Car, cell expressing car and use thereof

Abstract

Provided are an isolated CAR, a nucleic acid, an expression vector, a cell expressing the CAR, a pharmaceutical composition and a use thereof. The CAR comprises an extracellular binding domain that binds to CD84, a transmembrane domain and an intracellular signal domain, wherein the extracellular binding domain that binds to CD84 is an antibody that binds to CD84. The CAR can achieve a relatively high conversion positive rate in the preparation of CAR-T cells, and the prepared CAR-T cell has excellent tumor killing effect and the capability of promoting cytokine release.

Description

CAR, CAR-expressing cells, and uses thereof

[0001] This application claims the benefit of Chinese Patent Application No. 2023116404678, filed December 1, 2023. The entire disclosure of the aforementioned Chinese Patent Application is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of cellular immunotherapy technology, and in particular to CAR, cells expressing CAR and uses thereof. Background Art

[0003] Chimeric Antigen Receptor T-Cell (CAR-T) technology is a new adoptive immunotherapy that has emerged in recent years. It involves genetically modifying a patient's T cells ex vivo, expanding them, and then infusing them back into the patient, achieving targeted tumor killing. The CAR-T structure primarily consists of a single-chain antibody that specifically recognizes tumor-specific antigens on the extracellular surface, a transmembrane domain, and a tandem signaling domain for intracellular T cell activation (CD28-CD3z and 4-1BB-CD3z are currently the most commonly used). Adoptive cell therapy, which involves transferring genetically engineered chimeric antigen receptors (CARs) into T cells to directly target cancer cells and activate a specific immune response against tumors, has successfully transitioned from the laboratory to the clinic. CAR receptors can recognize specific antigens, leading to T cell activation, proliferation, and killing of target cells. Due to its enormous potential for continuous development of new targets and continued optimization, CAR-T cell therapy is an attractive alternative to traditional therapies (chemotherapy, radiotherapy, and stem cell transplantation).

[0004] CD84 (SLAMF5) is a member of the SLAM family of cell surface immune receptors and is widely expressed on most immune cell subsets. It has an affinity adhesion molecule function and can activate or inhibit lymphocytes depending on the type of lymphocyte or the degree of activation / differentiation. CD84-mediated signaling pathways can regulate various immune responses, including T cell cytokine secretion, natural killer cell cytotoxicity, monocyte activation, autophagy, interactions between cognate T and B lymphocytes, and B cell tolerance at the germinal center (GC) checkpoint (AMC, AJS, rpád Lányi b, et al. CD84 cell surface signaling molecule: An emerging biomarker and target for cancer and autoimmune disorders [J]. Clinical Immunology, 2019, 204: 43-49.).

[0005] Alterations in CD84 are associated with autoimmune and lymphoproliferative disorders. For example, specific allelic variants in CD84 are associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (ASH, 2022, Oral Session: 602). In chronic lymphocytic leukemia, CD84 mediates both intrinsic and stromal-induced survival of malignant cells. CD84 is overexpressed in acute myeloid leukemia (AML) cells, and its upregulation is associated with poor prognosis in AML patients (ASH, 2022, Oral Session: 602). CD84 is highly expressed on myeloid-derived suppressor cells, which secrete the cytokine macrophage migration inhibitory factor (MIF), which induces CD84 expression on cells in their microenvironment. Its activation leads to increased gene expression of regulatory differentiated monocytes / granulocytes-myeloid-derived suppressor cells (M-MDSCs and G-MDSCs, respectively), and upregulates PD-L1 expression on MDSCs, thereby jointly inhibiting T cell function (Lewinsky H, Gunes EG, David K, et al. CD84 is a regulator of the immunosuppressive microenvironment in Multiple Myeloma[J]. JCI Insight, 2021, 6(4).). In chronic lymphocytic leukemia (CLL), cell-cell interactions mediated by CD84 can upregulate PD-L1 expression in CLL cells and the microenvironment, as well as PD-1 expression on T cells (Hadas Lewinsky, et al. CD84 regulates PD-1 / PD-L1 expression and function in chronic lymphocytic leukemia. J Clin Invest.). The above research results indicate that leukemia cells can regulate immune checkpoints through CD84, and blocking the effect of CD84 may be a new strategy to reverse tumor-induced immunosuppression (Binsky-Ehrenreich I, Marom A, Sobotta MC, et al. CD84 is a survival receptor for CLL cells[J]. Oncogene, 2014, 33(8): 1006.).

[0006] Given that CD84 is widely expressed in various hematological 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), relevant studies have shown that inhibiting CD84 function has therapeutic effects on hematological malignancies, especially AML.

[0007] At present, no drugs targeting CD84 have been approved for marketing, so CD84-targeted CART therapy will bring hope for the treatment of CD84-positive tumors. The development of CD84-targeted drugs to treat AML has important clinical value.

[0008] SUMMARY OF THE INVENTION

[0009] 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.

[0010] 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, 23 and 76, 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.

[0011] In some embodiments, the present invention also provides anti-CD84 antibodies or antigen-binding fragments thereof that bind to the same or overlapping epitopes as exemplary antibodies of the present invention (e.g., antibodies having the VH and VL sequence combinations of the antibodies listed in Table 1 below) and / or compete for binding to CD84 and / or inhibit (e.g., competitively inhibit) exemplary antibodies of the present invention. 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.

