Targeted EPHA3 and its use

Abstract

Antigen-binding molecules and chimeric antigen receptors (CARs) capable of specifically recognizing or binding to at least EphA3 are disclosed. Methods of medical treatment and prevention are also disclosed.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, more specifically to antibody technology. More particularly, the present invention relates to antibodies or chimeric antigen receptors (CARs) that can specifically recognize at least EphA3 or specifically bind to EphA3. The present invention also relates to methods of medical treatment and prevention.

Background Art

[0002] Ephrin type-A receptor 3 (EphA3) has been found to be overexpressed or abnormally expressed in tumor cells derived from a wide variety of human solid tumors and leukemias, including colon cancer, breast cancer, chronic myeloid leukemia (CML), and glioblastoma multiforme (GBM). GBM is one of the most highly malignant solid brain tumors. Standard treatment consists of maximal surgical resection, radiotherapy, and combined chemotherapy and adjuvant chemotherapy with temozolomide. However, even when optimal treatment is performed, the median survival period after initial diagnosis is less than 15 months (1). Recent advances using checkpoint blockade have improved the outcomes of some human cancers, but GBM appears to be resistant to this treatment approach alone (2). Nevertheless, there is still a need to develop new treatment methods not only for GBM but also for a wider range of cancers.

Summary of the Invention

[0003] The present invention broadly targets anti-EphA3 binding agents, including human or humanized recombinant anti-EphA3 antibodies, and methods of using them. Certain forms of the present invention further provide chimeric antigen receptors (CARs) comprising antigen-binding domains that can specifically bind to EphA3, and methods of using them.

[0004] In a broad form, the present invention relates to EphA3 binding agents and CARs comprising one or more CDRs of the EphA3 monoclonal antibodies described herein.

[0005] In one embodiment, the present invention provides an EphA3 binder comprising at least one complementarity-determining region (CDR) having an amino acid sequence shown in SEQ ID NOs. 13-72 and / or Tables 4-7 or an amino acid sequence that is at least 70% identical thereto.

[0006] In some embodiments, the EphA3 binder is (a) A heavy chain immunoglobulin variable region (VH) polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 13-17, CDR having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 18-22, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 23-27, and / or (b) A light chain immunoglobulin variable region (VL) polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 28-32, CDR2 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 33-37, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 38-42. Includes.

[0007] In such embodiments, the VH polypeptide preferably comprises the amino acid sequence shown in SEQ ID NO: 153 or an amino acid sequence that is at least 70% identical thereto, and / or the VL polypeptide preferably comprises the amino acid sequence shown in SEQ ID NO: 154 or an amino acid sequence that is at least 70% identical thereto.

[0008] In some and some other embodiments of this embodiment, the EphA3 binder is (a) A VH polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 43-47, CDR2 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 48-52, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 53-57, and / or (b) A VL polypeptide comprising CDR1 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs. 58-62, CDR2 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs. 63-67, and CDR3 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs. 68-72. Includes.

[0009] In this regard, the VH polypeptide may contain the amino acid sequence shown in SEQ ID NO: 155 or an amino acid sequence that is at least 70% identical thereto, and / or the VL polypeptide may contain the amino acid sequence shown in SEQ ID NO: 156 or an amino acid sequence that is at least 70% identical thereto.

[0010] Preferably, the EphA3 conjugate is an antibody or antibody fragment. In one embodiment, the antibody or antibody fragment is a 3C3-1 or 2D4-1 monoclonal antibody, or a fragment thereof. In a particular embodiment, the EphA3 conjugate is a recombinant, human, or humanized antibody, or an antibody fragment.

[0011] In another embodiment, the present invention relates to a chimeric antigen receptor (CAR) comprising an antigen-binding domain having at least one CDR having an amino acid sequence shown in SEQ ID NOs. 13-72 and / or Tables 4-7 or an amino acid sequence that is at least 70% identical thereto, a transmembrane domain, and an intracellular signaling domain.

[0012] In some embodiments, the antigen-binding domain is (a) A heavy chain immunoglobulin variable region (VH) polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 13-17, CDR having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 18-22, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 23-27, and / or (b) A light chain immunoglobulin variable region (VL) polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 28-32, CDR2 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 33-37, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 38-42. It includes, consists of, or is essentially derived from.

[0013] In such embodiments, the VH polypeptide may include the amino acid sequence shown in SEQ ID NO: 153 or an amino acid sequence that is at least 70% identical thereto, and / or the VL polypeptide may include the amino acid sequence shown in SEQ ID NO: 154 or an amino acid sequence that is at least 70% identical thereto.

