Anti-CD84 antibodies and chimeric antigen receptors

Abstract

An antigen-binding domain, antibody or chimeric antigen receptor that binds to CD84.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to anti-CD84 antibodies and chimeric antigen receptors (CARs) and their use in therapy, particularly cancer therapy. The present invention also relates to methods for determining CD84 levels in a sample and for identifying subjects suitable for treatment with anti-CD84 antibodies or CARs. [Background technology]

[0002] 2. Background of the Invention The human CD84 protein is a "cluster of differentiation" protein that was first identified in 1993. Sequencing of CD84 identified it as a member of the Slam (Signaling Lymphocyte Activation Molecule) family.

[0003] The extracellular portion of the CD84 receptor contains a non-canonical IgV distal domain and an IgC2g proximal domain, which is shared by all members of the SLAM family and similar to CD2. CD84 functions as a homophilic binding molecule, with receptor:ligand interactions involving the IgV domain and independent of the cytoplasmic domain, and with specific differences in the homophilic interface that prevent binding of CD84 to other molecules in the SLAM family.

[0004] Human CD84 is expressed on a variety of immune cell types. Expression levels vary with cell type and its state of differentiation or activation. CD84 is found on myeloid precursor cells very early in hematopoietic differentiation, and is expressed on most T and B cells, but is more highly expressed on memory T and B lymphocytes, follicular helper T cells, and germinal center B cells. Myeloid antigen-presenting cells, such as monocytes and monocyte-derived dendritic cells (DCs), are also CD84 positive. Granulocytes also express significant levels of CD84, with expression highest on basophils and mast cells. Of all hematopoietic cells, platelets express the highest levels of CD84, which was originally proposed as an aggregation-inducing signaling receptor involved in stabilizing platelet-platelet interactions. However, studies with CD84-deficient mice have shown that CD84 does not play a significant role in hemostatic and thrombotic functions. CD84 expression is low on natural killer cells (NK) and absent on erythrocytes.

[0005] CD84 signals can activate or inhibit leukocyte functions, depending on the cell type and its stage of activation or differentiation. Signaling through CD84 regulates diverse immunological processes, including T cell cytokine secretion, natural killer cell cytotoxicity, monocyte activation, autophagy, cognate T:B interactions, and B cell tolerance at the germinal center level.

[0006] Mutations in CD84 are associated with autoimmune disorders. For example, certain allelic mutations in CD84 are associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (RA). Typical treatments for such autoimmune diseases often include disease-modifying antirheumatic drugs (DMARDS), which are not curative but may halt or slow symptoms and / or disease progression.

[0007] CD84 is expressed on certain cancers, especially hematological malignancies such as chronic lymphocytic leukemia (CLL). Summary of the Invention

[0008] Hematological malignancies are often treated with chemotherapy or radiation therapy, which have known adverse side effects, and therefore there remains a great need for improved therapies for hematological malignancies.

[0009] Summary of the Invention The inventors have determined that CD84 is overexpressed in a range of cancer cell lines derived from hematological malignancies, including Burkitt's lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), and histiocytic lymphoma.

[0010] The present inventors have studied and developed a range of antibodies and chimeric antigen receptors (CARs) that bind to CD84.

[0011] In particular, the inventors characterized the cellular expression of CAR and functionally characterized CAR-T cells, for example, through measuring CAR-T cell proliferation, cytokine production, and cytotoxicity against a range of target cancer cell lines. Furthermore, the inventors demonstrated that the chimeric antigen receptor of the present invention can induce cytotoxicity in cancer cells expressing CD84.

[0012] Thus, the present invention provides a) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-NYWIN (SEQ ID NO: 1); CDR2-DIYPVSGTTNYNEKFKR (SEQ ID NO: 2); and CDR3-GTGRFAY (SEQ ID NO: 3); or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVSTSSYSYMH (SEQ ID NO: 4); CDR2-FASNLES (SEQ ID NO: 5); and CDR3-QHSWEIPYT (SEQ ID NO: 6); or variants thereof each having up to three amino acid substitutions, additions or deletions; b) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-NYWLG (SEQ ID NO: 7); CDR2-DIYPGGGYTNYIEKFKG (SEQ ID NO: 8); and CDR3-YEGGYYGNYDAMDY (SEQ ID NO: 9), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASESVDNYGISFMN (SEQ ID NO: 10); CDR2-AASNQGS (SEQ ID NO: 11); and CDR3-QQSKAVPRT (SEQ ID NO: 12), or variants thereof, each with up to three amino acid substitutions, additions or deletions; c) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSSYA (SEQ ID NO: 13); CDR2-ISGSGGST (SEQ ID NO: 14); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO: 15), or a variant thereof with up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-NIESKD (SEQ ID NO: 16); CDR2-DDA (SEQ ID NO: 17); and CDR3-QVWDSSSDHVV (SEQ ID NO: 18), or variants thereof, each having up to three amino acid substitutions, additions or deletions; d) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSSYP (SEQ ID NO: 19); CDR2-ISYHGRNK (SEQ ID NO: 20); and CDR3-ARDRDATPGGTGVGNHGMAV (SEQ ID NO: 21), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-QSLLHSSGYNY (SEQ ID NO: 22); CDR2-MGS (SEQ ID NO: 23); and CDR3-MQGLQTPPT (SEQ ID NO: 24), or variants thereof, each having up to three amino acid substitutions, additions or deletions; e) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSDNA (SEQ ID NO: 25); CDR2-ISGTGRTT (SEQ ID NO: 26); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO: 27), or a variant thereof with up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-QSLVYSDGDTY (SEQ ID NO: 28); CDR2-KVS (SEQ ID NO: 29); and CDR3-MQGTHWPPNT (SEQ ID NO: 30), or variants thereof with up to 3 amino acid substitutions, additions or deletions, respectively; f) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 31); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO: 32); and CDR3-MRTSYYFDY (SEQ ID NO: 33), or a variant thereof with up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIFSSLA (SEQ ID NO: 34); CDR2-NAKTLAE (SEQ ID NO: 35); and CDR3-QHHYATPFT (SEQ ID NO: 36), or a variant thereof, each having up to three amino acid substitutions, additions or deletions; g) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-SYWIN (SEQ ID NO: 37); CDR2-DIYLGSGSTNYNEKFKS (SEQ ID NO: 38); and CDR3-SGGYLGY (SEQ ID NO: 39), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVSTSSYSYMH (SEQ ID NO: 40); CDR2-FASNLES (SEQ ID NO: 41); and CDR3-QHSWEIPYT (SEQ ID NO: 42), or a variant thereof with up to 3 amino acid substitutions, additions or deletions, respectively; h) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-NYWIG (SEQ ID NO: 43); CDR2-DIYPGGGYTNYNENFKG (SEQ ID NO: 44); and CDR3-STTYYSSYWCFDV (SEQ ID NO: 45), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and Light chain variable region (VL) having CDRs with the sequences: CDR1-KSSQSLLNSGNQANYLA (SEQ ID NO: 46); CDR2-GASTRES (SEQ ID NO: 47); and CDR3-QNDHSYPFT (SEQ ID NO: 48), or variants thereof each having up to three amino acid substitutions, additions or deletions; i) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RYWMS (SEQ ID NO: 49); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 50); and CDR3-PGPTVVATYWYFDV (SEQ ID NO: 51), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 52); CDR2-KVSSRFS (SEQ ID NO: 53); and CDR3-FQGSHVPRT (SEQ ID NO: 54), or variants thereof each having up to three amino acid substitutions, additions or deletions; j) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RYWIN (SEQ ID NO: 55); CDR2-DIYPGSGSTNYNEKFKS (SEQ ID NO: 56); and CDR3-DTTIAY (SEQ ID NO: 57), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVTTSRYSYMH (SEQ ID NO: 58); CDR2-FASNLES (SEQ ID NO: 59); and CDR3-QHSWEIPYT (SEQ ID NO: 60), or a variant thereof with up to 3 amino acid substitutions, additions or deletions, respectively; k) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GYNMN (SEQ ID NO: 131); CDR2-NIDPYYGGTNYNQKFKG (SEQ ID NO: 132); and CDR3-GLLSGSFPY (SEQ ID NO: 133), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and Light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIYSYLA (SEQ ID NO: 134); CDR2-NAKTLAE (SEQ ID NO: 135); and CDR3-QHHYGSPLT (SEQ ID NO: 136), or variants thereof each having up to three amino acid substitutions, additions or deletions; l) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RSWMS (SEQ ID NO: 137); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 138); and CDR3-FYDGYSIYWYFDV (SEQ ID NO: 139), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RSSQSIVHSNGDTYLE (SEQ ID NO: 140); CDR2-KVSNRFS (SEQ ID NO: 141); and CDR3-FQGSHVPRT (SEQ ID NO: 142), or variants thereof, each having up to three amino acid substitutions, additions or deletions; m) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 143); CDR2-HIWWDDVKRYNPALRS (SEQ ID NO: 144); and CDR3-IAVTYFFDF (SEQ ID NO: 145), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIFSSFA (SEQ ID NO: 146); CDR2-NARTLAE (SEQ ID NO: 147); and CDR3-QHHYASPFT (SEQ ID NO: 148), or variants thereof, each having up to three amino acid substitutions, additions or deletions; n) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 149); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO: 150); and CDR3-MSTSYYFDY (SEQ ID NO: 151), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-KASQSLFTSVA (SEQ ID NO: 152); CDR2-SASYRYT (SEQ ID NO: 153); and CDR3-QQHYSSPFT (SEQ ID NO: 154), or variants thereof each having up to three amino acid substitutions, additions or deletions; or o) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-IYAMN (SEQ ID NO: 155); CDR2-RIRSKSNNYARFYADSVKD (SEQ ID NO: 156); and CDR3-PLRSYFSMDY (SEQ ID NO: 157), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and Light chain variable region (VL) having CDRs with the sequences: CDR1-KASENVDTYVS (SEQ ID NO: 158); CDR2-GASNRYT (SEQ ID NO: 159); and CDR3-GQTYSYPWT (SEQ ID NO: 160), or variants thereof with up to 3 amino acid substitutions, additions or deletions, respectively. The present invention provides an antigen-binding domain comprising:

[0013] In some embodiments the antigen binding domain comprises: a) a VH domain having the sequence of SEQ ID NO: 61; and a VL domain having the sequence of SEQ ID NO: 62 or 108; b) a VH domain having the sequence of SEQ ID NO: 63; and a VL domain having the sequence of SEQ ID NO: 64 or 109; c) a VH domain having the sequence of SEQ ID NO: 65; and a VL domain having the sequence of SEQ ID NO: 66; d) a VH domain having the sequence of SEQ ID NO: 68; and a VL domain having the sequence of SEQ ID NO: 69; e) a VH domain having the sequence of SEQ ID NO: 71; and a VL domain having the sequence of SEQ ID NO: 72; f) a VH domain having the sequence of SEQ ID NO: 74; and a VL domain having the sequence of SEQ ID NO: 75; g) a VH domain having the sequence of SEQ ID NO: 76; and a VL domain having the sequence of SEQ ID NO: 77; h) a VH domain having the sequence of SEQ ID NO: 78; and a VL domain having the sequence of SEQ ID NO: 79; i) a VH domain having the sequence of SEQ ID NO: 80; and a VL domain having the sequence of SEQ ID NO: 81; j) a VH domain having the sequence of SEQ ID NO: 82; and a VL domain having the sequence of SEQ ID NO: 83; k) a VH domain having the sequence of SEQ ID NO: 161; and a VL domain having the sequence of SEQ ID NO: 162; l) a VH domain having the sequence of SEQ ID NO: 163; and a VL domain having the sequence of SEQ ID NO: 164; m) a VH domain having the sequence of SEQ ID NO: 165; and a VL domain having the sequence of SEQ ID NO: 166; n) a VH domain having the sequence of SEQ ID NO: 167; and a VL domain having the sequence of SEQ ID NO: 168; or o) a VH domain having the sequence of SEQ ID NO: 169; and a VL domain having the sequence of SEQ ID NO: 170; or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto, respectively. The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0014] Suitably, the antigen binding domain is a CD84 binding domain.

[0015] In some embodiments, the antigen binding domain is an scFv.

[0016] In another aspect, the present invention provides an antibody comprising the antigen-binding domain of the present invention.

[0017] In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising the antigen binding domain of the present invention.

[0018] Suitably, the antigen binding domain, antibody or CAR of the present invention binds to CD84.

[0019] In some embodiments, the CAR is an scFv CAR.

[0020] In some embodiments, the CAR comprises a CD8a or CD28 transmembrane domain. In a preferred embodiment, the CAR comprises a CD8a transmembrane domain.

[0021] The antigen-binding domain and the transmembrane domain may be linked by a spacer. In some embodiments, the spacer comprises a CD8a hinge or a CD28 hinge. In a preferred embodiment, the CAR comprises a CD8a hinge.

[0022] A CAR can comprise one or more, e.g., two or more, intracellular signaling domains. In a preferred embodiment, the CAR comprises a CD3-zeta signaling domain.

[0023] The CAR may comprise one or more, for example, two or three, costimulatory domains. In some embodiments, the CAR comprises one or more costimulatory domains selected from the group consisting of a 4-1BB costimulatory domain, a CD28 costimulatory domain, an OX40 costimulatory domain, and an ICOS costimulatory domain.

[0024] In a preferred embodiment, the CAR comprises a 4-1BB costimulatory domain. In some embodiments, the CAR comprises a CD28 costimulatory domain. In some embodiments, the CAR comprises an OX40 costimulatory domain. In some embodiments, the CAR comprises an ICOS costimulatory domain.

[0025] In a preferred embodiment, the CAR comprises a 4-1BB costimulatory domain and a CD3-zeta signaling domain.

[0026] In a preferred embodiment, the CAR comprises an antigen-binding domain of the invention, a CD8a transmembrane domain, a 4-1BB costimulatory domain and a CD3-zeta signaling domain.

[0027] In another aspect, the present invention provides a polynucleotide comprising one or more nucleotide sequences encoding an antigen binding domain, antibody or CAR of the present invention.

[0028] In another aspect, the present invention provides a vector comprising a polynucleotide of the present invention.

[0029] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral or lentiviral vector, preferably a lentiviral vector.

[0030] In another aspect, the present invention provides a cell comprising a polynucleotide or vector of the present invention.

[0031] In another embodiment, the present invention provides a cell comprising the antigen binding domain or CAR of the present invention.

[0032] The cells may comprise a further (second) CAR.

[0033] In another embodiment, the invention provides a cell comprising a first CAR and a second CAR, wherein the first CAR is a CAR of the invention.

[0034] In another embodiment, the invention provides a composition comprising a first cell and a second cell, wherein the first cell comprises a first CAR and the second cell comprises a second CAR, wherein the first CAR is a CAR of the invention.

[0035] In some embodiments, the first CAR and the second CAR are different. In some embodiments, the first CAR and the second CAR bind to different antigens.

[0036] In another embodiment, the invention provides a cell comprising a tandem CAR, wherein the tandem CAR comprises a first antigen binding domain (e.g., an scFv) and a second antigen binding domain (e.g., an scFv), wherein the first antigen binding domain (e.g., scFv) is an antigen binding domain (e.g., an scFv) of the invention.

[0037] In some embodiments, the first antigen binding domain (e.g., scFv) and the second antigen binding domain (e.g., scFv) are different. In some embodiments, the first antigen binding domain (e.g., scFv) and the second antigen binding domain (e.g., scFv) bind to different antigens.

[0038] In some embodiments, the second CAR is an anti-CD19 CAR, an anti-CD20 CAR, or an anti-CD22 CAR (e.g., for the treatment of a B-cell malignancy); an anti-CD33 CAR or an anti-CD123 CAR (e.g., for the treatment of AML); or an anti-CD7 CAR (e.g., for the treatment of T-ALL).

[0039] In some embodiments, the second antigen binding domain (e.g., scFv) is an anti-CD19 antigen binding domain (e.g., scFv), an anti-CD20 antigen binding domain (e.g., scFv) or an anti-CD22 antigen binding domain (e.g., scFv) (e.g., for the treatment of B-cell malignancies); an anti-CD33 antigen binding domain (e.g., scFv) or an anti-CD123 antigen binding domain (e.g., scFv) (e.g., for the treatment of AML); or an anti-CD7 antigen binding domain (e.g., scFv) (e.g., for the treatment of T-ALL).

[0040] In some embodiments, the cells are T cells or NK cells, preferably T cells. In some embodiments, the T cells are autologous or allogeneic T cells. In some embodiments, the NK cells are autologous or allogeneic NK cells.

[0041] The NK cells may be derived, for example, from any suitable tissue sample, cell line, or stem cell source. In some embodiments, the NK cells (preferably allogeneic NK cells) are derived from umbilical cord blood (CB), induced pluripotent stem cells (iPSCs), bone marrow (BM), human embryonic stem cells (hESCs), cell lines (e.g., NK92 or YT), or peripheral blood (PB) (e.g., autologous or allogeneic). Preferably, the NK cells are derived from CB.

[0042] In another aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding domain, antibody, CAR, polynucleotide, vector or cell of the present invention.

[0043] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in therapy.

[0044] In some embodiments, the therapy is treatment of cancer.

[0045] In another aspect, the present invention provides a method for treating a disease comprising administering to a subject in need thereof an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the present invention.

[0046] In some embodiments, the disease is cancer.

[0047] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for the manufacture of a medicament.

[0048] In some embodiments, the medicament is for treating a disease, preferably cancer.

[0049] In a preferred embodiment, the cancer is a hematological malignancy.

[0050] In a preferred embodiment, the cancer cells express CD84, for example, the hematological malignancy can be a hematological malignancy that expresses CD84.

[0051] In some embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome, T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), chronic myeloproliferative syndrome, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, dendritic cell neoplasm, and histiocytic sarcoma.

[0052] In some embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), acute myeloid leukemia (AML), and histiocytic sarcoma.

[0053] In preferred embodiments, the cancer is selected from the group consisting of CLL, B-cell lymphoma, B-ALL, T-ALL and AML.

[0054] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in the treatment of AML.

[0055] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in the treatment of T-ALL.

[0056] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in the treatment of B cell lymphoma.

[0057] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in treating Burkitt's lymphoma.

[0058] In another embodiment, the invention provides the use of an antigen binding domain or an antibody of the invention for determining the CD84 expression level in a sample, optionally for analysing the CD84 expression level in a sample from a subject.

[0059] In another embodiment, the present invention provides a method for identifying a subject suitable for treatment with an anti-CD84 CAR or antibody, comprising determining the CD84 expression level of a sample isolated from the subject, wherein the CD84 expression level is determined using an antigen binding domain or antibody of the present invention.