[0012] In some embodiments, the present invention provides methods for making the antibodies or antigen-binding fragments thereof of the present invention.

[0013] In some embodiments, the present invention provides a chimeric antigen receptor (CAR), immunoconjugate, or drug combination comprising an antibody of the present invention.

[0014] 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).

[0015] 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.

[0016] 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.

[0017] In some embodiments, the chimeric antigen receptor (CAR) provided by the present invention specifically comprises (e.g., sequentially connected) an extracellular binding region, a transmembrane region, and an intracellular signaling region (e.g., comprising a co-stimulatory domain) that binds to CD84, wherein the extracellular binding region that binds to CD84 is an antibody that binds to CD84, and the antibody that binds to CD84 comprises a VH (heavy chain variable region) and a VL (light chain variable region), wherein the VH comprises three HCDRs (heavy chain complementarity determining regions): HCDR1, HCDR2, and HCDR3, and the VL comprises three LCDRs (light chain complementarity determining regions): LCDR1, LCDR2, and LCDR3, wherein:

[0018] (1) the amino acid sequence of HCDR1 is shown in SEQ ID NO: 8, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 9, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 21, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 11, the amino acid sequence of LCDR2 is WAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 12;

[0019] (2) the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 13, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 14, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 15, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 16, the amino acid sequence of LCDR2 is YAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 17; or

[0020] (3) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:76, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is WAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:12; or

[0021] (4) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of LCDR2 is FAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 7.

[0022] 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.

[0023] The CD84 antibody provided by the present invention specifically recognizes and binds to the CD84 protein;

[0024] The CD84 antibody provided by the present invention specifically recognizes and binds to target cells expressing CD84 protein, and can be used to prepare anti-CD84 chimeric antigen receptors and immune effector cells expressing anti-CD84 chimeric antigen receptors;

[0025] The anti-CD84 chimeric antigen receptor immune effector cells (CAR-T cells) provided by the present invention specifically recognize, bind to and kill target cells positive for CD84 protein, and can efficiently stimulate target cells (such as 293T-CD84-LUC) to release cytokines (such as IL-2 and TNF-α). BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 shows the construction of CD84 overexpressing cell lines.

[0027] Figure 2 shows the light / heavy chain CDR sequences of chimeric antibodies.

[0028] FIG3 shows the affinity experiment of chimeric antibody to 239T-hCD84 cells.

[0029] FIG4A shows the sequence information of humanized antibody VH / VL. FIG4A shows the sequence information of humanized antibody VH / VL.

[0030] FIG4B shows the sequence information of humanized antibody VH / VL. FIG4B shows the sequence information of humanized antibody VH / VL.

[0031] Figure 4C shows the VH / VL sequence information of humanized antibodies.

[0032] Figure 4D shows the VH / VL sequence information of humanized antibodies.

[0033] FIG5 shows the affinity experiment between humanized antibodies and 293T-hCD84 cells.

[0034] FIG6 shows the affinity experiment between humanized antibodies and HEL cells.

[0035] Figure 7 shows the affinity experiment between humanized antibodies and U937 cells.

[0036] Figure 8 is a schematic diagram of an exemplary CAR-CD84 structure, wherein VL and VH refer to the VL and VH sequences of the anti-CD84 antibody.

[0037] FIG9A shows the CAR positive rate results of negative control human T cells detected by flow cytometry after 72 hours.

[0038] FIG9B shows the flow cytometry results of CAR positivity detection of human T cells infected with CD84 CAR-01 lentivirus 72 hours later.

[0039] Figure 9C shows the results of flow cytometry detection of CAR positivity 72 hours after human T cells were infected with CD84 CAR-02 lentivirus.

[0040] Figure 9D shows the results of flow cytometry detection of CAR positivity rate 72 hours after human T cells were infected with CD84 CAR-03 lentivirus.

[0041] Figure 9E shows the results of flow cytometry detection of CAR positivity rate 72 hours after human T cells were infected with CD84 CAR-04 lentivirus.

[0042] Figure 10A shows the killing efficiency (Lysis %) of T cells expressing CAR-CD84 and target cells 293T-CD84-LUC co-cultured for 18 hours when the effector-target ratio was 1:1, 1:2, and 1:4, respectively.

[0043] Figure 10B shows the killing efficiency (Lysis %) of T cells expressing CAR-CD84 and target cells THP-1-LUC co-cultured for 18 hours when the effector-target ratio was 1:1, 1:2, and 1:4, respectively.

[0044] Figure 11A shows the results of flow cytometry detection of cytokine release levels of T cells expressing CAR-CD84 after stimulation and activation by target cells 293T-CD84-LUC.

[0045] Figure 11B shows the results of flow cytometry detection of cytokine release levels of T cells expressing CAR-CD84 after stimulation and activation by target cells THP-1-LUC.

[0046] Detailed Description of the Invention

[0047] definition

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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)).

[0053] 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:

[0054] 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.

[0055] 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).

[0056] 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 (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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] Various aspects of the invention are described in further detail in the following subsections.

[0066] i. Anti-CD84 Antibodies of the Present Invention

[0067] 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.

[0068] Antibody CDR region

[0069] "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.

[0070] Table 1 VH and VL sequence combinations of exemplary antibodies

[0071] 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 2:

[0072] Table 2 Common CDR definition schemes

[0073] 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.

