After washing, the plate was inverted to remove residual liquid. 100 μL of a gradient concentration of the test antibody solution was added to each well and incubated at room temperature for 1 hour. The wells were aspirated and washed five times with wash buffer. 100 μL of Goat Anti-Human IgG Secondary Antibody (HRP) (Sino Biological SSA002) was added to each well and incubated for 0.5 hour at room temperature. The wells were aspirated and washed five times with wash buffer. Add 100 μL of TMB substrate solution to each well and incubate the plate at room temperature for 10 minutes or longer, until the solution reaches the desired color intensity. Add 100 μL of HCl stop solution to each well; within 20 minutes of adding the stop solution, measure the absorbance of each well at 450 nm using a microplate reader. BIS03, BIS05, and BIS07 were assayed separately, along with CD3 mAb, CD84 mAb, an isotype control (indicated in the figure), and PBS as controls.

[0193] The experimental results are shown in Figure 10, where A is a graph showing the binding results of the CD84 protein, and B is a graph showing the binding results of the CD3 protein. The experimental results show that the bispecific antibody configuration does not affect its binding to CD84 and CD3 proteins.

[0194] Example 9 Detection of binding of bispecific antibodies to different cells

[0195] This example is about the affinity determination of bispecific antibodies with different cells, 293T-hCD84, HEL, and THP-1, to detect the ability of bispecific antibodies to bind to CD84 proteins on different cell lines.

[0196] The specific operation is as follows: the pre-cultured cells (HEK293T-hCD84; HEL; THP-1) were digested and resuspended in FACS Buffer (PBS + 1% BSA) solution. After counting the cells, 2E5 cells / 100 μL were planted in a 96-well V-bottom plate. After the cell treatment was completed, 100 μL of antibody was added to each well according to the pre-set concentration, mixed with the cells, and incubated at 4 degrees for 1 hour. After the incubation, centrifuged at 1500 rpm for 5 minutes, removed the supernatant and retained the cell pellet, added 200 μL FACS buffer to resuspend the cells, and repeated centrifugation and washing under the same conditions twice. Then, 100 μL of secondary antibody Alexa Incubate with 647-conjugated AffiniPure™ Goat Anti-Human IgG (Jackson 109-605-098) for another 0.5 h, then centrifuge and wash twice. Resuspend the cells in 100 μL of FACS buffer and analyze them on the FACS analyzer. The number of cells in each sample should not be less than 10,000. BIS03, BIS05, and BIS07 were analyzed separately, and CD3 mAb, CD84 mAb, and isotype were used as controls.

[0197] The experimental results are shown in Figure 11, where Figure A shows the results of the experiment with HEK293T-hCD84 cells, Figure B shows the results of the experiment with HEL cells, and Figure C shows the results of the experiment with THP-1 cells. The table shows the specific EC50 test data. The experimental results show that BIS03, BIS05, and BIS07 have strong binding ability to HEK293T-hCD84, HEL, and THP-1 cell lines, indicating that the bispecific antibodies have strong binding ability to CD84 protein on cells overexpressing or tumor cells.

[0198] Example 10 Bispecific Antibody Mediates Immune Cell Killing of Tumor Cells

[0199] This embodiment is used to detect the toxicity of bispecific antibodies to immune cells against tumor cells (AML cells). The specific operation is as follows: the pre-cultured tumor cells (HEL; U937; THP-1; MOML-13) are digested and resuspended in a culture solution (PBS + 2% FBS). After cell counting, they are planted in a 96-well cell culture plate at a number of 1.5E4 cells / 100 μL per well and placed in a 37-degree incubator for 4-5 hours. PBMCs are counted and calculated according to a 10:1 ratio of PBMC to tumor cells. 1.5E5 cells / 50 μL per well are added to the cell culture plate. 50 μL of antibody is added to each well according to the pre-set concentration and placed in a 37-degree incubator for 24 hours. Aspirate 150 μl of supernatant and add it to a clean 96-well plate to reserve for subsequent ELISA and LDH experiments; add 50 μl of CTG (Promega, catalog number: G7573) to the remaining approximately 50 μl of the 96-well culture plate; gently tap to mix, aspirate 80 μl of the reaction solution, add it to a 96-well plate with a transparent white bottom, and detect the luminescence data using a microplate reader.

[0200] The experimental results are shown in Figure 12. The experimental results show that the bispecific antibody can significantly enhance the toxicity of immune cells against tumor cells (AML cells).