[0014] In some embodiments, the antigen-binding domain is (a) A VH polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 43-47, CDR2 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 48-52, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 53-57, and / or (b) A VL polypeptide comprising CDR1 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs. 58-62, CDR2 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs. 63-67, and CDR3 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs. 68-72. It includes, consists of, or is essentially derived from.

[0015] In such embodiments, the VH polypeptide may include the amino acid sequence shown in SEQ ID NO: 155 or an amino acid sequence that is at least 70% identical thereto, and / or the VL polypeptide may include the amino acid sequence shown in SEQ ID NO: 156 or an amino acid sequence that is at least 70% identical thereto.

[0016] In some embodiments, the antigen-binding domain includes a linker. In one particular embodiment, the linker includes, consists of, or is essentially the same as the amino acid sequence shown in SEQ ID NO: 158 or an amino acid sequence that is at least 70% identical thereto.

[0017] Preferably, the CAR further comprises a leader sequence or signal peptide sequence, for example, the leader sequence or signal peptide sequence of CD8 shown in SEQ ID NO: 157, or an amino acid sequence that is at least 70% identical thereto.

[0018] In certain embodiments, the transmembrane domain includes a CD8 transmembrane domain, for example, a CD8 transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 159 or an amino acid sequence that is at least 70% identical thereto.

[0019] Preferably, the intracellular T cell signaling domain includes a CD3 zeta intracellular signaling domain.

[0020] In certain embodiments, the intracellular signaling domain includes the CD3 amino acid sequence shown in SEQ ID NO: 162 or an amino acid sequence that is at least 70% identical thereto.

[0021] Preferably, the CAR further comprises one or more co-stimulatory domains, for example, a CD28 co-stimulatory domain having the amino acid sequence shown in SEQ ID NO: 161 or an amino acid sequence that is at least 70% identical thereto, and / or a CD137 co-stimulatory domain having the amino acid sequence shown in SEQ ID NO: 160 or an amino acid sequence that is at least 70% identical thereto.

[0022] In some embodiments, the EphA3 binder of the first embodiment or the CAR of the second embodiment is intended for use in the treatment or prevention of cancer in a subject, such as solid tumors like glioblastoma multiforme.

[0023] In this regard, the antibodies and antigen-binding molecules described above and elsewhere in this specification exhibit substantial antitumor activity and are particularly effective in reducing tumors. In some embodiments, the antigen-binding molecules of the present invention have the ability to substantially remove tumors from subjects having cancer.

[0024] In another embodiment, the present invention provides isolated nucleic acids encoding EphA3 binders and / or CARs as described above and elsewhere in this specification.

[0025] In another embodiment, the present invention lies in a gene construct comprising isolated nucleic acids as described above and elsewhere in this specification.

[0026] In yet another aspect, the present invention provides a host cell comprising nucleic acids and / or gene constructs as described above and elsewhere in this specification.

[0027] Preferably, the host cell is a T cell or contains T cells.

[0028] In another embodiment, the present invention relates to a method for producing an isolated EphA3 binder or CAR, comprising the steps of (i) culturing host cells according to the fifth embodiment, and (ii) isolating the EphA3 binder or CAR from the host cells cultured in step (i).

[0029] In another embodiment, the present invention provides an EphA3 binder or CAR prepared by the method of the sixth embodiment.

[0030] In another embodiment, the present invention is (i) EphA3 binder of the first embodiment, and / or (ii) CAR in the second form It is present in antibodies or antibody fragments that bind to and / or are produced against.

[0031] In another embodiment, the present invention provides a composition comprising an EphA3 binder according to the first or sixth embodiment, a CAR according to the second or sixth embodiment, a nucleic acid according to the third embodiment, a gene construct according to the fourth embodiment, and / or a host cell according to the fifth embodiment, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0032] In yet another embodiment, the present invention relates to a method for treating or preventing cancer in a subject, comprising the step of administering a therapeutically effective amount of an EphA3 binder of the first or sixth embodiment, a CAR of the second or sixth embodiment, a nucleic acid of the third embodiment, a gene construct of the fourth embodiment, a host cell of the fifth embodiment, and / or a composition of the ninth embodiment to the subject, thereby treating or preventing cancer in the subject.

[0033] In another aspect, the present invention provides the use of an EphA3 conjugate according to the first or sixth aspect, a CAR according to the second or sixth aspect, a nucleic acid according to the third aspect, a gene construct according to the fourth aspect, and / or a host cell according to the fifth aspect in the manufacture of a pharmaceutical product for the prevention and / or treatment of cancer in a subject.

[0034] With respect to the first, second, tenth, and eleventh embodiments, cancer preferably is or includes glioblastoma pleomorphicum.