[0074] Unless otherwise specified, the variable region and CDR sequences in the disclosed examples are subject to the "IMGT" numbering convention.

[0075] In a preferred experimental scheme, the CDRs of the antibodies of the present invention are shown in Table 3.

[0076] Table 3. Exemplary CDR sequence combinations of the present invention

[0077] 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:

[0078] (i) six CDR sequences contained in the VH and VL sequences of any of the antibodies listed in Table 1; or

[0079] (ii) 6 CDR sequences from any combination listed in Table 3; or

[0080] (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).

[0081] In one embodiment, the antibody or antigen-binding fragment thereof of the invention comprises:

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

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

[0084] (iii) the amino acid sequences of HCDR1, 2 and 3 of the heavy chain variable region as shown in SEQ ID NO: 43, and LCDR1, 2 and 3 of the light chain variable region as shown in SEQ ID NO: 42, or

[0085] (iv) the amino acid sequences of HCDR1, 2 and 3 of the heavy chain variable region as shown in SEQ ID NO: 30, and the amino acid sequences of LCDR1, 2 and 3 of the light chain variable region as shown in SEQ ID NO: 29.

[0086] Antibody variable region

[0087] "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 relatively 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, and 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).

[0088] 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 of the heavy and light chain variable regions of one of the antibodies in Table 1, 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.

[0089] 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 1, or VH consists of the amino acid sequence. In another embodiment, the antibody of the present invention comprises a variant of the VH sequence.

[0090] 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 1, or VL consists of the amino acid sequence. In yet another embodiment, the antibody of the present invention comprises a variant of the VL sequence.

[0091] In one embodiment, an antibody of the invention comprises:

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

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

[0094] (iii) VH comprising the amino acid sequence shown in SEQ ID NO: 43 or a variant thereof, and / or VL comprising the amino acid sequence shown in SEQ ID NO: 42 or a variant thereof, or

[0095] (iv) VH comprising the amino acid sequence shown in SEQ ID NO: 30 or a variant thereof, and / or VL comprising the amino acid sequence shown in SEQ ID NO: 29 or a variant thereof.

[0096] 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.

[0097] 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.

[0098] 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 1, or consists of said amino acid sequence pair. The present invention also provides variants of the antibody, for example, variants having at least 95-99% identity in VH, VL, or VH and VL, or comprising no more than 10 amino acid changes.

[0099] 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.

[0100] 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.

[0101] Antibody heavy and light chains

[0102] 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 contain 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] ii. Polynucleotides, vectors, and hosts

[0111] 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.

[0112] 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.

[0113] 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.

[0114] In a preferred embodiment, the nucleic acid of the invention encoding an antibody further comprises a nucleotide sequence encoding a heavy chain constant region.

[0115] 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).

[0116] 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.

[0117] 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).

[0118] iii. Chimeric Antigen Receptor

[0119] 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).

[0120] 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 KIR2DS2 hinge or a CD8 hinge.

[0121] 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.

[0122] 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.

[0123] iv. Immunoconjugates

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] v. Pharmaceutical compositions and pharmaceutical preparations

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] vi. Combination products

[0135] 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.

[0136] 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).

[0137] vii. Therapeutic / preventive / diagnostic uses

[0138] 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.

[0139] 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).

[0140] viii. Detection Application

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] Given that CD84 is widely expressed in various hematological malignancies, including 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). Related studies have shown that inhibiting CD84 function has the effect of treating malignant blood tumors, especially AML. This field urgently needs to develop new cell therapies to achieve therapeutic purposes by acting on CD84, and further solve the technical problems of the small number and poor effect of CAR and corresponding CAR-T cells in the prior art. The present invention provides a CAR (Chimeric Antigen Receptor), CAR-T cells (Chimeric Antigen Receptor T-Cell, CAR-T), and a method for preparing CAR-T cells and their applications.

[0146] A technical solution provided by the present invention is: a separated chimeric antigen receptor (CAR) molecule, which comprises an extracellular binding region, a transmembrane region and an intracellular signaling region (e.g., comprising a co-stimulatory domain) that binds to CD84 (e.g., sequentially connected), wherein the extracellular binding region that binds to CD84 is an antibody that binds to CD84, and the antibody that binds to CD84 comprises a VH (heavy chain variable region) and a VL (light chain variable region), wherein the VH comprises three HCDRs (heavy chain complementary determining regions): HCDR1, HCDR2 and HCDR3, and the VL comprises three LCDRs (light chain complementary determining regions): LCDR1, LCDR2 and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are selected from one of the following combinations:

[0147] (1) the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 8, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 9, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 21, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 11, the amino acid sequence of LCDR2 is WAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 12; or

[0148] (2) the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 13, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 14, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 15, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 16, the amino acid sequence of LCDR2 is YAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 17; or

[0149] (3) the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 8, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 76, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 10, the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 11, the amino acid sequence of LCDR2 is WAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 12; or

[0150] (4) The amino acid sequence of HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of LCDR1 is shown in SEQ ID NO: 6, the amino acid sequence of LCDR2 is FAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 7.

[0151] In one embodiment of the present invention, the antibody that binds to CD84 comprises one of the following VH and VL combinations:

[0152] (1) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 45, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 46, or

[0153] (2) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 53, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 54, or

[0154] (3) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 43, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 42, or

[0155] (4) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 30, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 29.