[0060] In some embodiments, the sample is a blood sample. [Brief description of the drawings]

[0061] [Figure 1] Figure 1. Bar graph showing CD84 (also known as LY9B, SLAMF5, hCD84, mCD84) gene expression profile of all tumor samples and paired normal tissues. Bar height represents median expression for a particular tumor type or normal tissue. From http: / / gepia.cancer-pku.cn / detail.php?gene=CD84. [Diagram 2]Figure 2. Box and whisker representation (10th–90th percentile) of CD84 mRNA expression measured by RNA sequencing (top) or Affymetrix microarrays (bottom) in cell lines derived from different tumor types. From https: / / portals.broadinstitute.org / ccle. The figure shows only the 20 tumor types with the highest CD84 expression. [Diagram 3] Figure 3. CD84 expression in different cell lines. The light grey histograms represent staining with an isotype-matched control antibody, and the dark grey histograms represent staining with a specific CD84 antibody. [Figure 4] Figure 4. CD84 expression in nine samples from patients diagnosed with chronic lymphocytic leukemia assessed by flow cytometry. The light grey histograms represent staining with an isotype-matched control antibody, and the dark grey histograms represent staining with a specific CD84 antibody. [Diagram 5] Figure 5. CD84 expression in 10 samples from patients diagnosed with acute myeloid leukemia was evaluated by flow cytometry. A representative example is shown for patient P04 with moderate CD84 expression and one for patient P10 with high CD84 expression. The light grey histograms represent staining with an isotype-matched control antibody, and the dark grey histograms represent staining with a specific CD84 antibody. [Figure 6] Figure 6. Map of the pCCL-EF1α_CAR84 plasmid vector showing the insertion of the CD84 CAR sequence (top). Scheme of the anti-CD84 second generation CAR construct (bottom). [Figure 7] Figure 7. IFN-γ secretion by CD84-targeted CAR-T cells co-cultured with Ramos cells at an effector:target ratio of 2:1 after 24 h of incubation. UT: non-transduced T cells. Statistical significance was determined by Kruskal-Wallis (multiple comparisons against UT). Mean ± SEM of four experiments is shown. ***p<0.001, **p<0.01, *p<0.05. [Figure 8]Figure 8. IL-2 secretion by CD84-targeted CART cells co-cultured with Ramos cells at an effector:target ratio of 2:1 after 24 hours of incubation. UT: non-transduced T cells. Statistical significance was determined by Kruskal-Wallis (multiple comparisons against UT). Mean ± SEM of 4 trials is shown. ***p<0.001, **p<0.01, *p<0.05. [Figure 9] Figure 9. Granzyme B secretion by CD84-targeted CART cells co-cultured with Ramos cells at an effector:target ratio of 2:1 after 24 hours of incubation. UT: non-transduced T cells. Statistical significance was determined by Kruskal-Wallis (multiple comparisons against UT). Mean ± SEM of 4 trials is shown. ***p<0.001, **p<0.01, *p<0.05. [Figure 10] Figure 10. TNF-γ secretion by CD84-targeted CART cells co-cultured with Ramos cells at an effector:target ratio of 2:1 after 24 hours of incubation. UT: non-transduced T cells. Statistical significance was determined by Kruskal-Wallis (multiple comparisons against UT). Mean ± SEM of 4 trials is shown. ***p<0.001, **p<0.01, *p<0.05. [Figure 11] Figure 11. Cytotoxicity assay of different CD84-targeted CART cells against Ramos-GFP+ cells at an effector:target ratio of 2:1 and after an incubation period of 24 hours. The percentage of target viable cells is shown relative to untreated cells (target cells alone). Mean ± SEM of eight independent experiments. UT: untransduced T cells. Statistical significance was determined by ordinary one-way ANOVA (multiple comparisons against UT): **p<0.001, *p<0.01. [Figure 12] Figure 12. Cytotoxicity assay of different CD84-targeted CART cells against K562-GFP+ cells after an incubation period of 24 h at an effector:target ratio of 2:1. The percentage of target viable cells is shown relative to untreated cells (target cells alone). Mean ± SEM of 8 independent experiments. UT: untransduced T cells. Statistical significance was determined by ordinary one-way ANOVA (multiple comparisons against UT): **p<0.001, *p<0.01. [Figure 13] Figure 13. Cytotoxicity assay of different CD84-targeted CART cells against Ramos-GFP+ cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1, after incubation periods of 24 and 48 hours. Percentage of target viable cells versus untreated cells (target cells alone) is shown. Mean ± SEM of 5 independent experiments. UT: untransduced T cells. Statistical significance was determined by ordinary one-way ANOVA (multiple comparisons versus UT): ***p<0.001, *p<0.05. [Figure 14] Figure 14. Cytotoxicity assay of different CD84-targeted CART cells against K562-GFP+ cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. The percentage of target viable cells is shown relative to untreated cells (target cells alone). UT: untransduced T cells. Mean ± SEM of 5 independent experiments. Statistical significance was determined by ordinary one-way ANOVA (multiple comparisons against UT): **p<0.01, *p<0.05, ns: not significant difference. [Figure 15] Figure 15. Cytotoxicity assay of different CD84-targeted CART cells against MOLM-13-GFP+ cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. Percentage of target viable cells versus untreated cells (target cells alone) is shown. Mean ± SEM of three independent experiments, UT: untransduced T cells. Statistical significance was determined by ordinary one-way ANOVA (multiple comparisons versus UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 16]Figure 16. Cytotoxicity assay of various CD84-targeted CART cells against NALM6-GFP+ cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. Percentage of target viable cells versus untreated cells (target cells alone) is shown. Mean ± SEM of three independent experiments. UT: untransduced T cells. Statistical significance was determined by ordinary one-way ANOVA (multiple comparisons versus UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 17] Figure 17. Cytotoxicity assay of different CD84-targeted CART cells against MOLT4-GFP+ cells at effector:target ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. The percentage of target viable cells relative to untreated cells (target cells alone) is shown. Mean ± SEM of 4 independent experiments. UT: untransduced T cells. Statistical significance was determined by ordinary one-way ANOVA (multiple comparisons versus UT): **p<0.01, *p<0.05, ns: not significant difference. [Figure 18] Figure 18. CD84 expression in peripheral blood mononuclear cells (PBMCs) assessed by flow cytometry. Light grey histograms represent staining with an isotype-matched control antibody, and dark grey histograms represent staining with a specific CD84 antibody. PBMCs were stained with specific antibodies against CD4, CD8, CD19, and CD14. [Figure 19] Figure 19. Cytotoxicity assay of different CD84-targeted CART cells against CFSE+ labeled PBMC and Ramos-GFP+ cells after a 16 hour incubation period. The percentage of target viable cells is shown relative to untreated cells (target cells alone). Mean ± SEM of three independent experiments from three different donors. UT: untransduced T cells. Statistical significance was determined by paired t-test (compared to UT): **p<0.01, ns=not significant difference. [Figure 20] Figure 20. SDS PAGE gel image of CD84 antigen. Reducing SDS PAGE with Coomassie blue staining. [Figure 21] Figure 21. CD84 expression assessed by flow cytometry on leukemic cells derived from peripheral blood from two patients (P01 and P02) diagnosed with T-ALL. The histograms represent CD84 expression on blast cells gated on CD34, the light grey histograms represent staining with an isotype-matched control antibody, and the dark grey ones represent staining with a specific CD84 antibody. [Figure 22] Figure 22. CAR T cell expansion 6-7 days after transduction, shown as fold increase relative to T cell numbers on day 0 of culture. The figure represents 5-12 independent expansions, each using a cell tail from a different healthy donor. UT: non-transduced T cells. [Figure 23] Figure 23. Cytotoxicity assay of different CD84-targeted CART cells against Ramos-GFPffLuc cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. The percentage of target viable cells relative to untreated cells (target cells alone) is shown. Mean ± SEM of 5 independent experiments. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA test (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 24] Figure 24. Cytotoxicity assay of different CD84-targeted CART cells against MOLM-13 GFPffLuc cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. The percentage of target viable cells relative to untreated cells (target cells alone) is shown. Mean ± SEM of at least 5 independent experiments. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA test (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Diagram 25]Figure 25. Cytotoxicity assay of different CD84-targeted CART cells against U937 GFPffLuc cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. The percentage of target viable cells relative to untreated cells (target cells alone) is shown. Mean ± SEM of at least three independent experiments. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA test (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 26] Figure 26. Cytotoxicity assay of different CD84-targeted CART cells against MOLT-4 GFPffLuc cells at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after incubation periods of 24 and 48 hours. The percentage of target viable cells relative to untreated cells (target cells alone) is shown. Mean ± SEM of at least 5 independent experiments. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA test (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 27] Figure 27. Cytotoxicity assay of different CD84-targeted CART cells against primary AML cells stained with CFSE after incubation periods of 24 and 48 hours at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1. The percentage of target viable cells is shown relative to untreated cells (target cells alone). UT: untransduced T cells. Statistical significance was determined by two-way ANOVA test (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 28]Figure 28. Cytotoxicity assay of different CD84-targeted CART cells against primary T-ALL cells stained with CFSE after incubation periods of 24 and 48 hours at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1. The percentage of viable target cells relative to untreated cells (target cells alone) is shown. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA test (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 29] Figure 29. IFN-γ secretion by CD84-targeted CAR-T cells co-cultured with Ramos, MOLM-13 or MOLT-4 cells as indicated at an effector:target ratio of 2:1 after 24 hours of incubation. UT: non-transduced T cells. Statistical analysis was determined by two-way ANOVA mixed effects analysis assuming sphericity and Dunnett's post-hoc test for multiple comparisons (vs. UT). Mean ± SD of at least three experiments is shown. ***p<0.001, **p<0.01, *p<0.05. [Diagram 30] Figure 30. Granzyme B secretion by CD84-targeted CAR-T cells co-cultured with Ramos, MOLM-13 or MOLT-4 cells as indicated at an effector:target ratio of 2:1 after 24 hours of incubation. UT: non-transduced T cells. Statistical analysis was determined by two-way ANOVA mixed effects analysis assuming sphericity and Dunnett's post-hoc test for multiple comparisons (vs. UT). Mean ± SD of at least three experiments is shown. ***p<0.001, **p<0.01, *p<0.05. [Diagram 31] Figure 31. TNF-γ secretion by CD84-targeted CAR-T cells co-cultured with Ramos, MOLM-13 or MOLT-4 cells as indicated at an effector:target ratio of 2:1 after 24 h of incubation. UT: non-transduced T cells. Statistical analysis was determined by two-way ANOVA mixed effects analysis assuming sphericity and Dunnett's post-hoc test for multiple comparisons (vs. UT). Mean ± SD of at least three experiments is shown. ***p<0.001, **p<0.01, *p<0.05. [Diagram 32]Figure 32. Proliferation of CART cells measured in vitro by CFSE assay at 4 days (flow cytometry images). Proliferation at day 0 and after 4 days in medium alone (control), when stimulated with IL-2 or in the presence of MOLM-13 AML cells. UT: non-transduced T cells. [Diagram 33] Figure 33. CD84, CD33 and CD123 expression assessed by flow cytometry on CD34+ HPSCs isolated from apheresis products of healthy donors for allogeneic stem cell transplantation. Light grey histograms represent staining with isotype-matched control antibodies, dark grey represent staining with specific antibodies. [Diagram 34] Figure 34. Cytotoxicity assay of various CD84-targeted CART cells against CD34+ HPSCs isolated from five different cord blood units at effector:target (E:T) cell ratios of 4:1 and 2:1 after a 24 hour incubation period. The percentage of target viable cells relative to untreated cells (target cells alone) is shown. Mean ± SEM of five independent experiments. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA (multiple comparisons versus UT): ***p<0.001, **p<0.01, *p<0.05. [Diagram 35] Figure 35. Cytotoxicity assay of different CD84-targeted CART cells against CD34+ HPSCs isolated from apheresis products of healthy donors for allogeneic stem cell transplantation at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1 after a 24 hour incubation period. The percentage of target viable cells relative to untreated cells (target cells alone) is shown. Mean ± SEM of three independent experiments. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Diagram 36]Figure 36. Cytotoxicity assay of different CD84-targeted CART cells against CD3+ T cells isolated from the same donor after a 24 hour incubation period at effector:target (E:T) cell ratios of 4:1, 2:1, 1:1 and 0.5:1. The percentage of target viable T cells relative to untreated cells (target T cells alone) is shown. Mean ± SEM of 5 independent experiments. UT: untransduced T cells. Statistical significance was determined by two-way ANOVA (multiple comparisons against UT): ***p<0.001, **p<0.01, *p<0.05. [Figure 37] Figure 37. Flow cytometric analysis of different T cell subsets on target T cells before and after co-culture with various CART84. The following T cell subsets were considered: CD45RA+ / CD62L+ naive (white), CD45RA- / CD62L+ central memory (dark grey), CD45RA- / CD62L- effector memory (black), and CD45RA+ / CD62L- effector T cells (light grey). [Figure 38] Figure 38. Efficacy of CART84 cells against MOLM-13 cells in vivo. OLM-13 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (A) and animal survival (B). Statistical analysis of bioluminescence quantification was performed with a two-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice. Statistical significance of survival was determined with the log-rank test with corrected p-values ​​for 3 comparisons. [Figure 39]Figure 39. Efficacy of CART84 cells on MOLM-13 cells in vivo. MOLM-13 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (A) and animal survival (B). Total numbers of MOLM-13 GFP-ffLuc cells (C), CD3+ T cells (D) and CART cells (E) assessed by flow cytometry in bone marrow (top) and spleen (bottom) of mice at the end of the experiment. Statistical analysis of bioluminescence quantification was performed with a two-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice. Statistical significance of survival was determined with a log-rank test with corrected p-values ​​for three comparisons. Statistical analysis of MOLM-13 and CD3+ T cell quantification was performed with a one-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice, and analysis of CART cell quantification was performed with a one-way ANOVA model with Tukey's multiple comparisons compared to UT-treated mice. [Diagram 40] Figure 40. Efficacy of CD84 CART cells on MOLM-13 cells in vivo. MOLM-13 disease progression was followed by bioluminescence every week. Bioluminescence quantification (A) and animal survival (B). Total numbers of MOLM-13 GFP-ffLuc cells (C), CD3+ T cells (D) and CART cells (E) assessed by flow cytometry in bone marrow (top) and spleen (bottom) of mice at the end of the experiment. Statistical analysis of bioluminescence quantification was performed with a two-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice. Statistical significance of survival was determined with a log-rank test with corrected p-values ​​for three comparisons. Statistical analysis of MOLM-13 and CD3+ cell quantification was performed with a one-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice, and analysis of CART cell quantification was performed with a one-way ANOVA model with Tukey's multiple comparisons compared to UT-treated mice. [Diagram 41]Figure 41. Efficacy of CD84 CART cells against MOLT-4 cells in vivo. MOLT-4 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (A) and animal survival (B). Statistical analysis of bioluminescence quantification was performed with a two-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice. Statistical significance of survival was determined with the log-rank test with corrected p-values ​​for 3 comparisons. [Diagram 42] Figure 42. Efficacy of CD84 CART cells against MOLT-4 cells in vivo. MOLT-4 disease progression was monitored weekly by bioluminescence. Bioluminescence quantification (A) and animal survival (B). Total numbers of MOLT-4 GFP-ffLuc cells (C), CD3+ T cells (D) and CART cells (E) detected by flow cytometry in bone marrow (top) and spleen (bottom) of mice at the end of the experiment. Statistical analysis of bioluminescence quantification was performed with a two-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice. Statistical significance of survival was determined with a log-rank test with corrected p-values ​​for three comparisons. Statistical analysis of MOLT-4 and CD3+ cell quantification was performed with a one-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice, and analysis of CART cell quantification was performed with a one-way ANOVA model with Tukey's multiple comparisons compared to UT-treated mice. [Diagram 43] Figure 43. CD84 expression on human primary cells assessed by flow cytometry. Light grey histograms represent staining with an isotype-matched control antibody, dark grey histograms represent staining with a specific CD84 antibody. [Diagram 44] Figure 44. Cytotoxicity assay of CART84 cells 152.3, 153.5 and UT against human primary cells measured 72 hours after addition of CART / UT (arrows) to cell cultures using the XCELLigence instrument. UT: non-transduced T cells. [Diagram 45]Figure 45. Efficacy of CD84 CART cells against Ramos cells in vivo. (A) Ramos disease progression was monitored weekly by bioluminescence. Statistical analysis of bioluminescence quantification was performed with a two-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice. (B) Total number of Ramos GFP-ffLuc cells assessed by flow cytometry in bone marrow of mice at termination. Statistical analysis was performed with a one-way ANOVA model with Dunnett's multiple comparisons compared to UT-treated mice. UT: non-transduced T cells. *p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] Detailed Description of the Invention The terms "comprising," "comprises," and "comprised of," as used herein, are synonymous with "including" or "includes" or "containing" or "contains," and indicate inclusion or open-endedness and do not exclude additional, unrecited members, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0063] CD84 CD84 (also known as LY9B and SLAMF5) is a membrane glycoprotein that is a member of the signalling lymphocyte activation molecule (SLAM) family, which itself is a subset of the larger CD2 cell surface receptor subgroup of the Ig superfamily.

[0064] The extracellular portion of the CD84 receptor contains a non-canonical IgV distal domain and an IgC2g proximal domain, a structure shared by all members of the SLAM family. CD84 functions as a homophilic binding molecule and is expressed on many immune cell types. The receptor:ligand interaction involves the IgV domain and is independent of the cytoplasmic domain. The homophilic interface contains specific differences that prevent binding of CD84 to other molecules in the SLAM family.

[0065] The present inventors have determined that CD84 is overexpressed in a variety of cell lines derived from hematological malignancies, including Burkitt's lymphoma, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), and histiocytic lymphoma.

[0066] Studies suggest that CD84 is overexpressed in chronic lymphocytic leukemia (CLL).

[0067] An example of the amino acid sequence of CD84 is as follows: TIFF2025500618000002.tif33162

[0068] Antigen-binding domain An antigen-binding domain can be a protein or peptide that has the ability to recognize and bind to an antigen. Antigen-binding domains include natural, synthetic, semi-synthetic or recombinantly produced binding partners for an antigen of interest.

[0069] Exemplary antigen binding domains include an antibody or antibody fragment or derivative, an extracellular domain of a receptor (eg, a TCR), a ligand of a cell surface molecule / receptor, or a receptor binding domain thereof.

[0070] In a preferred embodiment, the antigen-binding domain is an antibody or is derived from an antibody. The antibody-derived domain can be an antibody fragment or a genetically engineered product of one or more fragments of an antibody, which fragment is involved in binding to an antigen. Examples include variable region (Fv), complementarity determining region (CDR), Fab, single chain variable fragment (scFv), heavy chain variable region (VH), light chain variable region (VL) and camelid antibody (VHH).

[0071] The antigen-binding domain may be non-human (e.g., murine), chimeric, humanized or fully human.

[0072] In a preferred embodiment, the antigen-binding domain is a single chain variable fragment (scFv). The scFv can be, for example, a murine, human or humanized scFv.

[0073] The term "complementarity determining region" (CDR), with reference to an antibody or antigen-binding fragment thereof, refers to the highly variable loops in the variable region of the heavy or light chain of the antibody. CDRs can interact with the conformation of an antigen and largely determine antigen binding (although some framework regions are known to be involved in binding). The heavy and light chain variable regions each contain three CDRs.