[0035] In another embodiment, the present invention relates to a method for detecting EphA3 or cells expressing EphA3, comprising the step of forming a complex of an EphA3 conjugate according to the first embodiment or a CAR according to the second embodiment with EphA3, thereby detecting EphA3 or cells expressing EphA3.

[0036] Preferably, the method includes a first step of contacting EphA3 or cells expressing EphA3 with an EphA3 conjugate or CAR.

[0037] In certain embodiments, the cells are cancer cells or include cancer cells.

[0038] In another embodiment, the present invention provides an isolated protein comprising, essentially, or consisting of an amino acid sequence shown in any one of SEQ ID NOs: 13-156 and / or Tables 4-7, or an amino acid sequence that is at least 70% identical thereto.

[0039] A further aspect of the present invention provides human T cells expressing (a) a T cell receptor (TCR) that is activated by binding to a CMV antigen, and (b) a chimeric antigen receptor (CAR) that includes an antigen-binding domain that binds to an epitope on EphA3.

[0040] In some embodiments, the antigen-binding domain is an scFv that includes a heavy chain variable (VH) region and a light chain variable (VL) region.

[0041] In a similar embodiment, the present invention provides a T cell comprising (a) a T cell receptor (TCR) expressing a TCR specific to the CMV antigen, and (b) an antigen-binding molecule that binds to EphA3.

[0042] In yet another embodiment, the present invention lies in isolated nucleic acids comprising, consisting of, or essentially comprising nucleic acid sequences that are at least 70% identical to, or the nucleic acid sequences shown in any one of SEQ ID NOs: 1 to 12 and / or Table 3.

[0043] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” are understood to mean that they include the integer or group of integers described, but not that they exclude other integers or groups of integers.

[0044] In the context of amino acid sequences, "essentially consisting of" means that the amino acid sequence in question contains one, two, three, four, or five additional amino acids at its N-terminus and / or C-terminus.

[0045] As used herein, the indefinite articles “a” and “an” are used to refer to or include singular or plural elements or features and should not be construed as meaning or defining “one” or “single” elements or features. [Brief explanation of the drawing]