[0156] In one embodiment of the present invention, the antibody that binds to CD84 comprises one of the following VH and VL combinations:

[0157] (1) a VH comprising the amino acid sequence shown in SEQ ID NO: 45, and a VL comprising the amino acid sequence shown in SEQ ID NO: 46, or

[0158] (2) a VH comprising the amino acid sequence shown in SEQ ID NO: 53, and a VL comprising the amino acid sequence shown in SEQ ID NO: 54, or

[0159] (3) a VH comprising the amino acid sequence shown in SEQ ID NO: 43, and a VL comprising the amino acid sequence shown in SEQ ID NO: 42, or

[0160] (4) VH comprising the amino acid sequence shown in SEQ ID NO: 30, and VL comprising the amino acid sequence shown in SEQ ID NO: 29.

[0161] In one embodiment of the present invention, the CAR further comprises a signal peptide. Preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO: 75.

[0162] In one embodiment of the present invention, the antibody that binds to CD84 is a scFv antibody.

[0163] In one embodiment of the present invention, the linker in the scFv antibody includes an amino acid sequence based on SEQ ID NO: 61, for example, a single Gly can be added at both ends of the amino acid sequence shown in SEQ ID NO: 61, wherein n is an integer of 1-10, for example, n≤6, and the linker is preferably as shown in SEQ ID NO: 62.

[0164] In one embodiment of the present invention, the structure of the scFv antibody is VH-linker-VL or VL-linker-VH.

[0165] In one embodiment of the present invention, the amino acid sequence of the scFv antibody is shown in any one of SEQ ID NOs: 66 and 63-65.

[0166] In one embodiment of the present invention, the transmembrane region may include: the transmembrane region of CD4, CD28 or CD8α. Preferably, the CD8α includes the sequence shown in SEQ ID NO:67.

[0167] In one embodiment of the present invention, the CAR further comprises a hinge region, and the transmembrane region can be connected to the extracellular binding region of the binding CD84 via a hinge region (e.g., a hinge region from a human protein). Preferably, the hinge region includes a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a KIR2DS2 hinge, or a CD8 hinge. More preferably, the hinge region includes an amino acid sequence as shown in SEQ ID NO: 68.

[0168] In one embodiment of the present invention, the intracellular signaling region comprises CD3ζ, and the CD3ζ comprises, for example, an amino acid sequence as shown in SEQ ID NO:69. The CD3ζ chain provides T cell receptor activation signals and proliferation stimulation for genetically modified T cells. Preferably, the intracellular signaling region further comprises a costimulatory domain. More preferably, the number of the costimulatory domains is 1, 2 or 3. Further preferably, the costimulatory domain is a CD28 costimulatory domain and / or a 4-1BB costimulatory domain. The CD28 costimulatory signaling domain enhances the initial activation and proliferation of CAR-T cells and improves effector function, while the 4-1BB costimulatory signaling domain mainly improves the expansion and long-term persistence of CAR-T cells. Further preferably, the 4-1BB costimulatory domain comprises an amino acid sequence as shown in SEQ ID NO:70.

[0169] In one embodiment of the present invention, the CAR comprises a signal peptide (e.g., sequentially connected from the N-terminus to the C-terminus), a scFv antibody that binds to CD84, a hinge region, CD8α, a 4-1BB co-stimulatory domain, and CD3ζ.

[0170] In one embodiment of the present invention, the amino acid sequence of the CAR comprises a sequence as shown in any one of SEQ ID NOs: 74 and 71-73.

[0171] In one embodiment of the present invention, the intracellular signaling region co-expresses some small molecules (such as the pro-inflammatory cytokine IL-12), which can trigger signals induced by cytokines or block some signaling pathways that affect the function of CAR-T cells.

[0172] Another technical solution provided by the present invention is: an isolated nucleic acid, wherein the isolated nucleic acid encodes the CAR described in the present invention.

[0173] Another technical solution provided by the present invention is an expression vector comprising the isolated nucleic acid described herein. Preferably, the starting vector of the expression vector is a lentiviral plasmid expression vector, such as a shuttle plasmid. Preferably, the expression vector further comprises a psPAX2 plasmid and a pMD2.G plasmid, forming a vector system with the lentiviral plasmid expression vector.

[0174] Another technical solution provided by the present invention is: an isolated cell expressing the CAR described in the present invention. Preferably, the host cell of the cell is an immune effector cell. More preferably, the immune effector cell is a T lymphocyte, a macrophage, a NK cell, or a NKT cell.

[0175] Another technical solution provided by the present invention is: a pharmaceutical composition, comprising one or more of the CAR described in the present invention, the isolated nucleic acid described in the present invention, the expression vector described in the present invention, and the isolated cells described in the present invention, and optionally a pharmaceutical excipient.

[0176] Another technical solution provided by the present invention is a method for preparing the isolated cells described herein, comprising transfecting a host cell with the expression vector described herein. Preferably, the host cell is an immune effector cell. More preferably, the immune effector cell is a T lymphocyte, a NK cell, or an NKT cell.