[0074] "Heavy chain variable region" (VH) refers to the variable fragment of the heavy chain of an antibody and comprises the three CDRs interposed between adjacent stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold supporting the CDRs.

[0075] "Light chain variable region" (VL) refers to the variable fragment of the light chain of an antibody, which contains the three CDRs interposed between framework regions.

[0076] "Fv" refers to the minimum antibody fragment that retains a complete antigen-binding site and is composed of one light chain variable region and one heavy chain variable region bound together.

[0077] "Single-chain variable fragment" (scFv) refers to an engineered antibody consisting of a light chain variable region (VL) and a heavy chain variable region (VH) linked together either directly or via a peptide linker sequence.

[0078] An antigen-binding domain can specifically bind to an antigen, e.g., bind to the antigen but not to other peptides, or bind other peptides with lower affinity.

[0079] The binding affinity between two molecules (e.g., an antigen-binding domain and an antigen) is determined, for example, by the dissociation constant (K D ) can be quantified. D can be determined by measuring the rates of complex formation and dissociation between the antigen-binding domain and the antigen, for example, using techniques such as surface plasmon resonance (SPR). The rate constants corresponding to the association and dissociation of the complex are the association rate constant k, a (or k on ) and the dissociation rate constant k d (or k off ) is expressed as the formula K D =k d / k a From K D is k a and k d It is related to.

[0080] Suitably, the antigen binding domain is a CD84 binding domain.

[0081] TIFF2025500618000003.tif213162TIFF2025500618000004.tif245161TIFF2025500618000005.tif24616 0TIFF2025500618000006.tif246161TIFF2025500618000007.tif243162TIFF2025500618000008.tif54163

[0082] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-NYWIN (SEQ ID NO:1); CDR2-DIYPVSGTTNYNEKFKR (SEQ ID NO:2); and CDR3-GTGRFAY (SEQ ID NO:3); or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVSTSSYSYMH (SEQ ID NO:4); CDR2-FASNLES (SEQ ID NO:5); and CDR3-QHSWEIPYT (SEQ ID NO:6); or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0083] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-NYWLG (SEQ ID NO:7); CDR2-DIYPGGGYTNYIEKFKG (SEQ ID NO:8); and CDR3-YEGGYYGNYDAMDY (SEQ ID NO:9), or a variant thereof, each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RASESVDNYGISFMN (SEQ ID NO:10); CDR2-AASNQGS (SEQ ID NO:11); and CDR3-QQSKAVPRT (SEQ ID NO:12), or a variant thereof, each having up to three amino acid substitutions, additions or deletions.

[0084] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSSYA (SEQ ID NO: 13); CDR2-ISGSGGST (SEQ ID NO: 14); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO: 15), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-NIESKD (SEQ ID NO: 16); CDR2-DDA (SEQ ID NO: 17); and CDR3-QVWDSSSDHVV (SEQ ID NO: 18), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0085] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSSYP (SEQ ID NO: 19); CDR2-ISYHGRNK (SEQ ID NO: 20); and CDR3-ARDRDATPGGTGVGNHGMAV (SEQ ID NO: 21), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-QSLLHSSGYNY (SEQ ID NO: 22); CDR2-MGS (SEQ ID NO: 23); and CDR3-MQGLQTPPT (SEQ ID NO: 24), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0086] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSDNA (SEQ ID NO:25); CDR2-ISGTGRTT (SEQ ID NO:26); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO:27), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-QSLVYSDGDTY (SEQ ID NO:28); CDR2-KVS (SEQ ID NO:29); and CDR3-MQGTHWPPNT (SEQ ID NO:30), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0087] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO:31); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO:32); and CDR3-MRTSYYFDY (SEQ ID NO:33), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIFSSLA (SEQ ID NO:34); CDR2-NAKTLAE (SEQ ID NO:35); and CDR3-QHHYATPFT (SEQ ID NO:36), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0088] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-SYWIN (SEQ ID NO:37); CDR2-DIYLGSGSTNYNEKFKS (SEQ ID NO:38); and CDR3-SGGYLGY (SEQ ID NO:39), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVSTSSYSYMH (SEQ ID NO:40); CDR2-FASNLES (SEQ ID NO:41); and CDR3-QHSWEIPYT (SEQ ID NO:42), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0089] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-NYWIG (SEQ ID NO: 43); CDR2-DIYPGGGYTNYNENFKG (SEQ ID NO: 44); and CDR3-STTYYSSYWCFDV (SEQ ID NO: 45), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-KSSQSLLNSGNQANYLA (SEQ ID NO: 46); CDR2-GASTRES (SEQ ID NO: 47); and CDR3-QNDHSYPFT (SEQ ID NO: 48), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0090] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RYWMS (SEQ ID NO:49); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO:50); and CDR3-PGPTVVATYWYFDV (SEQ ID NO:51), or a variant thereof, each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO:52); CDR2-KVSSRFS (SEQ ID NO:53); and CDR3-FQGSHVPRT (SEQ ID NO:54), or a variant thereof, each having up to three amino acid substitutions, additions or deletions.

[0091] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RYWIN (SEQ ID NO:55); CDR2-DIYPGSGSTNYNEKFKS (SEQ ID NO:56); and CDR3-DTTIAY (SEQ ID NO:57), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVTTSRYSYMH (SEQ ID NO:58); CDR2-FASNLES (SEQ ID NO:59); and CDR3-QHSWEIPYT (SEQ ID NO:60), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0092] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GYNMN (SEQ ID NO: 131); CDR2-NIDPYYGGTNYNQKFKG (SEQ ID NO: 132); and CDR3-GLLSGSFPY (SEQ ID NO: 133), or a variant thereof, each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIYSYLA (SEQ ID NO: 134); CDR2-NAKTLAE (SEQ ID NO: 135); and CDR3-QHHYGSPLT (SEQ ID NO: 136), or a variant thereof, each having up to three amino acid substitutions, additions or deletions.

[0093] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RSWMS (SEQ ID NO: 137); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 138); and CDR3-FYDGYSIYWYFDV (SEQ ID NO: 139), or a variant thereof, each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RSSQSIVHSNGDTYLE (SEQ ID NO: 140); CDR2-KVSNRFS (SEQ ID NO: 141); and CDR3-FQGSHVPRT (SEQ ID NO: 142), or a variant thereof, each having up to three amino acid substitutions, additions or deletions.

[0094] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 143); CDR2-HIWWDDVKRYNPALRS (SEQ ID NO: 144); and CDR3-IAVTYFFDF (SEQ ID NO: 145), or a variant thereof, each having up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIFSSFA (SEQ ID NO: 146); CDR2-NARTLAE (SEQ ID NO: 147); and CDR3-QHHYASPFT (SEQ ID NO: 148), or a variant thereof, each having up to three amino acid substitutions, additions or deletions.

[0095] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 149); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO: 150); and CDR3-MSTSYYFDY (SEQ ID NO: 151), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-KASQSLFTSVA (SEQ ID NO: 152); CDR2-SASYRYT (SEQ ID NO: 153); and CDR3-QQHYSSPFT (SEQ ID NO: 154), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0096] In some embodiments, the antigen binding domain comprises a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-IYAMN (SEQ ID NO: 155); CDR2-RIRSKSNNYARFYADSVKD (SEQ ID NO: 156); and CDR3-PLRSYFSMDY (SEQ ID NO: 157), or a variant thereof, each with up to three amino acid substitutions, additions or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1-KASENVDTYVS (SEQ ID NO: 158); CDR2-GASNRYT (SEQ ID NO: 159); and CDR3-GQTYSYPWT (SEQ ID NO: 160), or a variant thereof, each with up to three amino acid substitutions, additions or deletions.

[0097] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 61; and a VL domain having the sequence of SEQ ID NO: 62 or 108, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto, respectively.

[0098] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 63; and a VL domain having the sequence of SEQ ID NO: 64 or 109, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto, respectively.

[0099] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 65; and a VL domain having the sequence of SEQ ID NO: 66, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto, respectively.

[0100] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 68; and a VL domain having the sequence of SEQ ID NO: 69, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0101] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 71; and a VL domain having the sequence of SEQ ID NO: 72, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0102] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 74; and a VL domain having the sequence of SEQ ID NO: 75, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0103] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 76; and a VL domain having the sequence of SEQ ID NO: 77, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0104] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 78; and a VL domain having the sequence of SEQ ID NO: 79, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0105] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 80; and a VL domain having the sequence of SEQ ID NO: 81, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0106] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 82; and a VL domain having the sequence of SEQ ID NO: 83, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0107] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 161; and a VL domain having the sequence of SEQ ID NO: 162, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0108] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 163; and a VL domain having the sequence of SEQ ID NO: 164, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0109] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 165; and a VL domain having the sequence of SEQ ID NO: 166, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0110] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 167; and a VL domain having the sequence of SEQ ID NO: 168, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0111] In some embodiments, the antigen binding domain comprises a VH domain having the sequence of SEQ ID NO: 169; and a VL domain having the sequence of SEQ ID NO: 170, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0112] The CD84 binding domain can be an scFv. The scFv can comprise the heavy chain variable region (VH) and the light chain variable region (VL) of an antibody linked by a short linker peptide, for example of about 10-25 amino acids. The scFv can be in the orientation (N-terminus to C-terminus) VH-VL or VL-VH. In some embodiments, the scFv is in the orientation (N-terminus to C-terminus) VH-VL. In some embodiments, the scFv is in the orientation (N-terminus to C-terminus) VL-VH.

[0113] Examples of linker sequences for linking the VH and VL domains include the following: TIFF2025500618000009.tif33161

[0114] The antigen-binding domain may comprise or consist of any one of SEQ ID NOs: 61, 63, 112-125 or 181-184, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0115] Suitably, the variants are capable of binding to at least CD84 and the corresponding antigen binding domains set forth as SEQ ID NOs: 61, 63, 112-125 or 181-184. For example, the variants are capable of specifically binding to CD84 with an affinity at least equivalent to the binding affinity between the corresponding antigen binding domains set forth as SEQ ID NOs: 61, 63, 112-125 or 181-184 and CD84.

[0116] TIFF2025500618000010.tif73161TIFF2025500618000011.tif250161TIFF2025500618000012.tif233161

[0117] In some embodiments the antigen binding domain comprises or consists of the sequence of SEQ ID NO: 61, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably retaining the ability to bind CD84.

[0118] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 63, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0119] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 112, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0120] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 113, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0121] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 114, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably retaining the ability to bind CD84.

[0122] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 115, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably retaining the ability to bind CD84.

[0123] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 116, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably retaining the ability to bind CD84.

[0124] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 117, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0125] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 118, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0126] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 119, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0127] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 120, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably retaining the ability to bind CD84.

[0128] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 121, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0129] In some embodiments the antigen binding domain comprises or consists of the sequence of SEQ ID NO: 122, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably retaining the ability to bind CD84.

[0130] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 123, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0131] In some embodiments the antigen binding domain comprises or consists of the sequence of SEQ ID NO: 124, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0132] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 125, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto, and preferably which retains the ability to bind CD84.

[0133] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 181, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0134] In some embodiments the antigen binding domain comprises or consists of the sequence of SEQ ID NO: 182, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0135] In some embodiments the antigen-binding domain comprises or consists of the sequence of SEQ ID NO: 183, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto, and preferably which retains the ability to bind CD84.

[0136] In some embodiments the antigen binding domain comprises or consists of the sequence of SEQ ID NO: 184, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto and preferably which retains the ability to bind CD84.

[0137] The antigen binding domain may be comprised in a chimeric antigen receptor (CAR).

[0138] Chimeric antigen receptors (CARs) "Chimeric antigen receptor" (CAR or CARs), as used herein, refers to an engineered receptor that can confer antigen specificity to cells (e.g., T cells, such as naive T cells, central memory T cells, effector memory T cells, or combinations thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs can also confer antigen specificity to other immune cells, such as NK cells (e.g., from umbilical cord blood (CB), induced pluripotent stem cells (iPSC), bone marrow (BM), human embryonic stem cells (hESC), cell lines (e.g., NK92 or YT), or peripheral blood (PB)) (Mehta et al. (2018) Front. Immunol. 9: 283; Liu et al. (2020) N. Engl. J. Med. 382: 545-553). CARs for use with NK cells may have other transmembrane domains (such as NKG2D or DAP12) and other costimulatory domains (such as NKG2D or 2B4), and they may incorporate genes for IL-2 or IL-15 within the CAR construct to provide constant cytokine support to the CAR-NK cells.

[0139] A CAR may comprise, for example, an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (endodomain).

[0140] In some embodiments, the CAR comprises a CD84 binding domain, a transmembrane domain and an intracellular signaling domain. The CAR may include one or more costimulatory domains.

[0141] The antigen binding domain of the CAR (e.g., the CD84 binding domain) can be an antigen binding domain as disclosed herein.

[0142] In another aspect, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of any one of SEQ ID NOs: 172-180, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0143] Suitably, the variants may function at least similarly to the corresponding CARs set forth as SEQ ID NOs: 172-180. For example, the variants may specifically bind to CD84 with a binding affinity at least equivalent to the binding affinity between the corresponding CARs set forth as SEQ ID NOs: 172-180 and CD84.

[0144] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of the sequence of SEQ ID NO: 172, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0145] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of a sequence of SEQ ID NO: 173, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.

[0146] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of the sequence of SEQ ID NO: 174, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.

[0147] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of a sequence of SEQ ID NO: 175, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.

[0148] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of the sequence of SEQ ID NO: 176, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0149] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of the sequence of SEQ ID NO: 177, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.

[0150] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of the sequence of SEQ ID NO: 178, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.

[0151] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of a sequence of SEQ ID NO: 179, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.

[0152] In another embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising or consisting of a sequence of SEQ ID NO: 180, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0153] Transmembrane domain The CAR may comprise a transmembrane domain.

[0154] The transmembrane domain may comprise a transmembrane sequence derived from any protein having a transmembrane domain, including any of type I, type II or type III transmembrane proteins. The transmembrane domain of CAR may also comprise an artificial hydrophobic sequence. The transmembrane domain of CAR may be selected so as not to dimerize.

[0155] Examples of transmembrane (TM) regions used in CAR constructs include a) the CD28 TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al., Blood, 2013, Nov 14;122(20):3461-72); b) the OX40 TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41); c) the 4-1BB TM region (Brentjens et al, CCR, 2007, Sep 15;13 (18 Pt 1):5426-35); d) the CD3-ζ TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41; Di Stasi et al. Blood, 2009, Jun 18;113(25):6392-402);e)CD8a TM region (Maher et al., Nat Biotechnol, 2002, Jan;20(1):70-5; Imai C et al., Leukemia, 2004, Apr;18(4):676-84; Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1) :5426-35; Milone et al., Mol Ther, 2009, Aug;17(8):1453-64);f) DAP12 TM region (Mueller, N. et al. J. Immunother. 2015, June 01; 38, 197));g)2B4 TM region (Altvater, B. et al. Clin. Cancer Res, 2009, July 22; (15) 4857-4866) and h) NKG2D TM region (Li, Y et al, Cell Stem Cell, 2018, Aug 2; (23):181-192).

[0156] Additional transmembrane domains will be apparent to those of skill in the art.

[0157] An example of the amino acid sequence of the CD8a transmembrane domain is: The file is TIFF2025500618000013.tif18161.

[0158] An example of the amino acid sequence of the CD28 transmembrane domain is: The file is TIFF2025500618000014.tif22161.

[0159] In some embodiments, the transmembrane domain comprises or consists of the sequence of SEQ ID NO: 127 or 128, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0160] Signal peptide The CAR can contain a signal peptide such that when the CAR is expressed in a cell, the protein is targeted to the endoplasmic reticulum and then to the cell surface.

[0161] The signal peptide may be recognized by a signal peptidase during or after translocation and cleaved to generate the mature protein.

[0162] In some embodiments, the signal peptide is the CD8a signal peptide.

[0163] Spacer The CAR may comprise a spacer that links the antigen binding domain to the transmembrane domain.

[0164] The spacer sequence may include, for example, an IgG1 Fc region, an IgG1 hinge, or a human or mouse CD8 stalk.

[0165] In some embodiments, the CAR comprises a CD8a stalk.

[0166] Intracellular signaling domains The intracellular domain can provide signaling in the CAR.

[0167] The intracellular signaling domain may comprise one or more immunoreceptor tyrosine-based activation motifs (ITAMs), which are conserved sequences of four amino acids that are repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system. This motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids (YxxL / I). The tyrosine residues of these motifs can be phosphorylated following interaction of the receptor molecules with their ligands, forming binding sites for other proteins involved in signaling pathways.

[0168] The intracellular signaling domain may comprise or consist of a CD3-ζ endodomain containing three ITAMs, which can transmit an activation signal to T cells after antigen binding.

[0169] CAR can include one or more costimulatory domains. For example, 4-1BB (also known as CD137) can be used together with CD3-zeta, or CD28, OX40 and / or ICOS can be used together with CD3-zeta to deliver proliferation / survival signals. Also, NKG2D, 2B4 (also known as CD244), DAP12 and / or DAP10 can be used together with CD3-zeta to deliver proliferation / survival signals.

[0170] In some embodiments, a CAR comprises a CD3-zeta signaling domain and lacks a costimulatory domain, in some embodiments, a CAR comprises a CD3-zeta signaling domain and one or more costimulatory domains.

[0171] In some embodiments, the CAR comprises one or more costimulatory domains selected from the group consisting of a 4-1BB costimulatory domain, a CD28 costimulatory domain, and an OX40 costimulatory domain.

[0172] In a preferred embodiment, the CAR comprises a 4-1BB costimulatory domain. In some embodiments, the CAR comprises a CD28 costimulatory domain. In some embodiments, the CAR comprises an OX40 costimulatory domain.

[0173] In a preferred embodiment, the CAR comprises a 4-1BB costimulatory domain and a CD3-zeta signaling domain.

[0174] An example of the amino acid sequence of the CD3-ζ signaling domain is: The file is TIFF2025500618000015.tif27161.

[0175] In some embodiments, the signaling domain comprises or consists of the sequence of SEQ ID NO: 129, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90% sequence identity thereto.

[0176] An example of the amino acid sequence of a 4-1BB costimulatory domain is: The file is TIFF2025500618000016.tif19161.

[0177] An example of the amino acid sequence of the CD28 costimulatory domain is: The file is TIFF2025500618000017.tif19161.

[0178] In some embodiments, the costimulatory domain comprises or consists of the sequence of SEQ ID NO: 130 or 171, or a variant thereof having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%, preferably at least 90%, sequence identity thereto.

[0179] Polynucleotides The polynucleotides of the present invention may comprise DNA or RNA, preferably DNA. They may be single-stranded or double-stranded. Preferably, the polynucleotide is an isolated polynucleotide. Those skilled in the art will understand that many different polynucleotides can code for the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, those skilled in the art will understand that, using conventional techniques, nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the present invention can be made to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is expressed.

[0180] Polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of the polynucleotides of the invention.

[0181] Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques.