[0201] Example 11 In vitro safety assessment of bispecific antibodies

[0202] This example conducts an in vitro safety assessment of a bispecific antibody. The specific operation is as follows: the prepared PBMCs are resuspended in a culture solution (PBS + 2% FBS) and added to a cell culture plate at a volume of 22.5E5 cells / 100μL per well; 100μL of antibody is added to each well according to the pre-set concentration and incubated in a 37-degree incubator for 72 hours. 150μl of supernatant is aspirated and added to a clean 96-well plate for subsequent ELISA and LDH experiments; 50μl of CTG (Promega, catalog number: G7573) is added to the remaining approximately 50μl of the 96-well culture plate; gently tap to mix, aspirate 80μl of the reaction solution, add it to a 96-well plate with a transparent white bottom, and detect the luminescence data with an enzyme marker. BIS03, BIS05, and BIS07 were detected respectively, and CD84 mab, Talquetamab, and Isotype were used as controls; Talquetamab is a CD3 / GPRC5D bispecific T cell binding antibody.

[0203] The experimental results are shown in Figure 13. The experimental results show that the bispecific antibody molecules can significantly stimulate the proliferation and activation of PBMC in vitro, and there is a certain risk of cytokine storm. + The T cell proliferation effect has little effect on the suicide of T cells themselves.

[0204] Example 12 Kinetic Detection of Bispecific Antibodies and CD84 and CD3 Proteins

[0205] This example tests the dynamics of the bispecific antibody and CD84 and CD3 proteins. TM A label-free biomolecular analyzer was used to measure the binding kinetics (KD) of two bispecific antibodies to CD3 and CD84 proteins using bio-layer interferometry (BLI). CD3 and CD84 proteins were diluted to 1 μg / ml and immobilized on a his biosensor. The antibodies were then diluted to 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 1.5625 nM, for a total of six concentrations, allowing them to bind to the proteins. The results were fitted using a 1:1 model to calculate the equilibrium dissociation constant (KD) for the samples.

[0206] The experimental results are shown in Figures 14 and 15. Figure 14 shows the kinetics of the bispecific antibody binding to CD84, while Figure 15 shows the kinetics of the CD3 protein binding. The KD values ​​of BIS03 and BIS05 for CD84 and CD3 were determined, demonstrating strong affinity for both.

[0207] Example 13 In vivo anti-tumor experiment of bispecific antibodies

[0208] This example is an in vivo anti-tumor experiment of a bispecific antibody. The specific operation is as follows: (1) Tail vein in situ tumor inoculation model: Take THP1-luciferase (AML cell line) leukemia cells with good growth status, centrifuge and wash once with sterile PBS, centrifuge again, resuspend and count; adjust the cell density with sterile PBS solution. THP1-luciferase cells were transplanted into the tail vein of mice (6-week-old female NSG mice, which had been acclimated to the mouse house for one week): After the mouse tail was disinfected with 75% alcohol, 1×10 6(200 μl) cell suspension, recorded as day 0; (2) PBMC treatment of mice: 5 days after mouse tumor cell transplantation, PBMC were injected into the tail vein at an E:T ratio of 10:1. Then all mice were randomly divided into three groups according to body weight, namely Control, BIS03 and BIS05, with 8 mice in each group. The drugs were injected through the tail vein on day 6, day 9, day 13, day 16, day 20 and day 23, respectively, with a dose of 10 μg / mouse (0.5 mpk). Imaging observation was performed on day 7, day 14, day 21 and day 28. The entire experimental period was 77 days; (3) The mice were weighed every week and the mouse weight curve was drawn; the mouse bioluminescence imaging was performed every week to monitor the mouse tumor burden; the mouse survival time was recorded and the survival curve was drawn.

[0209] The experimental results are shown in Figure 16. The experimental results showed that the survival time of mice was significantly increased, proving that the bispecific antibody molecule has a significant anti-tumor effect in vivo.

Claims

1. An antibody or antigen-binding fragment thereof that binds to CD84, comprising three complementary determining regions (HCDRs) of a heavy chain variable region and three complementary determining regions (LCDRs) of a light chain variable region, wherein: (i) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:1, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:2, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:3, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:4, LCDR2 comprises or consists of NAK, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:5; or (ii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:1, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:2, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:3, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:6, LCDR2 comprises or consists of FAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:7; or (iii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:10, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:11, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:12; or (iv) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 13, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 14, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 15, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises or consists of YAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 17; or (v) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:18, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:10, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:19, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:20; or (vi) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:21, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:11, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:12; or (vii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:22, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:23, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:24, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:25, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:26; or (viii) HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:22, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:23, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:24, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:27, LCDR2 comprises or consists of WAS, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:26; Alternatively, the antibody comprises a variant of one of the CDR sequence combinations of (i) to (viii), wherein the variant comprises at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the 6 CDR regions, and preferably the heavy chain CDR3 remains unchanged; Wherein, the fifth amino acid X in the amino acid sequence of SEQ ID NO:9 is N, D, Q.