[0177] Another technical solution provided by the present invention is: the use of the CAR according to the present invention, the isolated nucleic acid according to the present invention, the expression vector according to the present invention, the isolated cell according to the present invention, or the pharmaceutical composition according to the present invention in the preparation of a drug for treating a CD84-related disease. Preferably, the CD84-related disease is a CD84-related cancer. More preferably, the CD84-related cancer is a CD84-related hematological malignancy. Further preferably, the CD84-related 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's lymphoma (HL), and the acute myeloid leukemia is, for example, acute monocytic leukemia.

[0178] Another technical solution provided by the present invention is: a CAR as described in the present invention, an isolated nucleic acid as described in the present invention, an expression vector as described in the present invention, an isolated cell as described in the present invention, or a pharmaceutical composition as described in the present invention for treating a CD84-related disease. Preferably, the CD84-related disease is a CD84-related cancer. More preferably, the CD84-related cancer is a CD84-related hematological malignancy. Further preferably, the CD84-related 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's lymphoma (HL), and the acute myeloid leukemia is, for example, acute monocytic leukemia.

[0179] Another technical solution provided by the present invention is: a method for treating a CD84-related disease, the method comprising administering to a subject in need thereof an effective amount of a CAR as described in the present invention, an isolated nucleic acid as described in the present invention, an expression vector as described in the present invention, an isolated cell as described in the present invention, or a pharmaceutical composition as described in the present invention. Preferably, the CD84-related disease is a CD84-related cancer. More preferably, the CD84-related cancer is a CD84-related hematological malignancy. Further preferably, the CD84-related 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's lymphoma (HL), and the acute myeloid leukemia is, for example, acute monocytic leukemia.

[0180] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0181] The reagents and raw materials used in the present invention are commercially available.

[0182] The positive progress effect of the present invention is:

[0183] The CAR described in the present invention can achieve a high conversion positive rate in the preparation of CAR-T cells, and the prepared CAR-T cells have excellent tumor killing effect and the ability to promote cytokine release. DETAILED DESCRIPTION

[0184] 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.

[0185] Example 1 Construction of CD84 overexpressing cell line

[0186] 1.1 Construction of HEK293T-hCD84 and HEK293T-Cyno-CD84 Overexpressing Cells

[0187] 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 the HEK293T-hCD84 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 (Cat. No.: Biolegend 326002) as a positive control. The construction results are shown in Figure 1: Both HEK293T-hCD84 and HEK293T-Cyno-CD84 overexpression cell lines were successfully constructed.

[0188] Example 2 Screening and Preparation of Hybridoma Cells and Monoclonal Antibodies

[0189] 2.1 Preparation, screening and identification of hybridoma cells

[0190] 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.

[0191] 2.2 Preparation of chimeric antibody molecules and determination of binding activity to 239T-hCD84 cells

[0192] Chimeric antibody expression

[0193] 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.

[0194] Flow cytometry screening

[0195] The pre-cultured cells were digested and resuspended in FACS Buffer (PBS + 1% BSA) solution. After counting the cells, 2E5 cells / 100 μL were seeded 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 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 twice under the same conditions. Then, 100 μL of secondary antibody was added and incubated for another 0.5 hour, centrifuged and washed twice, and finally 100 μL FACS Buffer was added to resuspend the cells and tested on the machine. The number of cells in each sample should not be less than 10,000 during the test. The experimental results are shown in Table 4 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.

[0196] Table 4 Affinity test results of chimeric antibodies

[0197] Example 3 Humanization of the anti-CD84 antibody of the present invention

[0198] 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:

[0199] ① Determine the CDR sequence according to the Kabat and IMGT extended CDR division method;

[0200] ② Find the closest homologous sequence for each V / J region of the heavy and light chains in the human germline sequence database;

[0201] ③ Screening for the human germline that best matches the heavy and light chains and the lowest amount of back mutations;

[0202] ④ Constructing the CDR region of the chimeric antibody onto the human framework region;

[0203] ⑤ Using sequence and structural features, determine the amino acid positions in the framework region that maintain CDR function;

[0204] ⑥ Perform back mutation at the sequence position determined to be important;

[0205] ⑦Optimize amino acids at risk sites.

[0206] For the specific sequence information of the light chain variable region and heavy chain variable region of the obtained humanized antibody, please refer to Figures 4A to 4D.

[0207] Example 4 Purification and expression of the anti-CD84 humanized antibody of the present invention

[0208] 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 293 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.

[0209] Example 5 Detection of Binding of Humanized Antibodies to Overexpressing Cells

[0210] 5.1 Affinity determination of humanized antibodies to 293T-hCD84, HEL, and U937 cells

[0211] The pre-cultured cells were digested and resuspended in FACS Buffer (PBS + 1% BSA) solution. After cell counting, 2E5 cells / 100 μL were seeded 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 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 twice under the same conditions. Then, 100 μL of secondary antibody was added and incubated for another 0.5 hour, centrifuged and washed twice, and finally 100 μL FACS Buffer was added to resuspend the cells and tested on the machine. The number of cells in each sample should not be less than 10,000 during the test. 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.

[0212] Example 6 Construction of Chimeric Antigen Receptor Expression Vector

[0213] Construction method:

[0214] (1) The signal peptide (SEQ ID NO: 75), CD84 antibody light chain variable region, linker (SEQ ID NO: 62), CD84 antibody heavy chain variable region, hinge region (SEQ ID NO: 68), CD8α transmembrane domain (SEQ ID NO: 67), 4-1BB costimulatory signal domain (SEQ ID NO: 70), and CD3ζ intracellular signal region (SEQ ID NO: 69) were sequentially connected from N-terminus to C-terminus, wherein the CD84 antibody light chain variable region, linker (SEQ ID NO: 62) and CD84 antibody heavy chain variable region constituted scFv targeting CD84, and four chimeric antigen receptor expression cassettes were obtained respectively, and the Kozak sequence was introduced at the front end of each expression cassette; the structure is shown in Figure 8.