[0182] Longer polynucleotides are generally produced using recombinant means, for example, using polymerase chain reaction (PCR) cloning techniques. This involves creating a primer pair (e.g., about 15-30 nucleotides) that flank the target sequence desired to be cloned, contacting those primers with mRNA or cDNA obtained from an animal or human cell, performing PCR under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. These primers can be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0183] The polynucleotide may comprise a promoter and / or enhancer operably linked to one or more nucleotide sequences encoding the antigen binding domain, antibody or CAR of the present invention. The term "operably linked" as used herein may mean that two components are linked to each other in such a manner that both can perform their functions substantially unimpeded. For example, the promoter and / or enhancer may promote and / or enhance the expression of the antigen binding domain, antibody or CAR.

[0184] In some embodiments, the promoter is an EF1α promoter.

[0185] vector In some embodiments, the polynucleotide is a vector.

[0186] Preferably, the vector is a viral vector, such as a retroviral vector, a lentiviral vector, an adeno-associated viral (AAV) vector, or an adenoviral vector, hi some embodiments, the polynucleotide is a viral genome.

[0187] In another aspect, the present invention provides a viral vector comprising a polynucleotide of the present invention.

[0188] In some embodiments, the viral vector is in the form of a viral vector particle.

[0189] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, as an example, some vectors used in recombinant nucleic acid technology allow an entity such as a segment of nucleic acid (e.g., a heterologous DNA segment, such as a heterologous cDNA segment) to be transferred into a target cell. A vector can serve the purpose of maintaining a heterologous nucleic acid (DNA or RNA) in a cell, facilitating the replication of a vector comprising a nucleic acid segment, and / or facilitating the expression of a protein encoded by the nucleic acid segment.

[0190] Vectors comprising the polynucleotides used in the present invention can be introduced into cells using a variety of techniques known in the art, such as transfection, transduction and transformation.

[0191] Transfection refers to the general process of introducing a nucleic acid into a cell and includes the process of delivering a polynucleotide to a cell using a non-viral vector. Transduction can refer to the process of introducing a nucleic acid into a cell using a viral vector.

[0192] Retroviral and Retiviral Vectors Retroviral vectors may be derived or derived from any suitable retrovirus. Many different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0193] Retroviruses are broadly divided into two categories: "simple" and "complex". Retroviruses are further divided into seven groups. Five of these groups are oncogenic retroviruses. The remaining two groups are lentiviruses and spumaviruses.

[0194] The basic structure of retroviral and lentiviral genomes has many common features, such as 5' and 3' LTRs. Located between or within these are packaging signals that allow packaging of the genome, primer binding sites, integration sites that allow integration into the host cell genome, and the gag, pol, and env genes that code for packaging components (these are polypeptides required for the assembly of viral particles). Lentiviruses have additional features, such as the HIV rev and RRE sequences, that allow efficient transport of integrated proviral RNA transcripts from the nucleus to the cytoplasm of infected target cells.

[0195] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). These LTRs are responsible for the integration and transcription of the provirus. LTRs can also act as enhancer-promoter sequences and control the expression of viral genes.

[0196] The LTRs themselves are identical sequences divided into three elements: U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from repeated sequences at both ends of the RNA. U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of these three elements vary considerably among different retroviruses.

[0197] In a defective retroviral vector genome, gag, pol and env may be absent or non-functional.

[0198] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication may be removed from the virus, rendering the viral vector replication-defective.

[0199] Lentiviral vectors are part of the larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. In summary, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the etiological agent of human acquired immune deficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototypic "slow virus" Visna / Medivirus (VMV), as well as the related Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), and the recently described Feline Immunodeficiency Virus (FIV) and Bovine Immunodeficiency Virus (BIV).

[0200] The Lentiviridae family differs from retroviruses in that lentiviruses are capable of infecting both dividing and non-dividing cells (Lewis, P et al. (1992) EMBO J. 11: 3053-8; Lewis, PF et al. (1994) J. Virol. 68: 510-6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as those that make up muscle, brain, lung and liver tissue.

[0201] A lentiviral vector, as used herein, is a vector that comprises at least one component part derivable from a lentivirus, preferably which component part is involved in the biological mechanism by which the vector infects cells, expresses genes, or replicates.

[0202] The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (i.e., derived from a virus that does not essentially infect primates, especially humans). An example of a non-primate lentivirus may be any member of the lentivirus family that does not naturally infect primates.

[0203] As examples of lentivirus-based vectors, HIV-1 and HIV-2 based vectors are described below.

[0204] HIV-1 vectors contain cis-acting elements that are also found in simple retroviruses. Sequences spanning the gag open reading frame have been shown to be important for HIV-1 packaging. Thus, HIV-1 vectors often contain a relevant portion of gag with a mutated translation initiation codon. In addition, most HIV-1 vectors also contain a portion of the env gene that contains the RRE. Rev binds to the RRE and allows transport of full-length or singly spliced ​​mRNA from the nucleus to the cytoplasm. In the absence of Rev and / or the RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses, such as the Mason-Pfizer monkey virus, can be used to alleviate the requirement for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0205] Most HIV-2-based vectors are structurally similar to HIV-1 vectors. Like HIV-1-based vectors, HIV-2 vectors also require the RRE for efficient delivery of full-length or singly spliced ​​viral RNA.

[0206] Preferably, the viral vectors used in the present invention contain a minimal amount of the viral genome.

[0207] By "minimal viral genome" it is meant that the viral vector is engineered to remove non-essential elements and retain essential elements to provide the functions required to infect, transduce and deliver the nucleotide sequence of interest to the target host cell. Further details on this strategy can be found in WO1998 / 017815.

[0208] Preferably, the plasmid vector used to produce the viral genome in the host / packaging cell carries sufficient lentiviral genetic information to allow packaging of the RNA genome into viral particles in the presence of packaging components, and these viral particles are capable of infecting target cells but are incapable of independently replicating to produce infectious viral particles in the final target cell. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0209] However, the plasmid vectors used to produce the viral genome in the host cell / packaging cell also contain transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in the host cell / packaging cell. These regulatory sequences may be the native sequences associated with the viral sequences to be transcribed (i.e., the 5' U3 region) or may be a heterologous promoter, such as another promoter (e.g., the CMV promoter).

[0210] The vector may be a self-inactivating (SIN) vector, in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated in vivo with the same efficiency as wild-type vectors and can transduce non-dividing cells. Transcriptional inactivation of the long terminal repeats (LTRs) of the SIN provirus should prevent mobilization by replication-competent viruses. This should also allow regulated expression of genes from internal promoters by eliminating the cis-acting effects of the LTRs.

[0211] Vector can be integration defective.Integration defective lentiviral vector (IDLV) can be produced by, for example, packaging catalytically inactive integrase (such as HIV integrase with D64V mutation at catalytic site; Naldini, L. et al. (1996) Science 272: 263-7; Naldini, L. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 11382-8; Leavitt, AD et al. (1996) J. Virol. 70: 721-8) in vector, or modifying or deleting essential att sequence from vector LTR (Nightingale, SJ et al. (2006) Mol. Ther. 13: 1121-32), or combination of the above.

[0212] cell In another aspect, the present invention provides a cell comprising a polynucleotide, vector, antigen-binding domain or CAR of the present invention.

[0213] In some embodiments, the cell is a T cell, lymphocyte or stem cell, such as a hematopoietic stem cell, a cord blood stem cell (CB) or an induced pluripotent stem cell (iPSC).

[0214] For example, the cell may be selected from the group consisting of CD4 cells, CD8 cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, gamma / delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, double negative T cells, naive T cells, memory stem T cells, central memory T cells, effector memory T cells, effector T cells, cytokine-induced killer (CIK) cells, hematopoietic stem cells, and induced pluripotent stem cells (iPSCs).

[0215] In some embodiments, the cell is a T cell or a NK cell, preferably a T cell. In some embodiments, the T cell is an autologous or allogeneic T cell.

[0216] The cells may be isolated from a subject.

[0217] The cell of the present invention can be provided for use in adoptive cell transfer.As used herein, the term "adoptive cell transfer" refers to the administration of a cell population to a patient.Generally, the cell is the T cell that is genetically modified after being isolated from a subject and cultured in vitro before being administered to a patient.

[0218] Adoptive cell transfer can be allogeneic or autologous.

[0219] By "autologous cell transfer" it is understood that the starting cell population, which is then transduced with a polynucleotide or vector according to the invention, is obtained from the same subject as the subject to whom the transduced cell population is to be administered. Autologous transfer is advantageous as it avoids problems associated with immunological incompatibility and is available to the subject regardless of the availability of a genetically matched donor.

[0220] By "allogeneic cell transfer" it is understood that the starting cell population (which is then transduced with a polynucleotide or vector according to the invention) is obtained from a subject different from the subject to which the transduced cell population is to be administered. Preferably, the donor is genetically matched to the subject to which the cells are to be administered, to minimize the risk of immunological incompatibility. Alternatively, the donor may be mismatched and unrelated to the patient.

[0221] Treatment method In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in therapy.

[0222] In some embodiments, the therapy is treatment of cancer.

[0223] In a preferred embodiment, the cancer is a hematological malignancy.

[0224] In a preferred embodiment, the cancer cells express CD84, for example, the hematological malignancy can be a hematological malignancy that expresses CD84.

[0225] In some embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome, T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), chronic myeloproliferative syndrome, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, dendritic cell neoplasm, and histiocytic sarcoma.

[0226] In some embodiments, the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), acute myeloid leukemia (AML), and histiocytic sarcoma.

[0227] In preferred embodiments, the cancer is selected from the group consisting of CLL, B-cell lymphoma, B-ALL, T-ALL and AML.

[0228] In preferred embodiments, the cancer is selected from the group consisting of B-cell lymphoma, B-ALL, T-ALL and AML.

[0229] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in the treatment of AML.

[0230] In another aspect, the present invention provides an antigen-binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in the treatment of T-ALL.

[0231] In another aspect, the present invention provides an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention for use in the treatment of B cell lymphoma.

[0232] In another aspect, the present invention provides an antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the present invention for use in the treatment of Burkitt's lymphoma.

[0233] In some embodiments, the cancer is not a T cell lymphoma. In some embodiments, the cancer is not a mature T cell lymphoma. In some embodiments, the cancer is not CLL. In some embodiments, the cancer is not a solid tumor.

[0234] The treatment of mammals, especially humans, is preferred, with both human and veterinary treatments being within the scope of the present invention.

[0235] Pharmaceutical compositions and injection solutions Agents for use in the present invention may be administered alone but, particularly in human therapy, will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent.

[0236] The agents of the present invention, such as cells or vector particles, may be formulated as pharmaceutical compositions. These compositions may comprise, in addition to the agent, pharma- ceutically acceptable carriers, diluents, excipients, buffers, preservatives, or other materials known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials can be determined by one skilled in the art depending on the route of administration, e.g., intravenous or intraarterial.

[0237] Pharmaceutical compositions are generally in liquid form. Liquid pharmaceutical compositions generally contain a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. They may also contain saline solution, magnesium chloride, dextrose or other sugar solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. In some cases, surfactants such as Pluronic Acid (PF68) 0.001% can be used. In some cases, serum albumin can be used in the composition.

[0238] For injection, the active ingredient can be in the form of a pyrogen-free aqueous solution with suitable pH, isotonicity and stability.Those skilled in the art can prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection or lactated Ringer's injection.Preservatives, buffers, antioxidants and / or other additives can be included as necessary.

[0239] For delayed release, the agent may be included in a pharmaceutical composition formulated for slow release, such as microcapsules formed from biocompatible polymers, or liposome carrier systems, by methods known in the art.

[0240] It is preferable that cell therapy products be handled in accordance with the FACT-JACIE international standards for cell therapy.

[0241] Administration In some embodiments, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention is administered systemically to the subject.

[0242] In some embodiments, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention is administered locally to a subject.

[0243] The terms "systemic delivery" or "systemic administration" as used herein mean that an agent of the invention is administered to the circulatory system, e.g., to achieve widespread distribution of the agent. In contrast, local or regional administration limits delivery of an agent to a localized area.

[0244] In some embodiments, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention is administered intravascularly, intravenously or intraarterially.

[0245] In a preferred embodiment, the antigen binding domain, antibody, CAR, polynucleotide, vector, cell or pharmaceutical composition of the invention is administered intravenously.

[0246] dose Those skilled in the art can easily determine the appropriate dose of the agent of the present invention to be administered to a subject.Generally, the actual dose that is most suitable for each patient is determined by a physician, and it depends on a variety of factors, including the activity of the specific compound employed, the metabolic stability and duration of action of the compound, age, body weight, health condition, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of specific pathology, and the individual being treated.Of course, there may be individual cases where higher or lower dose ranges are appropriate, and such cases are within the scope of the present invention.

[0247] subject The term "subject," as used herein, refers to a human or non-human animal.

[0248] Examples of non-human animals include vertebrates, such as mammals, such as non-human primates (especially higher primates), dogs, rodents (e.g., mice, rats or guinea pigs), pigs and cows. The non-human animals may be companion animals.

[0249] Preferably, the subject is a human.

[0250] Mutants, derivatives, analogues, homologues and fragments In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses mutants, derivatives, analogs, homologs and fragments thereof.

[0251] In the context of the present invention, a "variant" of a given sequence is a sequence in which a specific sequence of residues (whether amino acid or nucleic acid residues) has been modified such that the subject polypeptide or polynucleotide retains at least one of its endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the native polypeptide or polynucleotide.

[0252] The term "derivative", as used herein in reference to a protein or polypeptide of the invention, encompasses any substitution, variation, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to the sequence, so long as the resulting protein or polypeptide retains at least one of its endogenous functions.

[0253] The term "analog" as used herein with respect to a polypeptide or polynucleotide includes any mimetic, i.e., a chemical compound that has at least one of the intrinsic functions of the polypeptide or polynucleotide it mimics.

[0254] Generally, amino acid substitutions can be made, for example from 1, 2 or 3 to 10 or 20 substitutions, as long as the modified sequence retains the required activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogues.

[0255] The protein used in the present invention may also have deletions, insertions or substitutions of amino acid residues, which produce silent mutations and result in functionally equivalent proteins.Intentional amino acid substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues, so long as the intrinsic function is maintained.For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; uncharged polar head group amino acids with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine and tyrosine.

[0256] Conservative substitutions can be made, for example, according to the table below: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

[0257] TIFF2025500618000018.tif49161

[0258] The term "homolog" as used herein means an entity that has a certain homology with a wild-type amino acid sequence or a wild-type nucleotide sequence. The term "homology" can be considered equivalent to "identity".

[0259] In the present context, a homologous sequence is intended to encompass an amino acid sequence that may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. In general, a homolog comprises the same active site etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0260] In the present context, a homologous sequence is taken to encompass a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95%, 96% or 97% or 98% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0261] Preferably, references to a sequence having a percent identity to any one of the SEQ ID NOs detailed herein refer to a sequence having the stated percent identity over the entire length of the referenced SEQ ID NO.

[0262] Homology comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

[0263] Percent homology can be calculated over contiguous sequences, i.e., one sequence is aligned with the other and each amino acid or nucleotide of one sequence is directly compared, residue by residue, with the corresponding amino acid or nucleotide of the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are only performed over relatively short lengths of residue.

[0264] Although this is a very simple and consistent method, it fails to take into account that, for example, in an otherwise identical sequence pair, a single insertion or deletion in an amino acid or nucleotide sequence may cause the following residue or codon to fall out of alignment, which may result in a large decrease in the percent homology when a global alignment is performed. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment, attempting to maximize local homology.

[0265] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, such that a sequence alignment with as few gaps as possible will score higher than one with many gaps, reflecting a higher relatedness between the two sequences being compared for the same number of identical amino acids or nucleotides. An "affine gap cost" is commonly used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will naturally produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0266] Thus, the calculation of maximum percent homology first requires the generation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0267] Although the final homology percentage can also be assessed in terms of identity, the alignment process itself is usually not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values ​​or a custom symbol comparison table if provided (see user manual for further details). Depending on the application, it is preferred to use the public default values ​​of the GCG package, or a default matrix such as BLOSUM62 in the case of other software.

[0268] Once the software has produced an optimal alignment, it can calculate percent homology, preferably percent sequence identity, which the software typically does as part of the sequence comparison and generates a numerical result.

[0269] "Fragments" are also variants, and the term generally refers to a selected region of a polypeptide or polynucleotide that is of interest for functionality or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0270] Such variants can be made using standard recombinant DNA techniques such as site-directed mutagenesis. If an insertion is to be made, a synthetic DNA can be made that encodes the inserted sequence along with 5' and 3' flanking regions that correspond to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction sites that correspond to the sites in the naturally occurring sequence, allowing the sequence to be cleaved with an appropriate enzyme and the synthetic DNA to be ligated to the cleavage site. The DNA can then be expressed in accordance with the invention to make the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences, and other known techniques may be used.

[0271] Codon optimization Polynucleotides used in the present invention may be codon optimized. Codon optimization has been previously described in WO1999 / 41397 and WO2001 / 79518. Different cells use certain codons differently. This codon bias corresponds to a bias in the relative abundance of certain tRNAs in a cell type. Expression can be increased by altering the codons in the sequence to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. In this way, even greater degrees of translational control are possible. Codon usage tables are known in the art for mammalian cells as well as for a variety of other organisms.

[0272] A person skilled in the art will understand that he can combine all features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0273] Preferred features and embodiments of the present invention are described below by way of non-limiting examples.

[0274] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology which are within the capabilities of one of ordinary skill in the art. Such techniques are explained in the literature. For example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and See, Physical Analysis of DNA, Academic Press. Each of these general texts is incorporated herein by reference. EXAMPLES

[0275] Example 1: Expression of CD84 in hematological malignancies We found that CD84 is overexpressed in various hematological malignancies. Using GEPIA, an interactive web server for analyzing RNA-sequencing expression data of 9,736 tumor and 8,587 normal samples from the TCGA and GTEx projects (http: / / gepia.cancer-pku.cn / index.html; Tang et al. GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res. 2017; 10.1093 / nar / gkx247), we found that CD84 is overexpressed in various hematological malignancies. CD84 expression was 4.2-fold higher in 47 lymphoid neoplasms, diffuse large B-cell lymphomas (represented as DLBC in Figure 1), than in 337 blood samples. CD84 expression was 10.1-fold higher in 173 acute myeloid leukemias (represented as LAML in Figure 1) than in 70 bone marrow samples.

[0276] We also investigated the Cancer Cell Line Encyclopaedia (CCLE) dataset, which contains mRNA expression data for a panel of over 1,100 cell lines derived from various types of solid and hematological tumors (https: / / portals.broadinstitute.org / ccle). Both RNAseq and Affymetrix analysis of mRNA expression showed that CD84 is expressed at high levels in cell lines derived from Burkitt's lymphoma, acute myeloid leukemia (AML), B-cell lymphoma, chronic myeloid leukemia (CML), B-cell acute lymphoblastic leukemia (B-ALL), and T-cell acute lymphoblastic leukemia (T-ALL), but not in T-cell lymphoma or solid tumors (Figure 2).