[0046] [Figure 1] Cloning strategy for EphA3(P29320|21-541)pcDNA3.4 for expression in Expi293F cells. [Figure 2] SDS-PAGE and Western blot analysis of EphA3 (P29320|21-541). Lane M1: Protein marker TaKaRa (catalog number 3452); Lane M2: Protein marker (GenScript, catalog number M00521); Lane 1: Reducing condition; Lane 2: Non-reducing condition; Lane P: Multiple-tag as positive control (GenScript, catalog number M0101); Primary antibody: Mouse anti-His mAb (GenScript, catalog number A00186). [Figure 3] Parental antibody clones selected for subcloning (Panel A). Mice were immunized with recombinant human EphA3 protein (PP29320|21-541). Hybridomas were generated, and five parental hybridoma clones were selected for subcloning based on EphA3 specificity determined by ELISA and FACS using LK63 tumor cells expressing EphA3 (Panel B). [Figure 4-1] Monoclonal antibodies prepared against EphA3 exhibited different binding efficiencies. Subclonal supernatants were screened for EphA3 binding efficiency using ELISA (Panel A) and FACS (Panel B). [Figure 4-2] Monoclonal antibodies prepared against EphA3 exhibited different binding efficiencies. Subclonal supernatants were screened for EphA3 binding efficiency using ELISA (Panel A) and FACS (Panel B). [Figure 4-3] Monoclonal antibodies prepared against EphA3 exhibited different binding efficiencies. Subclonal supernatants were screened for EphA3 binding efficiency using ELISA (Panel A) and FACS (Panel B). [Figure 5] EphA3 is expressed on glioma cell lines. Flow cytometry analysis using 3C3-1 anti-EphA3 was performed on cultured U87, D270, and U251 cell lines. [Figure 6] Schematic diagrams of the pD2109-FA301_Ires-RFP CAR construct (top) and the pD2109-FA302_Ires-RFP CAR construct (bottom), which have a 3C3-1 scFv binding domain and a CD28ζ or 4-1BBζ signaling domain. [Figure 7] RT-PCR and agarose gel electrophoresis of sequences spanning a CAR T fragment (546 bp) in HEK293T cells. Forward primer - CAGCGGCTACACCTTTACCA and reverse primer - CCGGAGAATCTATCCGGCAC primers. [Figure 8]Transduction of Jurkat cells using EphA3-CAR lentiviruses prepared for constructs FA301 and FA302 (RFP reporter) and pD2109 (GFP reporter). [Figure 9] Surface expression of EphA3-CAR in Jurkat cells. Cells were transduced with (left) FA301 lentivirus and (right) FA302 lentivirus, and incubated with plate-bound EphA3-his protein. To determine EphA3-CAR surface expression, cells were stained in or without αEphA3 primary Ab, and then stained with αHis-tagged Ab. [Figure 10] Jurkat cells expressing CAR are activated by EphA3. Jurkat cells transduced with FA301 and FA302 were incubated with gradually increasing concentrations of EphA3 protein bound to a plate. The cells were stained for CD69 expression using FACs, and the levels of RFP positivity in CAR-expressing cells were compared with those of RFP-negative cells. [Figure 11] Jurkat cells expressing CAR are activated by tumor cell lines expressing EphA3. Jurkat cells transduced with FA301 were incubated with Lk63 cells in a 1:10 ratio (Jurkat-CAR:Lk63). Jurkat cells were stained for CD69 expression with FACs, and levels in RFP-positive cells were compared to those in RFP-negative cells. [Figure 12] T cells enlarged by CMV were transduced using pD2109 lentivirus and FA301 lentivirus. Transduction efficiency was determined by FACS after 3 days. [Figure 13]In vitro comparison of the costimulatory domains of EphA3 CAR T cells. (A) Peripheral blood mononuclear cells were stimulated with CD3 / 28+ beads (increasing polyclonality), and the cells were transduced with EphA3 lentivirus FA305-BBζ or FA306-28ζ and cultured for 12 days. Untransduced (NT) T cells were maintained as a control. CAR expression was determined by surface expression of anti-mouse IgG (CAR) and analysis by FACS. (B) Characterization of effector function of EphA3 CAR T cells. Transduced T cells with CAR were incubated overnight with LK63 (EphA3+) target cells, and their function was examined using intracellular TNF. [Figure 14] CAR T cell generation. Peripheral blood mononuclear cells were stimulated using CD3 / 28+ beads (polyclonality enhancement) or a pool of 26 HLA class I and class II restricted T cell peptide epitopes derived from multiple CMV antigens. These cells were transduced with EphA3 lentivirus and cultured for 14 days. Untransduced (NT) T cells were maintained as a control. CAR expression and CMV specificity were determined by FACS analysis of peptide tetramers (VTE and ELK) against anti-mouse IgG (CAR) and HLA complex-CMV. [Figure 15] Comparison of EphA3 CAR T cell effector function and cytotoxicity in polyclonal CMV-specific T cells. (A) CAR-transduced T cells were incubated overnight with LK63(EphA3+) target cells, and their function was examined using intracellular IFN-γ, TNF, and CD107a cell surface recruitment. [Figure 16] Characterization of the in vitro cytotoxicity of EphA3 CAR T cells. (A) The ability of T cells expressing EphA3-CAR to specifically eliminate EphA3+ tumors was measured by real-time target-induced cytolysis of U251 (EphA3+) and U87 (EphA3-) glioma cell lines. (B and C) RTCA analysis of polyclonal CMV-specific EphA3-CAR at effector-target ratios of 1:1, 5:1, and 10:1 using the U251 target cell line. [Figure 17-1] EphA3 CAR T cells mediate a potent anti-GBM response in a xenograft model of GBM. (A) Schematic diagram of the experimental design. Luciferase-expressing glioma cell lines U251 (EphA3+) or U87 (EphA3-) were subcutaneously transplanted into the flanks of NRG mice (ectopic model). Tumor size was measured or determined by bioluminescence. When the tumor reached approximately 25 mm2, EphA3-CAR, NT (non-transduced) T cells, or CAR19 (non-specific CAR T cells) were administered intravenously to the mice. (B) Representative FACS plot analysis of CD4+ and CD8+ percentages, and (C) Ki67 expression in blood collected on day 17. [Figure 17-2] EphA3 CAR T cells mediate a potent anti-GBM response in a xenograft model of GBM. (D) U251 tumor burden was determined weekly to assess the effect of CAR-T cell therapy on tumor regression. (E) Comparison of CAR EphA3 treatment in U251 and U87-carrying mice. (F) In vivo imaging of U251 (left) and U87 (right) luminescent tumor xenograft mice. (G) Kaplan-Meier survival curves for mice treated with EphA3-CAR or administered with control cells (NT or CAR19). [Modes for carrying out the invention]