[0215] The scFv sequences used are as follows:

[0216] 1. scFv 01 of CD84-CAR 01

[0217] 2. scFv 02 of CD84-CAR 02

[0218] 3. scFv 03 of CD84-CAR 03

[0219] 4. scFv 04 of CD84-CAR 04

[0220] (2) After the whole gene synthesis of the chimeric antigen receptor expression cassette sequence, it was connected to the empty vector pCDH-EF1-MSC-T2A (Shanghai Newpu Biotechnology Co., Ltd.) through the XbaI / EcoRI restriction site to obtain a chimeric antigen receptor expression vector; 4 chimeric antigen receptor expression vectors were obtained and sequenced to verify their correctness.

[0221] The amino acid sequences of all CARs used in the examples are as follows:

[0222] CD84-CAR-01:

[0223] CD84-CAR-02:

[0224] CD84-CAR-03:

[0225] CD84-CAR-04:

[0226] Example 7 Preparation of strains expressing chimeric antigen receptors

[0227] method:

[0228] (1) Take out DH5α competent cells (Cat. No.: TransGen cd201-02) from a -80°C freezer and thaw on ice;

[0229] (2) Add 5 ng of plasmid to the competent medium, mix gently, and place on ice for 5 minutes;

[0230] (3) Heat shock at 42°C for 90 seconds and place on ice for 30 minutes;

[0231] (4) Add 0.5 mL of resistance-free LB and incubate at 37°C, 180 rpm for 30 minutes;

[0232] (5) Apply to an ampicillin-resistant plate;

[0233] (6) Incubate overnight at 37°C;

[0234] (7) Pick a single clone and culture it in ampicillin-resistant LB at 37°C and 200 rpm for 9-12 hours;

[0235] (8) Add glycerol to the bacterial solution to a final concentration of 10% and store the bacteria at -80°C for future use, which can be used for subsequent large-scale plasmid extraction.

[0236] (9) After the above-mentioned strains are cultured in LB in large quantities, the plasmid is extracted using a plasmid extraction kit (Cat. No.: Tiangen Biochemical DP103-03) to obtain a shuttle plasmid encoding a chimeric antigen receptor for use in infection. The extraction method can be carried out according to the instructions.

[0237] Example 8 Virus Packaging

[0238] Cells were transfected by PEI method. 293T cells (purchased from Thermo Fisher Scientific) were trypsinized 24 hours before transfection and 4×10 6 293T cells were plated in a 10 cm cell culture dish and cultured in DMEM medium containing 10% FBS in a 37°C 5% CO2 incubator for no more than 24 hours. Cells were transfected when they reached a density of 60-80%.

[0239] The specific steps are as follows:

[0240] (1) Place the plasmid, PEI, and DMEM medium at room temperature for 5 minutes;

[0241] (2) Take 450 μL of DMEM and place it in a 1.5 mL EP tube. Then add 50 μL of PEI (1 μg / μL) and mix well. Let it stand at room temperature for 5 minutes.

[0242] (3) Take 10 μg of plasmid (the shuttle plasmid constructed in Example 7), 10 μg of psPAX2, and 5 μg of pMD2.G (all viral packaging plasmids are commonly used plasmids in the field), add DMEM to 500 μL, mix well, and let stand at room temperature for 5 minutes;

[0243] (4) adding the prepared PEI-DMEM solution of step (2) to the plasmid-containing DMEM obtained in step (3), mixing well, and standing at room temperature for 20 minutes to obtain a DNA / PEI mixture;

[0244] (5) Slowly drip 1 mL of DNA / PEI mixture into the 293T culture dish, mix gently, and incubate in a 37°C incubator for 6-8 hours.

[0245] (6) Discard the original culture medium, replace with fresh culture medium, and continue incubating in a 37°C incubator;

[0246] (7) After 48 hours of culture medium replacement, the culture medium was collected and 10 mL of fresh culture medium was added to each dish to continue culturing. After 24 hours, the supernatant was collected again and mixed with the supernatant collected at 48 hours.

[0247] (8) Centrifugation at 4000 g for 10 minutes at 4°C to remove cell debris;

[0248] (9) Filter through a 0.45 μm filter to obtain the supernatant;

[0249] (10) The filtered viral supernatant was transferred to an ultracentrifuge tube and centrifuged at 25,000 rpm for 2 hours. The viral precipitate obtained after ultracentrifugation was resuspended in serum-free medium and gently pipetted until completely dissolved to obtain virus solutions using different vectors;

[0250] (11) Each virus solution was divided into aliquots and stored in a -80°C refrigerator, and 5-10 μL of virus concentrate was reserved for titer determination.

[0251] Example 9 Virus Titer Determination

[0252] (1) Digest 293T cells and adjust the cell density to 4×10 5 / mL, add 500 μL of cell suspension to a 24-well plate and culture in an incubator;

[0253] (2) After 8 hours of cell adherence culture, the virus concentrate was added to the well plate in a gradient of 1 / 3 / 5 μL. The well plate was placed in an incubator for 6 hours and then replaced with fresh DMEM complete medium.