[0277] We next evaluated CD84 expression on the surface of cell lines derived from different hematological malignancies. Table 1 shows the cell lines used, the hematological malignancies from which they were derived, and a qualitative assessment of CD84 expression based on cytometric analysis as shown in Figure 3.

[0278] [Table 1]

[0279] We analyzed the expression of CD84 in nine samples from CLL patients by flow cytometry. Table 2 shows the qualitative assessment of CD84 expression based on the cytometric analysis shown in Figure 4. The expression of CD84 in leukemic cells was evaluated by comparing lymphocytes (CD84 moderate ) and monocytes (CD84 high ) The light grey histograms represent staining with an isotype-matched control antibody, and the dark grey histograms represent staining with a specific CD84 antibody.

[0280] [Table 2]

[0281] Although certain genomic databases suggest that CD84 is overexpressed at the mRNA level in AML, we sought to determine whether CD84 was expressed on the surface of malignant cells in patients (n=10) diagnosed with AML (Table 3).

[0282] [Table 3]

[0283] Two representative examples of flow cytometry data are shown in Figure 5: samples from patient 04 and patient 10 show medium and high expression of CD84, respectively.

[0284] Example 2: CD84 Antibody Mouse Monoclonal Antibody The present inventors have studied and determined the sequences of two anti-CD84 mouse monoclonal antibodies (152-1D5 and 153-4D9; Engel et al. B-cell antigens section report, in Schlossman S (ed): Leucocyte Typing V. Oxford, UK, Oxford University Press, 1995, page 483; Palou et al. Genomic characterization of CD84 reveals the existence of five isoforms differing in their cytoplasmic domains. Tissue Antigens. 2000; 55(2):118-27).

[0285] Furthermore, we have generated a number of new anti-CD84 mouse monoclonal antibodies by immunizing BALB / c mice with human CD84 protein, as described in Methods. Table 3 summarizes the features of these antibodies, which were characterized as described in Methods. 300.19-CD84 + Binding to Raji and Ramos cells, lymphocytes and monocytes was performed using hybridoma supernatants supplemented with equal amounts / concentrations of anti-CD84 antibodies.

[0286] [Table 4]

[0287] The sequences corresponding to the variable region of the light chain (VL) and the variable region of the heavy chain (VH) were determined from different hybridomas using the Mouse Ig-Primer Set (Novagen). Sanger sequencing of this region was performed by AbsoluteAntibody (UK). Complementarity determining regions (CDRs) and framework regions (FRs) were identified using the Abysis (sequences defined by the Kabat numbering scheme), IMGT and IgBLAST databases.

[0288] Table 5 shows the VH and VL sequences of these antibodies, with the three CDRs of each sequence highlighted in bold and underlined.

[0289] [Table 5] TIFF2025500618000024.tif95165

[0290] Human scFv Phage display screening was used to identify novel human scFvs that bind to CD84. Three different scFvs were identified (R3-B3, R3-G7 and R3-H3); the sequences of the CDRs and FRs of the heavy (VH) and light (VL) chain variable domains are shown in Table 6.

[0291] [Table 6] TIFF2025500618000026.tif66165

[0292] Example 3: Generation of CD84 CAR We generated several anti-CD84 CAR constructs (CAR84). The complete CAR84 sequence was cloned into the third-generation lentivirus vector pCCL (Dull et al. A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 1998:72:8463-8471), which contains a signal peptide under the control of the EF1α promoter, a scFv specific for CD84, the hinge and transmembrane regions of CD8a, the costimulatory domain 4-1BB, and the signaling domain CD3-ζ (Figure 6).

[0293] Various variants of scFv domains were designed based on the above-mentioned antibodies. These scFvs differ in the order of the VH and VL sequences (some variants have only a VH chain) and in the linker used between VH and VL, using three (S3) or four (S4) motifs (Gly-Gly-Gly-Gly-Ser). The name given to each CAR variant reflects the design of the scFv. The sequences of the different scFv domains synthesized and cloned into the pCCL vector are shown in Table 7.

[0294] [Table 7] TIFF2025500618000028.tif250165TIFF2025500618000029.tif248165TIFF2025500618000030.tif21165

[0295] The complete CAR sequence is shown in Table 8.

[0296] [Table 8] TIFF2025500618000032.tif190165

[0297] Production of CD84 CART Lentiviruses (LV) containing various variants of the pCCL-EF1α-CD84 vector were produced in HEK293-T cells, and the number of transducing units was determined by limiting dilution. These lentiviruses were then used to transduce T cells isolated from whole blood. These CAR-T cells were then expanded in culture for 6–8 days.

[0298] The tables below (Tables 9-11) summarize the numbers of T cells obtained after three independent transductions with different CARs, as well as the percentage of these T cells that expressed CD84 CAR on their surface.

[0299] [Table 9]

[0300] [Table 10]

[0301] [Table 11]

[0302] The following CAR LVs did not result in efficient expansion of CAR-positive T cells: 152.1, 153.1, 153.2, B3.4 and B3.5. The 152.1 and 153.1 CARs were not used for further experiments.

[0303] Example 4: CD84 CART in in vitro cytokine production To evaluate the cytokine release capacity of each CART cell, the supernatants of effector-target cell co-cultures were harvested. high The cell line Ramos was used as target cells at an effector:target cell ratio of 2:1. Levels of IFN-γ (Figure 7), IL-2 (Figure 8), Granzyme-B (Figure 9) and TNF-α (Figure 10) in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA) after 24 h of co-culture. Untransduced T cells (UT) were used as negative control (co-cultured with target cells). Four independent experiments were performed.

[0304] These results indicate that CART cells with R3-B3 scFv CAR binding domain lack cytokine release activity and therefore were not used in the following experiments. On the other hand, CART cells with R3-H3 scFv CAR binding domain released a large amount of cytokines, and these two CART cells (H3.4 and H3.5) were the ones with the greatest inflammatory profile (based on TNF-α secretion), followed by CART cells with 152-1D5 antibody CAR binding domain, which also showed a high cytokine release profile. 152-1D5 CART cells showed a similar profile regardless of the scFv design (VH-VL order and linker length). CART cells based on 153-4D9 and R3-G7 antibodies had similar profiles and showed lower cytokine release compared to R3-H3 and 152-1D5 CART cells, except for 153.3 CART cells, which secreted almost no cytokines.

[0305] Example 5: In vitro cytotoxicity of anti-CD84 CART To evaluate the efficacy of CART84 cells in vitro, we performed cytotoxicity assays of each CART cell against several GFP-expressing target cell lines with different CD84 expression levels and of both lymphoid (Ramos, NALM6 and MOLT-4 cell lines) and myeloid origin (K562 and MOLM-13 cell lines). CART cytotoxicity was assessed by determining the percentage of live GFP-positive cells by cytometry after 24 h of coculture. Effector:target cell ratios of 4:1, 2:1, 1:1 and 0.5:1 were tested.

[0306] First, the present inventors investigated CD84 highAll CARs were tested against the Ramos cell line (Figure 11). Results are shown for an effector:target ratio of 2:1. R3-H3 and 152-1D5 CART cells showed the highest cytotoxic activity against this cell line, while 153-4D9 and R3-G7 CART cells showed lower cytotoxic activity. 153.3 CART cells showed the lowest cytotoxic activity of all CARs based on the 153-4D9 construct.

[0307] Next, we tested all CARs against K562, a myeloid cell line with low CD84 expression (Figure 12). Results are shown for an effector:target ratio of 2:1. CARs based on the 152-1D5 and 153-4D9 antibodies showed statistically significant cytotoxic activity compared to UT cells.

[0308] Based on the cytotoxicity against Ramos and K562, we selected the following CART cells for further characterization: 152.3, 153.4, 153.5, G7.5 and H3.5. We evaluated the cytotoxicity of these CART cells against several cell lines at four effector:target ratios (4:1, 2:1, 1:1 and 0.5:1) after 24 and 48 hours of co-culture. Ramos is an aggressive B cell lymphoma cell line (Figure 13), K562 is an acute myeloid leukemia cell line with low CD84 expression (Figure 14), while MOLM-13 is an acute myeloid leukemia cell line with moderate CD84 expression (Figure 15). NALM-6 is a B-cell acute lymphoblastic leukemia cell line (FIG. 16), and MOLT-4 is a T-cell acute lymphoblastic leukemia cell line (FIG. 17).

[0309] As shown for the 2:1 ratio (Figure 11), the selected CARs showed statistically significant cytotoxic activity against Ramos cells compared to UT cells for each effector:target ratio (Figure 13). In the case of K562, the same pattern as shown in Figure 12 was observed, with CARs based on the 152-1D5 and 153-4D9 antibodies showing statistically significant cytotoxic activity compared to UT cells (Figure 14).

[0310] All selected CARs exhibited cytotoxic activity against MOLM-13, with CARs based on the 152.1D5 and 153.4D9 antibodies exhibiting higher cytotoxic effects than those based on the R3-G7 and R3-H3 antibodies (Figure 15).

[0311] CARs based on the 152.1D5 and 153.4D9 antibodies showed higher cytotoxic activity against the NALM-6 cell line than CARs based on the R3-G7 and R3-H3 scFv (Figure 16). In the MOLT-4 cell line, CARs based on the 152-1D5 antibody showed the highest statistically significant cytotoxic activity compared to UT cells, followed by CARs based on the 153-4D9 and R3-G7 antibodies (Figure 17).

[0312] Example 6: CD84 expression in peripheral blood mononuclear cells (PBMCs) We evaluated CD84 expression in different PBMC cell populations by flow cytometry and the results obtained were similar to those described previously. Monocytes show high CD84 expression similar to that seen in the Ramos cell line. B cells show intermediate CD84 expression. Both CD4 and CD8 T cells have clearly distinct subpopulations (positive and negative) with respect to CD84 expression, with intermediate levels of CD84 expression in the positive population (Figure 18).

[0313] Example 7: In vitro cytotoxicity of CD84 CART against PBMCs We evaluated the cytotoxic activity of CD84 CART against PBMCs. In each experiment, both effector cells (i.e., CART84 cells) and target cells (PBMCs) were obtained from the same donor. The average of three independent experiments from three different donors is shown. CART cells did not show statistically significant cytotoxic activity against their own PBMCs. However, these CART cells showed statistically significant cytotoxic activity against Ramos cells in a parallel experiment (Figure 19).

[0314] Example 8: Methods of Examples 1-20 Generation of mouse monoclonal anti-CD84 antibody BALB / c mice were immunized three to four times at 3-week intervals with 300.19 cells stably transfected with full-length CD84 DNA (de la Fuente MA et al. CD84 leukocyte antigen is a new member of the Ig superfamily. Blood. 1997; 15;90(6):2398-405). The first intraperitoneal (ip) injection consisted of 20 × 10 6 The first consisted of 20 × 10 cells in 300 μl of PBS. 6 The final injection consisted of 30 x 10 cells in 300 μl PBS. 6 Three days after the final booster immunization, the mice were euthanized, and splenocytes were harvested for cell fusion.

[0315] NS1 myeloma cells (European Collection of Cell Cultures, Salisbury, UK) and spleen cells were incubated at 37°C in a ratio of 4:1 (spleen cells:NS1), centrifuged at room temperature for 10 min, and fused by adding 1 mL of warm PEG solution to the cell pellet with constant mixing. Cells were gently resuspended in RPMI culture medium, centrifuged, and incubated at 37°C, 5% CO2 in a humidified incubator.

[0316] Ten days after the fusion, 50 μl of hybridoma supernatant was screened by flow cytometry analysis with 300.19-CD84 cells and non-transfected 300.19 cells as a negative control. Hybridomas that were positive for 300.19-CD84 cells and negative for 300.19 cells were transferred to 24-well plates and grown to confluence before being transferred to T75 flasks. Single hybridoma clones producing the antibody of interest were then isolated by limiting dilution. For each hybridoma, 10 single clones were retested by flow cytometry with 300.19-CD84 cells. This cloning protocol was repeated with one of the positive clones.

[0317] For each antibody, the isotype (class and subclass) was determined by ELISA, in which the antibody was added to anti-IgG-coated plates and then the plates were incubated with anti-mouse HRP antibody. To determine which of the two CD84 extracellular domains was recognized by the antibody, flow cytometry analysis was performed using COS cells expressing chimeric CD84 extracellular domains in which the sequences of domains 1 (D1) and 2 (D2) were human or mouse.

[0318] Identification of fully human anti-CD84 scFv Human CD84 produced by mammalian cells (confirmed by SDS-PAGE; Figure 20) was used as an antigen, and 5.37 × 10 10 Panning was performed on the human naive phage display LiAb SFMax library (Proteogenix, France) which contains a high diversity of scFv variants.

[0319] In the biopanning round, tubes were coated with antigen, blocked, washed, and incubated with the phage library. After washing, elution of phage binders was performed with glycine HCl and then neutralized.

[0320] To determine the concentration of eluted phages, they were added to E. coli TG1 cells, which were then poured onto plates and incubated upside down. Calculation of PFU (plaque forming units) was based on the number of plaques (i.e., dead TG1) on the plates.

[0321] To amplify the eluted phages, they were added to E. coli TG1 cells, which were then infected with helper phages and cultured. After phages were precipitated with PEG / NaCl, they were resuspended and the amplified phages were used for the next biopanning round for ELISA analysis.

[0322] This analysis showed significant enrichment with each round (Table 12), and since the output from round 3 already showed excellent enrichment, further biopanning rounds were not performed to prevent a decrease in phage diversity.

[0323] [Table 12]

[0324] Round 3 phages were selected for monoclonal ELISA analysis. Single E. coli TG1 clones were picked and cultured with helper phages. After centrifugation, the supernatant containing the phages was collected.

[0325] Plates were coated with either antigen or buffer, washed, blocked, and washed again. Phage were then added to the plates and incubated. Plates were washed and then anti-phage horseradish peroxidase (HRP) antibody was added, followed by washing and incubation with TMB and then HCl. Plates were read at 450 nm.

[0326] After sequencing of the positive clones, three different unique sequences were identified. The three unique clones identified were then reassayed by ELISA to ensure the specificity of the clones. All phages were assayed at the same concentration. The plates were covered with either antigen (Ag) or buffer (NC). These results revealed that the three clones specifically bound to the CD84 antigen (Table 13).

[0327] [Table 13]

[0328] Donors, cell lines Blood buffy coats from healthy donors were obtained from the reference blood bank Banc de Sang i Teixits, Barcelona (Spain).

[0329] Ramos, Raji, NALM6, K562, MOLM-13, Kasumi-6, THP-1, and U937 were purchased from the American Type Culture Collection (ATCC). Ramos, Raji, NALM6, K562, NS1, and THP-1 cell lines were cultured in RPMI medium (ThermoFisher) supplemented with 10% fetal bovine serum (FBS, Merck) and penicillin-streptomycin (Labclinics), and 300.19 cells were also supplemented with 1% L-glutamine (Gibco) and 0.1% 2-β-mercaptoethanol (Sigma). HEK293T and COS cell lines were cultured in DMEM medium (Gibco) supplemented with 10% FBS (Merck) and penicillin-streptomycin (Labclinics) with 1% L-glutamine (Gibco). All cell lines were grown at 37° C. and 5% CO2.

[0330] Lentivirus production HEK293-T cells were transfected with our transfer vector pCCL-EF1α-CD84 together with packaging plasmids pRSV-Rev (Addgene, 12253), pMDLg / Prre (Addgene, 12251), and envelope plasmid pCMV-VSV-G (Addgene, 12259) using linear polyethylenimine (PEI, MW 25000, Polysciences Inc 23966-1). Lentiviral supernatants were collected 48 hours later and concentrated using a LentiX-Concentrator (Clontech, Takara) according to the manufacturer's protocol. The concentrated lentivirus was stored at -80°C until use.

[0331] Lentivirus titration The number of transducing units (TU / mL) was determined by limiting dilution.

[0332] HEK293T cells were seeded 24 h prior to transduction, and a 1:10 dilution of viral supernatant was prepared and added to DMEM medium (Gibco) supplemented with polybrene (Sigma-Aldrich) at 8 mg / mL. After 48 h, cells were treated with trypsin and labeled with AffiniPure F(ab')2 fragment goat anti-mouse immunoglobulin G (IgG) allophycocyanin (APC) conjugate (JacksonImmuno Research Laboratories, 115-136-072). Viral titers were calculated using the dilution corresponding to 2%–20% positive cells.

[0333] T cell transduction and CART expansion cultures T cells were isolated from whole blood during density gradient centrifugation on Ficoll-Paque™ by RosetteSep™ (RosetteSep Human T cell Enrichment cocktail from StemCell). T cells were cultured in X-Vivo 15 Serum Free Cell Medium (Lonza) supplemented with 5% AB human serum (Sigma H4522), penicillin-streptomycin (ThermoFisher, 100mg / mL), and 50IU / mL of IL-2 (Miltenyi). Cells were then activated using beads conjugated with CD3 and CD28 mAb (Dynabeads Human T-Activator CD3 / CD28 Gibco, 11131D). After 24 hours, the cells were transduced with lentivirus in the presence of 8ug / mL of polybrene (Sigma-Aldrich). Expansion culture was allowed for 6-8 days before experiments were performed.

[0334] Flow cytometry CAR84 was detected with recombinant CD84-His protein (R&D, 1855-CD) and secondary His Tag APC-conjugated antibody (R&D, IC050A) and biotin SP-conjugated AffiniPure F(ab')2-fragment goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, 115-065-072) or biotin SP-conjugated AffiniPure F(ab')2-fragment goat anti-human IgG (Jackson ImmunoResearch Laboratories, 109-065-006) with BV421-conjugated streptavidin (BD Horizon, 563259). The following mAbs against human proteins were used: CD3-APC, CD4-PE-Cy7, CD4-Alexa Fluor-488, CD19-PE, CD8-APC-H7, PD-1-PE-Cy7, TIM-3-BB515, CD69-PerCP-Cy™5.5, LAG-3-BV-605, CD62L-FITC, CCR7-PerCP-Cy™5.5, CD84-PE, CD4- PE-Cy™7, CD45RA-APC (Becton Dickinson), CD84-APC and CD84-PE (BioLegend). Samples were run on flow cytometers BD FACSCanto II (BD Biosciences), LSR II Fortessa 4L with HTS (BD), and Attune NxT 4L cytometer (ThermoFisher). The following mAbs were used for the ELISA: anti-mouse IgG-HRP (anti-mouse IgG-peroxidase antibody made in goat, Sigma, catalog: A3673-1ML) and anti-mouse IgG coating made in goat (anti-mouse IgG antibody (sigma, catalog: M2650-1ML). Data were analyzed using FlowJo software 10.7.1.

[0335] In vitro cytotoxicity assay The cytotoxicity of CART cells was assessed at various time points using different effector:target ratios. Target cells used in these assays were modified with a lentiviral vector to overexpress GFP-firefly luciferase (GFP-ffLuc) as previously described (Shah et al. Antigen Presenting Cell-Mediated Expansion of Human Umbilical Cord Blood Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity. PLoS One. 2013; 8(10)).

[0336] survival GFP + The survival rate of tumor cells was analyzed by flow cytometry using the following formula: % of live cells = 100 × (x GFP + T cells at time point) + Number of cells / x time point GFP + The number of cells was calculated using the number of cells alone.