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises: (i) the amino acid sequence of SEQ ID NO:30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) the amino acid sequence of SEQ ID NO:43, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iii) the amino acid sequence of SEQ ID NO:45, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iv) the amino acid sequence of SEQ ID NO:53, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein: The light chain variable region comprises: (i) the amino acid sequence of SEQ ID NO:29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (ii) the amino acid sequence of SEQ ID NO:38, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iii) the amino acid sequence of SEQ ID NO:46, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto; or (iv) the amino acid sequence of SEQ ID NO:54, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy chain variable region and a light chain variable region selected from the group consisting of (i) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:30, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:29, or (ii) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:43, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:38, or (iii) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:45, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:46, or (iv) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:53, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

54.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is an antibody or antigen-binding fragment thereof in the form of IgG1, IgG2, IgG3, or IgG4, preferably a human IgG1 Fc region.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a murine antibody, or a chimeric antibody, or a humanized antibody, or wherein the antigen-binding fragment is an antibody fragment selected from the following: Fab, Fab', Fab'-SH, Fv, single-chain antibody, scFv, (Fab')2 fragment, single domain antibody, diabody (dAb) or linear antibody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody is a multispecific antibody.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody is a bispecific antibody, which further comprises an antigen-binding fragment that binds to a second antigen.

9. The antibody or antigen-binding fragment thereof according to claim 8, wherein the second antigen is a tumor-associated antigen or an immune cell antigen, preferably a T cell antigen.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the T cell antigen is selected from T cell receptor (TCR), CD3, CD4, CD8, CD16, CD25, CD28, CD38, CD44, CD62L, CD69, ICOS, 41-BB (CD137) and NKG2D.

11. A bispecific antibody or an antigen-binding fragment thereof, comprising a first antigen-binding fragment that binds to CD84 and a second antigen-binding fragment that binds to CD3, wherein the first antigen-binding fragment comprises a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and the second antigen-binding fragment comprises a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein the VL1 comprises LCDR 1-3 in the amino acid sequence shown in SEQ ID NO: 29, 38, 46 or 54, and the VH1 comprises HCDR 1-3 in the amino acid sequence shown in SEQ ID NO: 30, 43, 45 or 53; and / or the VL2 comprises LCDR 1-3 in the amino acid sequence shown in SEQ ID NO: 62, respectively, and the VH2 comprises HCDR 1-3 in the amino acid sequence shown in SEQ ID NO: 61, respectively.

12. The bispecific antibody according to any one of claims 8 to 11, wherein the second antigen-binding region is a scFv, the scFv comprises VL2 and VH2, and the scFv is optionally connected to the N / C terminus of the VL1 or VH1 or the Fc N / C terminus via a linker.

13. The bispecific antibody according to claim 12, wherein the linker comprises an amino acid sequence selected from (G4S)n and GS(G4S)n, wherein n is an integer selected from 1-5.

14. An isolated nucleic acid encoding the anti-CD84 antibody or antigen-binding fragment thereof of any one of the preceding claims.

15. A vector comprising the nucleic acid according to claim 14, preferably the vector is an expression vector.

16. A host cell comprising the nucleic acid according to claim 14 or the vector according to claim 15, preferably, the host cell is a mammalian cell.

17. A method for preparing an anti-CD84 antibody or an antigen-binding fragment thereof, the method comprising culturing a host cell comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 under conditions where the nucleic acid is expressed, optionally isolating the antibody or antigen-binding fragment thereof, and optionally the method further comprises recovering the anti-CD84 antibody or antigen-binding fragment thereof from the host cell.

18. An immunoconjugate comprising the CD84 antibody or antigen-binding fragment thereof of any one of the preceding claims 1-13 conjugated to a therapeutic or diagnostic agent.

19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13 or the immunoconjugate according to claim 18, and optionally a pharmaceutically acceptable excipient.

20. A method for preventing or treating a tumor in a subject, the method comprising administering to the subject an effective amount of an anti-CD84 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, or an immunoconjugate according to claim 18, or a pharmaceutical composition according to claim 19, preferably, the tumor is a hematological malignancy; preferably, the hematological malignancy is acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute lymphocytic leukemia (ALL), multiple myeloma (MM), hairy cell leukemia (HCL) or Hodgkin lymphoma (HL).

21. A method for detecting CD84 in a sample, the method comprising: (a) contacting a sample with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13; and (b) detecting formation of a complex between the antibody or antigen-binding fragment thereof and CD84; optionally, the antibody is detectably labeled.

Images