[0254] (3) After 48 hours of culture, centrifuge, resuspend and adjust the cell density to 1×10 6 / mL, add biotinylated CD84 antibody to 50 μL cell suspension, incubate for 30 minutes, wash 1-2 times with PBS, add streptavidin-APC antibody and incubate for 30 minutes, wash once with PBS and detect by flow cytometry.

[0255] Example 10 Preparation of T cells expressing chimeric antigen receptors targeting human CD84 antigen

[0256] method:

[0257] (1) Peripheral blood from healthy donors was collected using anticoagulant tubes, and human peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation.

[0258] (2) Enrichment of CD4+ / CD8+ T cells by magnetic bead separation (Cat. No.: Miltenyi Biotec, CliniMACS CD4 Reagent 200-070-132; CliniMACS CD8 Reagent 200-070-115);

[0259] (3) T cells were activated using T Cell TransAct human (Cat. No.: Miltenyi Biotec, 130-111-160) and stimulated for 24 hours;

[0260] (4) T cells were transduced with 4 different anti-CD84 CAR lentiviral vectors;

[0261] (5) 4-6 days after lentiviral transduction, T cells were stained with biotinylated CD84 antibody (Cat. No.: ACRO, CD4-H82E5) followed by streptavidin PE, and CAR expression was evaluated by flow cytometry using FlowJo for flow cytometric analysis (same as in Example 5).

[0262] result:

[0263] As shown in FIG9A to FIG9E , the positive rates of the control group and CD84 CAR 01-04 after lentiviral transduction were 0.23%, 59.0%, 60.7%, 69.2%, and 72.4%, respectively.

[0264] Example 11 Detection of tumor killing effect of CAR-T cells

[0265] The tumor killing effect of T cells expressing chimeric antigen receptors targeting human CD84 antigen was detected by in vitro co-culture. The target cells were luciferase-labeled 293T-CD84-LUC (cells from Xiangyao Biotechnology Co., Ltd.) and THP-1-LUC (human monocytic leukemia cells, Nanjing Kebai Biotechnology Co., Ltd.).

[0266] method:

[0267] (1) Chimeric antigen receptor T cells and tumor target cells 293T-CD84-LUC and THP-1-LUC were collected 72 hours after infection and counted. The cell density was adjusted to 1×10 6 / ml, and co-cultured in 96-well plates at effector-target ratios of 1:1, 1:2, and 1:4, respectively. The control cells were CD4+CD8+T cells that were not infected with the virus and were recorded as Control T cells.

[0268] (2) Place in a 37°C, 5% CO2 incubator for 18 to 24 hours.

[0269] (3) Mix the cells in each well by pipetting and transfer them to a new 96-well microtiter plate. Centrifuge the plate at 1800 rpm for 10 minutes to remove any bubbles. Add 10 μL of luciferase to each well, protect from light, and analyze within five minutes.

[0270] result

[0271] As shown in Figures 10A and 10B, the killing effects of CART cells on 293T-CD84-LUC and THP-1-LUC were observed when the effector-target ratios were 1:1, 1:2, and 1:4, respectively. The results showed that all four CAR-T cells were able to effectively kill tumors, among which the CAR-T cells constructed with CD84 CAR-04 had significantly better killing ability against target cells 293T-CD84-LUC and THP-1-LUC than the CAR-T cells constructed with the other three CARs.

[0272] Example 12 Determination of the ability of CAR-T cells to promote the release of cytokines IL2 and TNF-α

[0273] Flow cytometry CBA was used to detect the expression levels of cytokines IL2 and TNF-α

[0274] method:

[0275] (1) Chimeric antigen receptor T cells and tumor target cells 293T-CD84-LUC and THP-1-LUC were collected 72 hours after infection, counted, and the cell density was adjusted to 1×10 6 / ml, and co-incubated in 96-well plates at CAR-positive cell effector-target ratios of 1:1, 1:2, and 1:4;

[0276] (2) After 18 hours, the supernatant was collected and the expression levels of IL-2 and TNF-α were detected using a CBA kit (purchased from BD Pharmingen) according to the instructions.

[0277] result

[0278] As shown in Figures 11A and 11B, the release of IL-2 and TNF-a when CART cells were co-incubated with 293T-CD84-LUC and THP-1-LUC at effector-target ratios of 1:1, 1:2, and 1:4, respectively, indicated that all four CAR-T cells could efficiently stimulate the target cells to release IL-2 and TNF-α. Among them, CD84 CAR-04 had the strongest ability to stimulate the target cells 293T-CD84-LUC and THP-1-LUC to release IL-2 and TNF-α, which was significantly better than CAR-T cells constructed with other CARs, indicating that CD84 CAR-04 had the best effect in killing CD84-positive tumor cells.