[0337] In vitro proliferation assay Proliferation of CART84 cells in response to CD84 antigen was measured using a CFSE assay (Castella, M. et al. (2019) Mol. Ther. - Methods Clin. Dev. 12: 134-144). CART cells were stained with CellTrace CFSE (Invitrogen, ThermoFisher, 15598431) and then co-cultured with or without stimulation for 96 h. Proliferation was analyzed by flow cytometry and the proliferation index (PI) was calculated (PI = sum of the number of cells of different generations / number of cells).

[0338] In vitro cytokine production IFN-γ, TNF-α, IL-6, and IL-1β cytokines were quantified by enzyme-linked immunosorbent assay (DuoSet ELISA, R&D systems) according to the manufacturer's protocol.

[0339] All publications mentioned in the above specification are incorporated herein by reference.Various modifications and variations of the disclosed antigen-binding domains, antibodies, chimeric antigen receptors, uses and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.Although the present invention has been disclosed in terms of certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the disclosed modes for carrying out the invention that are apparent to those skilled in the art are intended to be included within the scope of the following claims.

[0340] Example 9: Engineering of CD84 CAR We generated several anti-CD84 CAR constructs based on the antibodies GYCD84.1-7, GYCD84.1-226 and GYCD84.3-89. The complete CAR84 sequence was cloned into the third-generation lentivirus vector pCCL (Dull et al. A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 1998:72:8463-8471), which includes a signal peptide, a CD84-specific scFv, a CD8α hinge and transmembrane region, a costimulatory domain 4-1BB and a signaling domain CD3-ζ under the control of the EF1α promoter (Figure 6).

[0341] Based on the antibody GYCD84.1-7, two different scFvs were designed that differ in the order of the VH and VL sequences. All of these CARs contain a linker of four (S4) motifs (Gly-Gly-Gly-Gly-Ser). The name given to each CAR variant reflects the design of the scFv. The sequences of the different scFv domains synthesized and cloned into the pCCL vector are shown in Table 14.

[0342] [Table 14] TIFF2025500618000039.tif46159

[0343] Example 10: CD84 Expression in Hematological Malignancies We analyzed the CD84 expression of leukemic cells of patients diagnosed with AML or T-ALL by flow cytometry. Tables 15 and 16 summarize the qualitative evaluation of CD84 expression on AML cells and T-ALL cells, respectively. Two representative examples of flow cytometry analysis of primary leukemic cells of AML samples are shown in Figure 5. Analysis of T-ALL samples (patient 01 and patient 02) is shown in Figure 21.

[0344] [Table 15]

[0345] [Table 16]

[0346] Example 11: Production of CD84 CART Lentiviruses (LV) containing the CART84 1.7.3, 1.226.5 and 3.89.5 pCCL-EF1α-CD84 vectors were produced in HEK293-T cells, and the number of transducing units per mL was determined by limiting dilution. The lentiviruses were used to transduce T cells isolated from whole blood, and then these CAR-T cells were expanded in culture for 6–8 days.

[0347] Table 17 summarizes the numbers of T cells obtained after three independent transductions, as well as the percentage of T cells that expressed CD84 CAR on their surface.

[0348] [Table 17]

[0349] Further analysis of CART84 1.7.3, 1.226.5 and 3.89.5 expansion cultures, as well as 152.3, 153.5 and H3.5 expansion cultures 6-7 days after transduction, is shown in Figure 22 and is expressed as fold increase relative to day 0 T cell numbers.

[0350] Example 12: CD84 CART in vitro cytotoxicity Based on cytotoxicity, the results obtained against Ramos, K562, MOLM-13, NALM6 and MOLT-4 cells, CART84 152.3, 153.5 and H3.5 were selected for further characterization and compared with 1.7.3 and 1.226.5 and 3.89.5. We evaluated their cytotoxicity against Ramos, MOLM-13, U937 and MOLT-4 cells after 24 or 48 hours of co-culture at effector:target cell ratios of 4:1, 2:1, 1:1 and 0.5:1 by determining the percentage of live GFP positive cells by cytometry. CD84 expression on these cells is shown in Figure 3.

[0351] All CART84 cells were Ramos cells (CD84 high ), which was statistically significant compared to UT (Figure 23). The cytotoxic effect increased over time in all CART cells, but this effect was lower in the cases of H3.5 and 1.226.5, especially at the lower ratios (1:1 and 0.5:1).

[0352] MOLM-13(CD84 moderate ) and U937(CD84 low) Against AML cells, CART84 152.3, 153.5, 1.7.3, 1.226.5 and 3.89.5 showed high cytotoxicity, which was statistically significant compared to UT (Figures 24 and 25, respectively). CART84 H3.5 showed lower cytotoxic activity than other CART cells against leukemia cells of both cell lines. The cytotoxic effect increased over time for all CARTs. All CART cells except H3.5 showed high cytotoxicity against U937 cells despite their low CD84 expression.

[0353] Finally, we investigated the effect of MOLT-4 cells (CD84 high When the cytotoxicity of CART84 152.3, 153.5, 1.7.5, 1.226.5 and 3.89.5 against UT, the same pattern as seen with U937 cells was observed, i.e., all CART cells except H3.5 showed a high cytotoxic effect that increased over time and was statistically significant compared to UT (Figure 26).

[0354] Example 13: In vitro cytotoxicity of CD84 CART against primary leukemic blast cells derived from AML and T-ALL We evaluated the cytotoxicity of CART84 cells 152.3, 153.5, G7.5 and H.3.5 against primary AML cells from patient samples obtained from the Haematology Department of Hospital Clinic de Barcelona (HCB). CD84 expression was evaluated on primary blasts, which were then stained with CFSE and co-cultured with effector cells (CART / UT) at effector:target cell ratios of 4:1, 2:1, 1:1 and 0.5:1 for 24 and 48 hours. After this period, cells were stained with LIVE / Dead™ Fixable Aqua to distinguish live / dead cells. CART cells 152.3 and 153.5 showed the highest cytotoxic activity against AML blasts, which was statistically significant compared to the effect of UT. CART H3.5 and G7.5 showed a low cytotoxic effect. The cytotoxic effect was induced by all CARTs and increased over time (Figure 27).

[0355] We also evaluated the cytotoxicity of CART84 cells 152.3, 153.5, 1.7.3, 1.226.5 and 3.89.5 against primary T-ALL patient samples obtained from the Haematology Department of Hospital Clinic de Barcelona (HCB). The experiments were performed as described above for AML samples. The cytotoxicity of CART 152.3, 153.5 and 1.7.3 was higher than that of CART 1.226.5 and 3.89.5 (Figure 28).

[0356] Example 14: In vitro cytokine production of CD84 CART To evaluate the cytokine release capacity of each CART cell, the supernatants of the effector-target cell cocultures were collected. Ramos, MOLM-13 and MOLT-4 were used as target cells at an effector:target cell ratio of 2:1. The levels of IFN-γ (Figure 29), Granzyme-B (Figure 30) and TNF-α (Figure 31) in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA) after 24 hours of coculture. Untransduced T cells (UT) were used as a negative control (cocultured with target cells). Four independent experiments were performed. IFN-γ and Granzyme B are cytotoxic cytokines and TNF-α is a proinflammatory cytokine. CART84 H3.5 showed the highest cytokine production of all CART84 tested when cocultured with Ramos. The cytokine profile of CART84 1.7.3 after coculture with MOLM-13 and MOLT-4 cells was similar to H3.5. All other CART cells tested produced similar levels of cytokines. These results suggest that both H3.5 and 1.7.3 CART cells may exhibit tonic signaling, i.e., constitutive or chronic activation in the absence of ligand.

[0357] Example 15: In vitro expansion of CD84 CART A CFSE proliferation assay was performed to evaluate the ability of each CART84 cell to proliferate specifically to the binding example of the antigen CD84. Briefly, CART cells were stained with CellTrace™ CFSE on day 0. Four conditions were tested: day 0 cells (UT or CART cells); day 4 cells cultured alone in medium; day 4 cells stimulated with a non-specific stimulus of IL-2 (50UI / mL); and day 4 cells stimulated with a specific antigen, i.e., MOLM-13 cells, at an effector:target ratio of 0.5:1. We were able to demonstrate that CART84 152.3, 153.5 and 1.226.5 proliferate more when exposed to the CD84 antigen than when incubated with medium alone or with IL-2 (Figure 32). In contrast, CART H3.5, 1.7.3 and 3.89.5 proliferated similarly upon stimulation with MOLM-13 or IL-2, suggesting that these CART84 may exhibit sustained signaling.

[0358] Example 16: In vitro cytotoxicity of CD84 CART against hematopoietic progenitor stem cells CD84 is expressed on malignant cells from several hematological malignancies. These include AML, where expression of CD84 is shared among myeloid leukemic blasts and, to a lesser extent, among hematopoietic progenitor stem cells (HPSCs). As well as CD33 and CD123, both of which are targets of CART agents in development for the treatment of AML. Figure 33 shows the expression of CD84, CD33 and CD123 on HPSCs from apheresis products.

[0359] To evaluate the potential myelotoxicity of CART84, we tested their cytotoxicity against HPSCs from two different sources: CD34 cells obtained from umbilical cord blood units (CBU, from BST) and apheresis products (also from BST) from healthy donors mobilized with G-CSF for allogeneic stem cell transplantation. + We investigated the CD34 expression in apheresis samples of hPSCs. + Although CD34 cells are an ideal model to study bone marrow toxicity, to study this type of on-target / off-tumor toxicity, CD34 cells from CBU +It is more common to use cells.

[0360] Purified CD34 from CBU + Cells were co-cultured for 24 h with CART84 152.3, 153.5, 1.7.3, 1.229 and 3.89.5 at effector:target cell ratios of 4:1 and 2:1. CART84 152.3, 153.5, 1.7.3 and 1.226.5 were co-cultured with CD34 + The cytotoxic activity of CART84 3.89.5 against the cells was the highest. Figure 34 summarizes the results from five independent experiments.

[0361] CD34 from apheresis products + In the cytotoxicity assay against hPSCs, buffy coats from three different donors were used to generate CART84. Unexpectedly, the cytotoxicity of CART84 against these cells was relatively low. After 48 hours, the cytotoxicity of CART increased, but so did the cytotoxicity of nonspecific UT (Figure 35).

[0362] Example 17: In vitro T cell toxicity of CD84 CART Since CD84 is expressed on T cells, we investigated the potential toxicity of CART84 to T cells. T cells were directly isolated from donor whole blood, half of which were activated and transduced to generate CART cells, and the other half were cryopreserved until CART cells were available (i.e., 6-7 days). T cells were stained with CFSE and then co-cultured with CART84. Figure 36 shows the results of five independent experiments. CART84 152.3, 153.5, 1.7.3 and 3.89.5 showed moderate cytotoxic effects on their own T cells. On the other hand, the cytotoxic activity induced by 1.226.5 was low and not statistically significant compared to UT.

[0363] To further understand which specific T cell fractions are targeted by CART84, the effect on the following T cell subsets was analyzed: naïve, central memory, effector memory, and effector CD4 / CD8 T cells. Different T cell subpopulations were examined by flow cytometry: 1) target T cells before the assay, and 2) viable T cells after co-culture with different CART84s, as shown in Figure 37. As expected since CD84 is highly expressed on memory T cells, it was observed that the central memory T cell fraction was most affected by CART84, especially 152.3, 153.5, and 1.7.3. Furthermore, all T cell subsets were present after co-culture with CART84.

[0364] Example 18: In vivo efficacy of anti-CD84 CART in AML models Using MOLM-13 AML cells engineered to express GFP-firefly luciferase (GFPffLuc), we tested the efficacy of several CART84 cell lines in three independent experiments in NOD scid gamma (NSG) immunodeficient mice. The CART84 with the best in vitro efficacy and safety profile were selected for in vivo testing: 152.3, 153.5, H3.5, 1.7.3 and 1.226.5.

[0365] In the first and second experiments, 1 × 10 6 or 0.25 x 10 6 MOLM-13 GFP-ffLuc cells were each injected intravenously (iv) into NSG mice (Figures 38 and 39). After 6 days, 4 × 10 6 ~5×10 6 Mice were injected iv with UT, CART84 cells 152.3, 153.5, H3.5 or 1.7.3. Mice treated with CART 152.3, 153.5 and 1.7.3 showed a statistically significant increase in survival compared to mice treated with UT.

[0366] In the experiment shown in Figure 40, 1 × 10 5MOLM-13 GFP-ffLuc cells were injected i.v., and 2 and 11 days later, 5 × 10 6 and 3 × 10 6 The mice were injected with a dose of 152.3 and 153.5 cells. CART 152.3 and 153.5 suppressed tumor progression as monitored by bioluminescence, which was statistically significant compared to mice treated with UT. Furthermore, mice treated with CART 152.3 and 153.5 showed a statistically significant increase in survival compared to mice treated with UT.

[0367] At the end of the experiment, the mice were euthanized, and bone marrow and spleens were collected and transfected with tumor cells (MOLM-13-GFPffLuc), human CD3 + The presence of T cells or CART cells was analyzed by flow cytometry to quantify. In both experiments, CART84-treated mice had significantly fewer tumor cells in both bone marrow and spleen compared to UT-treated mice (Figures 39C and 40C). Human T cells expanded in both bone marrow and spleen of mice treated with UT, likely due to xeno-GvHD (Figures 39D and 40D). CART84 cells were found in both bone marrow and spleen, most prominently in the case of CART84 152.3 (Figures 39E and 40E).

[0368] In summary, 152.3 and 153.5 were able to consistently inhibit AML disease progression and increased survival in treated animals.

[0369] Example 19: In vivo efficacy of CART84 in T-ALL models We investigated the efficacy of CART84 cells in two independent experiments in NOD scid gamma (NSG) mice using MOLT-4 T-ALL cells engineered to express GFP-ffLuc. The following CART84 clones were tested: 152.3, 153.5, 1.7.3, and 1.226.5.

[0370] In the experiment shown in Figure 41, NSG mice were injected with 0.75 × 10 6 5 days after injection of 3 x 10 MOLT-4 GFP-ffLuc cells 6 Mice were intravenously injected with 100 mg of CART or UT. CART84 152.3 statistically significantly inhibited tumor progression as monitored by bioluminescence compared to mice treated with UT. Furthermore, mice treated with CART 152.3 and 153.5 showed a statistically significant increase in survival compared to mice treated with UT.

[0371] In the experiment shown in Figure 42, 4 × 10 5 Inject 5 × 10 MOLT-4 GFP-ffLuc cells i.v. and 2 days later 6 CART or UT. Treatment with CART84 152.3 and 153.5 elicited statistically significant efficacy in both inhibiting tumor progression as monitored by bioluminescence and increasing survival rates compared to UT.

[0372] At the end of the experiment, the mice were euthanized and the bone marrow and spleen were cultured using 100% PBS containing tumor cells (MOLT-4), human CD3 + The presence of T cells and CART cells was analyzed by flow cytometry to quantify. In this case, there was no statistically significant difference in the number of tumor cells found in the bone marrow and spleen of animals from different groups (Figure 42C). Human T cells proliferated in both the bone marrow and spleen of mice treated with UT, likely due to xeno-GvHD (Figure 42D). CART cells were found in both the bone marrow and spleen, especially in the case of 152.3 cells (Figure 42E).

[0373] CART84 152.3 and 153.5 were the most effective in suppressing T-ALL disease and prolonging survival in both T-ALL experiments.

[0374] Example 20: In vitro cytotoxicity of CD84 CART against human primary cells To evaluate potential on-target / off-tumor toxicities, we investigated CD84 expression on human primary cells and subsequently investigated potential cytotoxic effects induced by CART84. The following human cells were examined: human coronary artery endothelial cells (HCAEC), human small airway epithelial cells (HsaEpC), human cardiomyocytes (HCM), human renal epithelial cells (HREpC) and human uterine fibroblasts (HUF). Using flow cytometry, we were able to demonstrate that CD84 is not expressed on the surface of these cells (Figure 43).

[0375] In the XCELLigence assay, we found that CART84 152.3 and 153.5 did not show cytotoxicity against HsaEpC, HCM, HREpC or HUF. However, some cytotoxic effect was seen against HCAEC despite the absence of CD84 expression, suggesting that this effect was not antigen-specific. To confirm this, we compared the cytotoxic activity of CART84 with that of CART targeting CD123 (as this antigen is expressed on the surface of endothelial cells). The cytotoxicity induced by CART cells against CD123 was significantly higher than that induced by CART84 152.3 or 153.5 (Figure 44).

[0376] Example 21: Methods of Examples 9 to 20 Generation of CD123 CAR We generated an anti-CD123 CAR construct based on the IL3scfv-IgG4(L235E)-CD28gg-ζ(26292) CAR from US2014 / 0271582. The CAR sequence was cloned into the third-generation lentivirus vector pCCL (Dull et al. A Third-Generation Lentivirus Vector with a Conditional Packaging System. J. Virol. 1998: 72:8463-8471), which contains a signal peptide (GMCSFRα), 26292 scFv (VH-linker-VL), IgG4-Fc hinge and CD28 transmembrane region, costimulatory domain CD28 and signaling domain CD3-ζ under the control of the EF1α promoter.

[0377] Flow cytometry The positive CAR fraction of T cells was detected with Biotin-SP (long spacer) AffiniPure goat anti-mouse IgG, F(ab')2 fragment goat anti-mouse IgG (Jackson ImmunoResearch) or Biotin SP-conjugated AffiniPure F(ab')2-fragment goat anti-human IgG (Jackson ImmunoResearch), washed, and then incubated with BV421 / PE-conjugated streptavidin (eBioscience) and the antibodies required to interrogate each panel of proteins as described below.

[0378] CAR T cell phenotype was examined using the following mAbs against human proteins: CD197-BV510 (CCR7), CD62L-FITC, CD4-Pecy7, CD8-APCH7, CD45RA-APC (Becton Dickinson).

[0379] The following mAbs against human proteins were used to investigate the different cell populations present in the AML / T-ALL samples: CD11-APC, CD19-Fitc, HLA-DR-A450, CD45-PerCPCy5.5, CD34-PerCPCy5.5, CD3-APC, CD3-APCH7, CD33-Fitc, CD14-Fitc, CD14-APCH7, CD13-PE, CD13-BV421 (Becton Dickinson), CD84-APC, CD84-PE (BioLegend), CD45-A750, CD117-PeCy7 (Beckman Coulter), CD123-PECy7 (eBiosciences).

[0380] The following mAbs against human proteins were used to investigate the different mononuclear cell populations present in peripheral blood: CD3-BV421, CD14-APCH7, CD19-FITC, CD33-APC, CD34-PerCPCy5.5, CD38-APC, CD84-PE (BioLegend), CD123-PECy7 (eBiosciences) and HLA-DR-BV450 (Becton Dickinson).