[0279] All sequence information is as follows Table 5:

[0280] Table 5: Sequence information

[0281] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An isolated CAR, comprising an extracellular binding region for binding to CD84, a transmembrane region and an intracellular signaling region, characterized in that: The extracellular binding region that binds to CD84 is an antibody that binds to CD84, and the antibody that binds to CD84 comprises VH and VL, wherein the VH comprises three HCDRs: HCDR1, HCDR2 and HCDR3, and the VL comprises three LCDRs: LCDR1, LCDR2 and LCDR3, and the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2 and the LCDR3 are selected from one of the following combinations: (1) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:9, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:21, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is WAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:12; or (2) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:13, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:14, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:15, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:16, the amino acid sequence of LCDR2 is YAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:17; or (3) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:8, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:76, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:10, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:11, the amino acid sequence of LCDR2 is WAS, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:12; or (4) The amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, the amino acid sequence of LCDR1 is shown in SEQ ID NO:6, the amino acid sequence of LCDR2 is FAS, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

7.

2. The CAR according to claim 1, wherein: The CD84-binding antibody comprises one of the following VH and VL combinations: (1) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:45, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:46, or (2) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:53, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:54, or (3) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:43, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:42, or (4) VH comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:30, and VL comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:29; Preferably, the antibody that binds to CD84 comprises one of the following VH and VL combinations: (1) a VH comprising the amino acid sequence shown in SEQ ID NO:45, and a VL comprising the amino acid sequence shown in SEQ ID NO:46, or (2) a VH comprising the amino acid sequence shown in SEQ ID NO: 53, and a VL comprising the amino acid sequence shown in SEQ ID NO: 54, or (3) a VH comprising the amino acid sequence shown in SEQ ID NO: 43, and a VL comprising the amino acid sequence shown in SEQ ID NO: 42, or (4) VH comprising the amino acid sequence shown in SEQ ID NO:30, and VL comprising the amino acid sequence shown in SEQ ID NO:

29.

3. The CAR according to claim 1 or 2, wherein: The antibody binding to CD84 is a scFv antibody.

4. The CAR according to claim 3, wherein: The linker in the scFv antibody comprises an amino acid sequence based on SEQ ID NO:61, wherein n is an integer from 1 to 10, for example, n≤6, and the linker is preferably as shown in SEQ ID NO:62; Preferably, the structure of the scFv antibody is VH-linker-VL or VL-linker-VH; More preferably, the amino acid sequence of the scFv antibody is shown in any one of SEQ ID NOs: 66 and 63-65.

5. The CAR according to any one of claims 1 to 4, wherein: The structure of the CAR includes one or more of the following groups: (1) The transmembrane region comprises: the transmembrane region of CD4, CD28 or CD8α; preferably, the CD8α comprises the amino acid sequence shown in SEQ ID NO: 67; (2) a hinge region; the transmembrane region is connected to the extracellular binding region that binds to CD84 via the hinge region; preferably, the hinge region comprises a human Ig hinge, a KIR2DS2 hinge or a CD8 hinge; more preferably, the hinge region comprises the amino acid sequence shown in SEQ ID NO: 68; (3) The intracellular signaling region comprises CD3ζ, and the CD3ζ comprises, for example, the amino acid sequence shown in SEQ ID NO: 69; preferably, the intracellular signaling region further comprises a costimulatory domain; more preferably, the number of the costimulatory domains is 1, 2 or 3; further preferably, the costimulatory domain is a CD28 costimulatory domain and / or a 4-1BB costimulatory domain; further preferably, the 4-1BB costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 70; (4) A signal peptide; preferably, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:

75.

6. The CAR according to any one of claims 1 to 5, wherein: The CAR comprises a signal peptide, a scFv antibody that binds to CD84, a hinge region, CD8α, a 4-1BB co-stimulatory domain and CD3ζ; preferably, the amino acid sequence of the CAR comprises a sequence as shown in any one of SEQ ID NOs: 74 and 71-73.

7. An isolated nucleic acid, characterized in that The isolated nucleic acid encodes the CAR according to any one of claims 1-6.

8. An expression vector, characterized in that The expression vector comprises the isolated nucleic acid as described in claim 7; preferably, the starting vector of the expression vector is a lentiviral plasmid expression vector, such as a shuttle plasmid; preferably, the expression vector also comprises a psPAX2 plasmid and a pMD2.G plasmid.

9. An isolated cell, characterized in that The isolated cells express the CAR according to any one of claims 1 to 6; preferably, the host cells of the cells are immune effector cells; more preferably, the immune effector cells are T lymphocytes, macrophages, NK cells or NKT cells.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises one or more of the CAR according to any one of claims 1 to 6, the isolated nucleic acid according to claim 7, the expression vector according to claim 8, and the isolated cell according to claim 9, and optionally a pharmaceutical excipient.

11. A method for preparing the isolated cell according to claim 9, characterized in that: The method comprises transfecting a host cell with the expression vector according to claim 8; preferably, the host cell is an immune effector cell; more preferably, the immune effector cell is a T lymphocyte, a NK cell or a NKT cell.

12. Use of the CAR according to any one of claims 1 to 6, the isolated nucleic acid according to claim 7, the expression vector according to claim 8, the isolated cell according to claim 9 or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating a CD84-related disease; preferably, the CD84-related disease is a CD84-related cancer; more preferably, the CD84-related cancer is a CD84-related hematological malignancy; further preferably, the CD84-related hematological malignancy is acute myeloid leukemia, chronic lymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, chronic myeloid leukemia, myelodysplastic syndrome, acute lymphocytic leukemia, multiple myeloma, hairy cell leukemia or Hodgkin's lymphoma, and the acute myeloid leukemia is, for example, acute monocytic leukemia.

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