[0381] The following mAbs against proteins were used for in vivo experiments performed in the MOLM-13 model: anti-human CD3-APCH7, anti-human CD33-APC, anti-human CD45-PerCP-Cy5.5, anti-mouse CD45-PECy7, streptavidin-BV421 (Becton Dickinson). The following mAbs against proteins were used for in vivo experiments performed in the MOLT-4 model: anti-human CD3-APC, anti-human CD45-PerCP-Cy5.5, anti-mouse CD45 Pe-Cy7, streptavidin-BV421 (Becton Dickinson).

[0382] All samples were run on a BD FACSCanto™ II, LSRFortessa™ 4L (DB Biosciences) or Attune™ NxT (Invitrogen) flow cytometer and analyzed with FlowJo v10.8.0 software (BD Biosciences).

[0383] In vitro cytotoxicity assay against primary leukemic blast cells from AML and T-ALL patients Primary blast cells were stained with CFSE (ThermoFisher) and co-cultured with effector cells (CART / UT) at effector:target cell ratios of 4:1, 2:1, 1:1 and 0.5:1 for 24 and 48 hours. After this period, the co-cultured cells were stained with LIVE / Dead™ Fixable Aqua (ThermoFisher) to distinguish live / dead cells. The percentage of remaining live blast cells was determined by flow cytometry and calculated using the following formula: % live cells = 100 x (number of negative LIVE / Dead positive CFSE cells with CART cells at time x / number of negative LIVE / Dead positive CFSE cells alone at time x).

[0384] CD34 + Cell isolation Hematopoietic stem cells (CD34 + ) were obtained from the umbilical cord blood unit (CBU, blood bank Banc de Sang i Teixits de Barcelona, ​​BST) and apheresis products from healthy donors modified with G-CSF for allogeneic stem cell transplantation (also obtained from BST).

[0385] CD34 + Cells were first isolated by Ficoll-Paque density gradient centrifugation and then CD34 isolation by magnetic separation using a human CD34 microbead kit (Miltenyi Biotec, positive selection). + Hematopoietic progenitor stem cells were isolated from the CBU by purification. CD34 cells were isolated from the apheresis product. + To isolate the cells, the cells were directly purified with CD34 magnetic beads.

[0386] CD34 + In vitro cytotoxicity assay against cells CD34 + To assess CD84 CART cytotoxicity against CD34 cells, + Cells were co-cultured with effector cells (CART / UT) at effector:target cell ratios of 4:1 and 2:1 for 24 and 48 hours. After this period, the co-cultured cells were stained with APC-conjugated CD3 and PE-conjugated CD34 antibodies to distinguish effector from target cells and labeled with LIVE / Dead Fixable Aqua to distinguish live / dead cells.

[0387] In vitro cytotoxicity assay against purified T cells T cells were isolated using immune cell isolation reagent from RosetteSep™ during Ficoll-Paque density gradient centrifugation from donor buffy coats. Half of the T cells obtained from this process were activated and transduced to generate CART cells, and the other half were cryopreserved until CART cells were available (i.e., 8 days) for use as target cells. To perform cytotoxicity assays, T cells were thawed and stained with CFSE, and then co-cultured with CART cells at various effector:target ratios. After 24 hours, both effector and target cells were fixed and stained with LIV / Dead Fixable Aqua cell staining kit.

[0388] The percentage of remaining live T cells was determined by flow cytometry and calculated using the following formula: % live cells = 100 x (number of negative LIVE / Dead positive CFSE cells with CART cells at time x / number of negative LIVE / Dead positive CFSE cells alone at time x).

[0389] To investigate the remaining live T cell subsets, these were also stained with human mAbs from the phenotypic panel as described in the flow cytometry section.

[0390] In vivo efficacy assay Eight to 12 week old non-diabetic-Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice (Charles River) were bred and housed under pathogen-free conditions in the Animal Facility of the Faculty of Medicine and Health Sciences of the University of Barcelona. 6 GFPffLuc-expressing MOLM-13 or GFPffLuc-MOLT-4 cells were resuspended in saline and injected intravenously (IV) into each NSG mouse on day 0. Two to nine days after tumor cell injection, 3–5 × 10 6 Mice were intravenously (iv) injected with UT or CD84 CART. Tumor progression was monitored by bioluminescence using a Xenogen IVIS 50 Imaging System (PerkinElmer). To measure bioluminescence, 100uL of XenoLight D-Luciferin (Perkin Elmer Ref.122799) was administered intraperitoneally to each mouse, and tumor burden was monitored weekly. Visualization and total luminescence flux were calculated using Living Image software (PerkinElmer). Mice were euthanized when they displayed signs of humane endpoint criteria. Bone marrow and spleens were removed and the presence of tumor cells, T cells, and CART cells was quantified by flow cytometry.

[0391] All procedures were performed in accordance with the Institutional Animal Care Committee of the Faculty of Medical and Health Sciences of the University of Barcelona.

[0392] Primary human cells The following primary human cells were obtained from PromoCell: human coronary artery endothelial cells (HCAEC), human small airway epithelial cells (HSAEpC), human uterine fibroblasts (HUF), human cardiomyocytes (HCM) and human kidney epithelial cells (HREpC). Cells were cultured at 37°C and 5% CO2 with specific media and supplements (also from PromoCell) as indicated in the datasheets.

[0393] In vitro cytotoxicity assay using adherent cells To evaluate the toxicity of CART cells against adherent cells, we used the xCELLigence instrument, which allows real-time measurement of live cell proliferation by impedance. Target cells were seeded in RTCA E-plates (16 wells) for xCELLigence, and the cell index was monitored for 24 hours using the xCELLigence RTCA Multiple Plate Monitoring System. At this point, 100 mL of growth medium or T cells (untransduced T cells or CART cells at an effector:target ratio of 4:1) were added to each well. Data obtained from each well (cell index) was normalized to 1, and cell proliferation was monitored for 72 hours. Data were analyzed using xCELLigence RTCA Software Lite 2.0.0.1301.

[0394] Example 22: In vivo efficacy of CART84 in B cell lymphoma models The efficacy of several CART84 cells was examined in in vivo experiments in NOD scid gamma (NSG) mice using Ramos (Burkitt's lymphoma) cells engineered to express GFP-ffLuc. The following CART84 cells were examined: 152.3, 153.5, and H3.5.

[0395] In the experiment shown in Figure 45, 4 × 10 5 Ramos GFP-ffLuc cells were injected i.v. into NSG mice on day 0. Two days later, 5 × 10 6 CART or UT cells were injected iv. Mice treated with CART 152.3, 153.5 and H3.5 showed a trend towards slower proliferation (Figure 45A). At the end of the experiment, the bone marrow of euthanized mice was collected and analyzed by flow cytometry to quantify the presence of tumor cells. CART84-treated mice had statistically significantly lower numbers of Ramos GFP-ffLuc cells in the bone marrow when compared to UT-treated mice (Figure 45B).

[0396] Embodiment Various features and embodiments of the present invention are described below with reference to the following numbered paragraphs.

[0397] 1.a) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-NYWIN (SEQ ID NO: 1); CDR2-DIYPVSGTTNYNEKFKR (SEQ ID NO: 2); and CDR3-GTGRFAY (SEQ ID NO: 3); or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVSTSSYSYMH (SEQ ID NO: 4); CDR2-FASNLES (SEQ ID NO: 5); and CDR3-QHSWEIPYT (SEQ ID NO: 6); or variants thereof each having up to three amino acid substitutions, additions or deletions; b) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-NYWLG (SEQ ID NO: 7); CDR2-DIYPGGGYTNYIEKFKG (SEQ ID NO: 8); and CDR3-YEGGYYGNYDAMDY (SEQ ID NO: 9), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASESVDNYGISFMN (SEQ ID NO: 10); CDR2-AASNQGS (SEQ ID NO: 11); and CDR3-QQSKAVPRT (SEQ ID NO: 12), or variants thereof, each with up to three amino acid substitutions, additions or deletions; c) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSSYA (SEQ ID NO: 13); CDR2-ISGSGGST (SEQ ID NO: 14); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO: 15), or a variant thereof with up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-NIESKD (SEQ ID NO: 16); CDR2-DDA (SEQ ID NO: 17); and CDR3-QVWDSSSDHVV (SEQ ID NO: 18), or variants thereof, each having up to three amino acid substitutions, additions or deletions; d) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSSYP (SEQ ID NO: 19); CDR2-ISYHGRNK (SEQ ID NO: 20); and CDR3-ARDRDATPGGTGVGNHGMAV (SEQ ID NO: 21), or a variant thereof with up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-QSLLHSSGYNY (SEQ ID NO: 22); CDR2-MGS (SEQ ID NO: 23); and CDR3-MQGLQTPPT (SEQ ID NO: 24), or variants thereof, each having up to three amino acid substitutions, additions or deletions; e) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GFTFSDNA (SEQ ID NO: 25); CDR2-ISGTGRTT (SEQ ID NO: 26); and CDR3-AKWDCSDGRCYWAY (SEQ ID NO: 27), or a variant thereof with up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-QSLVYSDGDTY (SEQ ID NO: 28); CDR2-KVS (SEQ ID NO: 29); and CDR3-MQGTHWPPNT (SEQ ID NO: 30), or variants thereof with up to 3 amino acid substitutions, additions or deletions, respectively; f) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 31); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO: 32); and CDR3-MRTSYYFDY (SEQ ID NO: 33), or a variant thereof with up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIFSSLA (SEQ ID NO: 34); CDR2-NAKTLAE (SEQ ID NO: 35); and CDR3-QHHYATPFT (SEQ ID NO: 36), or a variant thereof, each having up to three amino acid substitutions, additions or deletions; g) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-SYWIN (SEQ ID NO: 37); CDR2-DIYLGSGSTNYNEKFKS (SEQ ID NO: 38); and CDR3-SGGYLGY (SEQ ID NO: 39), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVSTSSYSYMH (SEQ ID NO: 40); CDR2-FASNLES (SEQ ID NO: 41); and CDR3-QHSWEIPYT (SEQ ID NO: 42), or a variant thereof with up to 3 amino acid substitutions, additions or deletions, respectively; h) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-NYWIG (SEQ ID NO: 43); CDR2-DIYPGGGYTNYNENFKG (SEQ ID NO: 44); and CDR3-STTYYSSYWCFDV (SEQ ID NO: 45), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and Light chain variable region (VL) having CDRs with the sequences: CDR1-KSSQSLLNSGNQANYLA (SEQ ID NO: 46); CDR2-GASTRES (SEQ ID NO: 47); and CDR3-QNDHSYPFT (SEQ ID NO: 48), or variants thereof each having up to three amino acid substitutions, additions or deletions; i) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RYWMS (SEQ ID NO: 49); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 50); and CDR3-PGPTVVATYWYFDV (SEQ ID NO: 51), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RSSQSIVHSNGNTYLE (SEQ ID NO: 52); CDR2-KVSSRFS (SEQ ID NO: 53); and CDR3-FQGSHVPRT (SEQ ID NO: 54), or variants thereof each having up to three amino acid substitutions, additions or deletions; or j) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RYWIN (SEQ ID NO: 55); CDR2-DIYPGSGSTNYNEKFKS (SEQ ID NO: 56); and CDR3-DTTIAY (SEQ ID NO: 57), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASQSVTTSRYSYMH (SEQ ID NO: 58); CDR2-FASNLES (SEQ ID NO: 59); and CDR3-QHSWEIPYT (SEQ ID NO: 60), or a variant thereof with up to 3 amino acid substitutions, additions or deletions, respectively; k) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-GYNMN (SEQ ID NO: 131); CDR2-NIDPYYGGTNYNQKFKG (SEQ ID NO: 132); and CDR3-GLLSGSFPY (SEQ ID NO: 133), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and Light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIYSYLA (SEQ ID NO: 134); CDR2-NAKTLAE (SEQ ID NO: 135); and CDR3-QHHYGSPLT (SEQ ID NO: 136), or variants thereof each having up to three amino acid substitutions, additions or deletions; l) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RSWMS (SEQ ID NO: 137); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 138); and CDR3-FYDGYSIYWYFDV (SEQ ID NO: 139), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RSSQSIVHSNGDTYLE (SEQ ID NO: 140); CDR2-KVSNRFS (SEQ ID NO: 141); and CDR3-FQGSHVPRT (SEQ ID NO: 142), or variants thereof, each having up to three amino acid substitutions, additions or deletions; m) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 143); CDR2-HIWWDDVKRYNPALRS (SEQ ID NO: 144); and CDR3-IAVTYFFDF (SEQ ID NO: 145), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-RASENIFSSFA (SEQ ID NO: 146); CDR2-NARTLAE (SEQ ID NO: 147); and CDR3-QHHYASPFT (SEQ ID NO: 148), or variants thereof, each having up to three amino acid substitutions, additions or deletions; n) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-TSGMGVG (SEQ ID NO: 149); CDR2-HIWWDDVKRYNPALKS (SEQ ID NO: 150); and CDR3-MSTSYYFDY (SEQ ID NO: 151), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and A light chain variable region (VL) having CDRs with the sequences: CDR1-KASQSLFTSVA (SEQ ID NO: 152); CDR2-SASYRYT (SEQ ID NO: 153); and CDR3-QQHYSSPFT (SEQ ID NO: 154), or variants thereof each having up to three amino acid substitutions, additions or deletions; or o) a heavy chain variable region (VH) having the complementarity determining regions (CDRs) with the sequences: CDR1-IYAMN (SEQ ID NO: 155); CDR2-RIRSKSNNYARFYADSVKD (SEQ ID NO: 156); and CDR3-PLRSYFSMDY (SEQ ID NO: 157), or a variant thereof having up to three amino acid substitutions, additions or deletions, respectively; and Light chain variable region (VL) having CDRs with the sequences: CDR1-KASENVDTYVS (SEQ ID NO: 158); CDR2-GASNRYT (SEQ ID NO: 159); and CDR3-GQTYSYPWT (SEQ ID NO: 160), or variants thereof with up to 3 amino acid substitutions, additions or deletions, respectively. An antigen-binding domain comprising:

[0398] 2.a) a VH domain having the sequence of SEQ ID NO: 61; and a VL domain having the sequence of SEQ ID NO: 62 or 108; b) a VH domain having the sequence of SEQ ID NO: 63; and a VL domain having the sequence of SEQ ID NO: 64 or 109; c) a VH domain having the sequence of SEQ ID NO: 65; and a VL domain having the sequence of SEQ ID NO: 66; d) a VH domain having the sequence of SEQ ID NO: 68; and a VL domain having the sequence of SEQ ID NO: 69; e) a VH domain having the sequence of SEQ ID NO: 71; and a VL domain having the sequence of SEQ ID NO: 72; f) a VH domain having the sequence of SEQ ID NO: 74; and a VL domain having the sequence of SEQ ID NO: 75; g) a VH domain having the sequence of SEQ ID NO: 76; and a VL domain having the sequence of SEQ ID NO: 77; h) a VH domain having the sequence of SEQ ID NO: 78; and a VL domain having the sequence of SEQ ID NO: 79; i) a VH domain having the sequence of SEQ ID NO: 80; and a VL domain having the sequence of SEQ ID NO: 81; or j) a VH domain having the sequence of SEQ ID NO: 82; and a VL domain having the sequence of SEQ ID NO: 83; k) a VH domain having the sequence of SEQ ID NO: 161; and a VL domain having the sequence of SEQ ID NO: 162; l) a VH domain having the sequence of SEQ ID NO: 163; and a VL domain having the sequence of SEQ ID NO: 164; m) a VH domain having the sequence of SEQ ID NO: 165; and a VL domain having the sequence of SEQ ID NO: 166; n) a VH domain having the sequence of SEQ ID NO: 167; and a VL domain having the sequence of SEQ ID NO: 168; or o) a VH domain having the sequence of SEQ ID NO: 169; and a VL domain having the sequence of SEQ ID NO: 170; or a variant thereof having at least 90% sequence identity thereto, respectively. 2. The antigen-binding domain of paragraph 1, comprising:

[0399] 3. An antibody comprising the antigen-binding domain of paragraph 1 or 2.

[0400] 4. In some cases, a) the CAR is an scFv CAR; b) the CAR comprises a CD8a transmembrane domain; c) the CAR comprises a 4-1BB or CD28 costimulatory domain; and / or d) the CAR comprises a CD3-zeta signaling domain; A chimeric antigen receptor (CAR) comprising the antigen-binding domain of paragraph 1 or 2.

[0401] 5. A polynucleotide comprising one or more nucleotide sequences encoding the antigen-binding domain of paragraph 1 or 2, the antibody of paragraph 3, or the CAR of paragraph 4.

[0402] 6. A vector comprising the polynucleotide of paragraph 5.

[0403] 7. A cell comprising the polynucleotide of paragraph 5 or the vector of paragraph 6.

[0404] 8. A cell comprising the antigen-binding domain of paragraph 1 or 2, or the CAR of paragraph 4.

[0405] 9. A cell comprising a first CAR and a second CAR, wherein the first CAR is a CAR of paragraph 4, and optionally, the second CAR is an anti-CD19 CAR, an anti-CD20 CAR, an anti-CD22 CAR, an anti-CD33 CAR, an anti-CD123 CAR; or an anti-CD7 CAR.

[0406] 10. A cell according to any one of paragraphs 7 to 9, which is a T cell or a NK cell, optionally an autologous or allogeneic cell.

[0407] 11. A pharmaceutical composition comprising the antigen-binding domain of paragraph 1 or 2, the antibody of paragraph 3, the CAR of paragraph 4, the polynucleotide of paragraph 5, the vector of paragraph 6, or the cell of any one of paragraphs 7 to 10.

[0408] 12. The antigen-binding domain of paragraph 1 or 2, the antibody of paragraph 3, the CAR of paragraph 4, the polynucleotide of paragraph 5, the vector of paragraph 6, the cell of any one of paragraphs 7 to 10, or the pharmaceutical composition of paragraph 11, for use in a therapy, optionally the therapy being the treatment of cancer.

[0409] 13. The antigen binding domain, antibody, CAR, polynucleotide, vector, cell, or pharmaceutical composition for use according to paragraph 12, wherein the cancer is a hematological malignancy, optionally a CD84 expressing hematological malignancy, and / or the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), B cell lymphoma, diffuse large B cell lymphoma (DLBCL), Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome, T cell acute lymphoblastic leukemia / lymphoma (T-ALL), chronic myeloproliferative syndrome, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, dendritic cell neoplasm and histiocytic sarcoma.

[0410] 14. Use of an antigen-binding domain of paragraph 1 or 2, or an antibody of paragraph 3, to determine the CD84 expression level in a sample, optionally to analyze the CD84 expression level in a sample derived from a subject.

[0411] 15. A method for identifying a subject suitable for treatment with an anti-CD84 CAR or antibody, comprising determining a CD84 expression level in a sample isolated from the subject, wherein the CD84 expression level is determined using an antigen-binding domain of paragraph 1 or 2, or an antibody of paragraph 3.

Claims

1. b) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - NYWLG (SEQ ID NO: 7); CDR2 - DIYPGGGYTNYIEKFKG (SEQ ID NO: 8); and CDR3 - YEGGYYGNYDAMDY (SEQ ID NO: 9), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASESVDNYGISFMN (SEQ ID NO: 10); CDR2 - AASNQGS (SEQ ID NO: 11); and CDR3 - QQSKAVPRT (SEQ ID NO: 12), or variants thereof with up to three amino acid substitutions, additions or deletions, each; a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - NYWIN (SEQ ID NO: 1); CDR2 - DIYPVSGTTNYNEKFKR (SEQ ID NO: 2); and CDR3 - GTGRFAY (SEQ ID NO: 3); or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASQSVSTSSYSYMH (SEQ ID NO: 4); CDR2 - FASNLES (SEQ ID NO: 5); and CDR3 - QHSWEIPYT (SEQ ID NO: 6); or variants thereof each having up to three amino acid substitutions, additions or deletions; c) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GFTFSSYA (SEQ ID NO: 13); CDR2 - ISGSGGST (SEQ ID NO: 14); and CDR3 - AKWDCSDGRCYWAY (SEQ ID NO: 15), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - NIESKD (SEQ ID NO: 16); CDR2 - DDA (SEQ ID NO: 17); and CDR3 - QVWDSSSDHVV (SEQ ID NO: 18), or variants thereof with up to three amino acid substitutions, additions or deletions, each; d) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GFTFSSYP (SEQ ID NO: 19); CDR2 - ISYHGRNK (SEQ ID NO: 20); and CDR3 - ARDRDATPGGTGVGNHGMAV (SEQ ID NO: 21), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - QSLLHSSGYNY (SEQ ID NO: 22); CDR2 - MGS (SEQ ID NO: 23); and CDR3 - MQGLQTPPT (SEQ ID NO: 24), or variants thereof with up to three amino acid substitutions, additions or deletions, each; e) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GFTFSDNA (SEQ ID NO: 25); CDR2 - ISGTGRTT (SEQ ID NO: 26); and CDR3 - AKWDCSDGRCYWAY (SEQ ID NO: 27), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - QSLVYSDGDTY (SEQ ID NO: 28); CDR2 - KVS (SEQ ID NO: 29); and CDR3 - MQGTHWPPNT (SEQ ID NO: 30), or variants thereof with up to three amino acid substitutions, additions or deletions, each; f) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - TSGMGVG (SEQ ID NO: 31); CDR2 - HIWWDDVKRYNPALKS (SEQ ID NO: 32); and CDR3 - MRTSYYFDY (SEQ ID NO: 33), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASENIFSSLA (SEQ ID NO: 34); CDR2 - NAKTLAE (SEQ ID NO: 35); and CDR3 - QHHYATPFT (SEQ ID NO: 36), or variants thereof with up to three amino acid substitutions, additions or deletions, each; g) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - SYWIN (SEQ ID NO: 37); CDR2 - DIYLGSGSTNYNEKFKS (SEQ ID NO: 38); and CDR3 - SGGYLGY (SEQ ID NO: 39), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - RASQSVSTSSYSYMH (SEQ ID NO: 40); CDR2 - FASNLES (SEQ ID NO: 41); and CDR3 - QHSWEIPYT (SEQ ID NO: 42), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; h) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - NYWIG (SEQ ID NO: 43); CDR2 - DIYPGGGYTNYNENFKG (SEQ ID NO: 44); and CDR3 - STTYYSSYWCFDV (SEQ ID NO: 45), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - KSSQSLLNSGNQANYLA (SEQ ID NO: 46); CDR2 - GASTRES (SEQ ID NO: 47); and CDR3 - QNDHSYPFT (SEQ ID NO: 48), or variants thereof with up to three amino acid substitutions, additions or deletions, each; i) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - RYWMS (SEQ ID NO: 49); CDR2 - EINPDSSTINYTPSLKD (SEQ ID NO: 50); and CDR3 - PGPTVVATYWYFDV (SEQ ID NO: 51), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RSSQSIVHSNGNTYLE (SEQ ID NO: 52); CDR2 - KVSSRFS (SEQ ID NO: 53); and CDR3 - FQGSHVPRT (SEQ ID NO: 54), or variants thereof with up to three amino acid substitutions, additions or deletions, each; j) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - RYWIN (SEQ ID NO: 55); CDR2 - DIYPGSGSTNYNEKFKS (SEQ ID NO: 56); and CDR3 - DTTIAY (SEQ ID NO: 57), or variants thereof with up to three amino acid substitutions, additions, or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - RASQSVTTSRYSYMH (SEQ ID NO: 58); CDR2 - FASNLES (SEQ ID NO: 59); and CDR3 - QHSWEIPYT (SEQ ID NO: 60), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; k) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GYNMN (SEQ ID NO: 131); CDR2 - NIDPYYGGTNYNQKFKG (SEQ ID NO: 132); and CDR3 - GLLSGSFPY (SEQ ID NO: 133), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASENIYSYLA (SEQ ID NO: 134); CDR2 - NAKTLAE (SEQ ID NO: 135); and CDR3 - QHHYGSPLT (SEQ ID NO: 136), or variants thereof with up to three amino acid substitutions, additions, or deletions, each; l) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RSWMS (SEQ ID NO: 137); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 138); and CDR3-FYDGYSIYWYFDV (SEQ ID NO: 139), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RSSQSIVHSNGDTYLE (SEQ ID NO: 140); CDR2 - KVSNRFS (SEQ ID NO: 141); and CDR3 - FQGSHVPRT (SEQ ID NO: 142), or variants thereof with up to three amino acid substitutions, additions or deletions, each; m) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - TSGMGVG (SEQ ID NO: 143); CDR2 - HIWWDDVKRYNPALRS (SEQ ID NO: 144); and CDR3 - IAVTYFFDF (SEQ ID NO: 145), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - RASENIFSSFA (SEQ ID NO: 146); CDR2 - NARTLAE (SEQ ID NO: 147); and CDR3 - QHHYASPFT (SEQ ID NO: 148), or variants thereof with up to three amino acid substitutions, additions, or deletions, each; n) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - TSGMGVG (SEQ ID NO: 149); CDR2 - HIWWDDVKRYNPALKS (SEQ ID NO: 150); and CDR3 - MSTSYYFDY (SEQ ID NO: 151), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - KASQSLFTSVA (SEQ ID NO: 152); CDR2 - SASYRYT (SEQ ID NO: 153); and CDR3 - QQHYSSPFT (SEQ ID NO: 154), or variants thereof each having up to three amino acid substitutions, additions or deletions; or o) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - IYAMN (SEQ ID NO: 155); CDR2 - RIRSKSNNYARFYADSVKD (SEQ ID NO: 156); and CDR3 - PLRSYFSMDY (SEQ ID NO: 157), or a variant thereof with up to three amino acid substitutions, additions, or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - KASENVDTYVS (SEQ ID NO: 158); CDR2 - GASNRYT (SEQ ID NO: 159); and CDR3 - GQTYSYPWT (SEQ ID NO: 160), or variants thereof with up to 3 amino acid substitutions, additions or deletions, each. A chimeric antigen receptor (CAR) comprising an antigen-binding domain comprising:

2. the antigen-binding domain b) a VH domain having the sequence of SEQ ID NO: 63; and a VL domain having the sequence of SEQ ID NO: 64 or 109; a) a VH domain having the sequence SEQ ID NO: 61; and a VL domain having the sequence SEQ ID NO: 62 or 108; c) a VH domain having the sequence of SEQ ID NO: 65; and a VL domain having the sequence of SEQ ID NO: 66; d) a VH domain having the sequence of SEQ ID NO: 68; and a VL domain having the sequence of SEQ ID NO: 69; e) a VH domain having the sequence of SEQ ID NO: 71; and a VL domain having the sequence of SEQ ID NO: 72; f) a VH domain having the sequence of SEQ ID NO: 74; and a VL domain having the sequence of SEQ ID NO: 75; g) a VH domain having the sequence of SEQ ID NO: 76; and a VL domain having the sequence of SEQ ID NO: 77; h) a VH domain having the sequence of SEQ ID NO: 78; and a VL domain having the sequence of SEQ ID NO: 79; i) a VH domain having the sequence of SEQ ID NO: 80; and a VL domain having the sequence of SEQ ID NO: 81; j) a VH domain having the sequence of SEQ ID NO: 82; and a VL domain having the sequence of SEQ ID NO: 83; k) a VH domain having the sequence of SEQ ID NO: 161; and a VL domain having the sequence of SEQ ID NO: 162; l) a VH domain having the sequence of SEQ ID NO: 163; and a VL domain having the sequence of SEQ ID NO: 164; m) a VH domain having the sequence of SEQ ID NO: 165; and a VL domain having the sequence of SEQ ID NO: 166; n) a VH domain having the sequence of SEQ ID NO: 167; and a VL domain having the sequence of SEQ ID NO: 168; or o) a VH domain having the sequence of SEQ ID NO: 169; and a VL domain having the sequence of SEQ ID NO: 170; or a variant thereof having at least 90% sequence identity thereto, respectively. The CAR of claim 1, comprising:

3. a) the CAR is an scFv CAR; b) the CAR comprises a CD8a transmembrane domain; c) the CAR comprises a 4-1BB or CD28 costimulatory domain; and / or d) the CAR comprises a CD3-ζ signaling domain; The CAR according to claim 1.

4. A polynucleotide comprising one or more nucleotide sequences encoding the CAR of claim 1.

5. A vector comprising the polynucleotide of claim 4.

6. A cell comprising the polynucleotide of claim 4.

7. A cell comprising the vector described in claim 5.

8. A cell comprising the CAR described in claim 1.

9. A cell comprising a first CAR and a second CAR, wherein the first CAR is the CAR of claim 1, and optionally the second CAR is an anti-CD19 CAR, an anti-CD20 CAR, an anti-CD22 CAR, an anti-CD33 CAR, an anti-CD123 CAR; or an anti-CD7 CAR.

10. 8. The cell of claim 6 or 7, which is a T cell or a NK cell, optionally an autologous or allogeneic cell.

11. A pharmaceutical composition comprising the CAR of claim 1, the polynucleotide of claim 4, the vector of claim 5, or the cell of claim 6 or 7.

12. 8. A CAR according to claim 1, a polynucleotide according to claim 4, a vector according to claim 5, a cell according to claim 6 or 7 for use in a therapy, optionally a therapy which is the treatment of cancer.

13. A pharmaceutical composition according to claim 11 for use in a therapy, optionally in the treatment of cancer.

14. 13. The CAR, polynucleotide, vector, or cell for use according to claim 12, wherein the cancer is a hematological malignancy, optionally a CD84-expressing hematological malignancy, and / or the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome, T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), chronic myeloproliferative syndrome, chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia, dendritic cell neoplasm, and histiocytic sarcoma.

15. A pharmaceutical composition for use according to claim 13, wherein the cancer is a hematological malignancy, optionally a CD84-expressing hematological malignancy, and / or the cancer is selected from the group consisting of chronic lymphocytic leukemia (CLL), B-cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), myelodysplastic syndrome, T-cell acute lymphoblastic leukemia / lymphoma (T-ALL), chronic myeloproliferative syndrome, chronic myeloid leukemia (CML), chronic myelomonocytic leukemia, dendritic cell neoplasm and histiocytic sarcoma.

16. b) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - NYWLG (SEQ ID NO: 7); CDR2 - DIYPGGGYTNYIEKFKG (SEQ ID NO: 8); and CDR3 - YEGGYYGNYDAMDY (SEQ ID NO: 9), or variants thereof each having up to three amino acid substitutions, additions, or deletions; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASESVDNYGISFMN (SEQ ID NO: 10); CDR2 - AASNQGS (SEQ ID NO: 11); and CDR3 - QQSKAVPRT (SEQ ID NO: 12), or variants thereof with up to three amino acid substitutions, additions or deletions, each; a) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - NYWIN (SEQ ID NO: 1); CDR2 - DIYPVSGTTNYNEKFKR (SEQ ID NO: 2); and CDR3 - GTGRFAY (SEQ ID NO: 3); or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASQSVSTSSYSYMH (SEQ ID NO: 4); CDR2 - FASNLES (SEQ ID NO: 5); and CDR3 - QHSWEIPYT (SEQ ID NO: 6); or variants thereof each having up to three amino acid substitutions, additions or deletions; c) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GFTFSSYA (SEQ ID NO: 13); CDR2 - ISGSGGST (SEQ ID NO: 14); and CDR3 - AKWDCSDGRCYWAY (SEQ ID NO: 15), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - NIESKD (SEQ ID NO: 16); CDR2 - DDA (SEQ ID NO: 17); and CDR3 - QVWDSSSDHVV (SEQ ID NO: 18), or variants thereof with up to three amino acid substitutions, additions or deletions, each; d) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GFTFSSYP (SEQ ID NO: 19); CDR2 - ISYHGRNK (SEQ ID NO: 20); and CDR3 - ARDRDATPGGTGVGNHGMAV (SEQ ID NO: 21), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - QSLLHSSGYNY (SEQ ID NO: 22); CDR2 - MGS (SEQ ID NO: 23); and CDR3 - MQGLQTPPT (SEQ ID NO: 24), or variants thereof with up to three amino acid substitutions, additions or deletions, each; e) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GFTFSDNA (SEQ ID NO: 25); CDR2 - ISGTGRTT (SEQ ID NO: 26); and CDR3 - AKWDCSDGRCYWAY (SEQ ID NO: 27), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - QSLVYSDGDTY (SEQ ID NO: 28); CDR2 - KVS (SEQ ID NO: 29); and CDR3 - MQGTHWPPNT (SEQ ID NO: 30), or variants thereof with up to three amino acid substitutions, additions or deletions, each; f) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - TSGMGVG (SEQ ID NO: 31); CDR2 - HIWWDDVKRYNPALKS (SEQ ID NO: 32); and CDR3 - MRTSYYFDY (SEQ ID NO: 33), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASENIFSSLA (SEQ ID NO: 34); CDR2 - NAKTLAE (SEQ ID NO: 35); and CDR3 - QHHYATPFT (SEQ ID NO: 36), or variants thereof with up to three amino acid substitutions, additions or deletions, each; g) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - SYWIN (SEQ ID NO: 37); CDR2 - DIYLGSGSTNYNEKFKS (SEQ ID NO: 38); and CDR3 - SGGYLGY (SEQ ID NO: 39), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - RASQSVSTSSYSYMH (SEQ ID NO: 40); CDR2 - FASNLES (SEQ ID NO: 41); and CDR3 - QHSWEIPYT (SEQ ID NO: 42), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; h) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - NYWIG (SEQ ID NO: 43); CDR2 - DIYPGGGYTNYNENFKG (SEQ ID NO: 44); and CDR3 - STTYYSSYWCFDV (SEQ ID NO: 45), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - KSSQSLLNSGNQANYLA (SEQ ID NO: 46); CDR2 - GASTRES (SEQ ID NO: 47); and CDR3 - QNDHSYPFT (SEQ ID NO: 48), or variants thereof with up to three amino acid substitutions, additions or deletions, each; i) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - RYWMS (SEQ ID NO: 49); CDR2 - EINPDSSTINYTPSLKD (SEQ ID NO: 50); and CDR3 - PGPTVVATYWYFDV (SEQ ID NO: 51), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RSSQSIVHSNGNTYLE (SEQ ID NO: 52); CDR2 - KVSSRFS (SEQ ID NO: 53); and CDR3 - FQGSHVPRT (SEQ ID NO: 54), or variants thereof with up to three amino acid substitutions, additions or deletions, each; or j) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - RYWIN (SEQ ID NO: 55); CDR2 - DIYPGSGSTNYNEKFKS (SEQ ID NO: 56); and CDR3 - DTTIAY (SEQ ID NO: 57), or variants thereof with up to three amino acid substitutions, additions, or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - RASQSVTTSRYSYMH (SEQ ID NO: 58); CDR2 - FASNLES (SEQ ID NO: 59); and CDR3 - QHSWEIPYT (SEQ ID NO: 60), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; k) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - GYNMN (SEQ ID NO: 131); CDR2 - NIDPYYGGTNYNQKFKG (SEQ ID NO: 132); and CDR3 - GLLSGSFPY (SEQ ID NO: 133), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RASENIYSYLA (SEQ ID NO: 134); CDR2 - NAKTLAE (SEQ ID NO: 135); and CDR3 - QHHYGSPLT (SEQ ID NO: 136), or variants thereof with up to three amino acid substitutions, additions, or deletions, each; l) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1-RSWMS (SEQ ID NO: 137); CDR2-EINPDSSTINYTPSLKD (SEQ ID NO: 138); and CDR3-FYDGYSIYWYFDV (SEQ ID NO: 139), or variants thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - RSSQSIVHSNGDTYLE (SEQ ID NO: 140); CDR2 - KVSNRFS (SEQ ID NO: 141); and CDR3 - FQGSHVPRT (SEQ ID NO: 142), or variants thereof with up to three amino acid substitutions, additions or deletions, each; m) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - TSGMGVG (SEQ ID NO: 143); CDR2 - HIWWDDVKRYNPALRS (SEQ ID NO: 144); and CDR3 - IAVTYFFDF (SEQ ID NO: 145), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - RASENIFSSFA (SEQ ID NO: 146); CDR2 - NARTLAE (SEQ ID NO: 147); and CDR3 - QHHYASPFT (SEQ ID NO: 148), or variants thereof with up to three amino acid substitutions, additions, or deletions, each; n) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - TSGMGVG (SEQ ID NO: 149); CDR2 - HIWWDDVKRYNPALKS (SEQ ID NO: 150); and CDR3 - MSTSYYFDY (SEQ ID NO: 151), or a variant thereof with up to three amino acid substitutions, additions or deletions, each; and a light chain variable region (VL) having CDRs with the sequences: CDR1 - KASQSLFTSVA (SEQ ID NO: 152); CDR2 - SASYRYT (SEQ ID NO: 153); and CDR3 - QQHYSSPFT (SEQ ID NO: 154), or variants thereof each having up to three amino acid substitutions, additions or deletions; or o) a heavy chain variable region (VH) having complementarity determining regions (CDRs) with the sequences: CDR1 - IYAMN (SEQ ID NO: 155); CDR2 - RIRSKSNNYARFYADSVKD (SEQ ID NO: 156); and CDR3 - PLRSYFSMDY (SEQ ID NO: 157), or a variant thereof with up to three amino acid substitutions, additions, or deletions, each; and A light chain variable region (VL) having CDRs with the sequences: CDR1 - KASENVDTYVS (SEQ ID NO: 158); CDR2 - GASNRYT (SEQ ID NO: 159); and CDR3 - GQTYSYPWT (SEQ ID NO: 160), or variants thereof with up to 3 amino acid substitutions, additions or deletions, each. An antigen-binding domain comprising:

17. An antibody comprising the antigen-binding domain of claim 16.

18. Use of an antigen-binding domain described in claim 16 or an antibody described in claim 17 for determining the CD84 expression level of a sample, optionally for analyzing the CD84 expression level of a sample derived from a subject.

19. 18. A method for identifying a subject suitable for treatment with an anti-CD84 CAR or antibody, comprising determining the CD84 expression level of a sample isolated from the subject, wherein the CD84 expression level is determined using the antigen-binding domain of claim 16 or the antibody of claim 17.

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