[0047] [A brief explanation of arrays] Sequence ID 1 Clone 3C3-1 Heavy Chain CDR1 Nucleotide Sequence Sequence ID 2: Clone 3C3-1 Heavy Chain CDR2 Nucleotide Sequence Sequence ID 3: Clone 3C3-1 heavy chain CDR3 nucleotide sequence Sequence ID 4: Clone 3C3-1 light chain CDR1 nucleotide sequence Sequence ID 5: Clone 3C3-1 light chain CDR2 nucleotide sequence Sequence ID 6: Clone 3C3-1 light chain CDR3 nucleotide sequence Sequence ID 7 Clone 2D4-1 Heavy Chain CDR1 Nucleotide Sequence Sequence ID 8 Clone 2D4-1 Heavy Chain CDR2 Nucleotide Sequence Sequence ID 9 Clone 2D4-1 Heavy Chain CDR3 Nucleotide Sequence Sequence ID 10 Clone 2D4-1 Light Chain CDR1 Nucleotide Sequence Sequence ID 11 Clone 2D4-1 Light Chain CDR2 Nucleotide Sequence Sequence ID 12 Clone 2D4-1 Light Chain CDR3 Nucleotide Sequence Sequence ID 13 Clone 3C3-1 Heavy Chain CDR1 Amino Acid Sequence (Chothia) Sequence ID No. 14 Clone 3C3-1 Heavy Chain CDR1 Amino Acid Sequence (AbM) Sequence ID 15 Clone 3C3-1 Heavy Chain CDR1 Amino Acid Sequence (Kabat) Sequence ID No. 16 Clone 3C3-1 Heavy Chain CDR1 Amino Acid Sequence (Contact) Sequence ID No. 17 Clone 3C3-1 Heavy Chain CDR1 Amino Acid Sequence (IMGT) Sequence ID No. 18 Clone 3C3-1 Heavy Chain CDR2 Amino Acid Sequence (Chothia) Sequence ID 19 Clone 3C3-1 Heavy Chain CDR2 Amino Acid Sequence (AbM) Sequence ID No. 20 Clone 3C3-1 Heavy Chain CDR2 Amino Acid Sequence (Kabat) Sequence ID No. 21 Clone 3C3-1 Heavy Chain CDR2 Amino Acid Sequence (Contact) Sequence ID No. 22 Clone 3C3-1 Heavy Chain CDR2 Amino Acid Sequence (IMGT) Sequence ID 23 Clone 3C3-1 Heavy Chain CDR3 Amino Acid Sequence (Chothia) Sequence ID No. 24 Clone 3C3-1 Heavy Chain CDR3 Amino Acid Sequence (AbM) Sequence ID No. 25 Clone 3C3-1 Heavy Chain CDR3 Amino Acid Sequence (Kabat) Sequence ID No. 26 Clone 3C3-1 Heavy Chain CDR3 Amino Acid Sequence (Contact) Sequence ID No. 27 Clone 3C3-1 Heavy Chain CDR3 Amino Acid Sequence (IMGT) Sequence ID No. 28 Clone 3C3-1 Light Chain CDR1 Amino Acid Sequence (Chothia) Sequence ID No. 29 Clone 3C3-1 Light Chain CDR1 Amino Acid Sequence (AbM) Sequence ID No. 30 Clone 3C3-1 Light Chain CDR1 Amino Acid Sequence (Kabat) Sequence ID 31 Clone 3C3-1 Light Chain CDR1 Amino Acid Sequence (Contact) Sequence ID 32 Clone 3C3-1 Light Chain CDR1 Amino Acid Sequence (IMGT) Sequence ID 33 Clone 3C3-1 Light Chain CDR2 Amino Acid Sequence (Chothia) Sequence ID No. 34 Clone 3C3-1 Light Chain CDR2 Amino Acid Sequence (AbM) Sequence ID 35 Clone 3C3-1 Light Chain CDR2 Amino Acid Sequence (Kabat) Sequence ID 36 Clone 3C3-1 Light Chain CDR2 Amino Acid Sequence (Contact) Sequence ID 37 Clone 3C3-1 Light Chain CDR2 Amino Acid Sequence (IMGT) Sequence ID 38 Clone 3C3-1 Light Chain CDR3 Amino Acid Sequence (Chothia) Sequence ID 39 Clone 3C3-1 Light Chain CDR3 Amino Acid Sequence (AbM) Sequence ID No. 40 Clone 3C3-1 Light Chain CDR3 Amino Acid Sequence (Kabat) Sequence ID No. 41 Clone 3C3-1 Light Chain CDR3 Amino Acid Sequence (Contact) Sequence ID No. 42 Clone 3C3-1 Light Chain CDR3 Amino Acid Sequence (IMGT) Sequence ID 43 Clone 2D4-1 Heavy Chain CDR1 Amino Acid Sequence (Chothia) Sequence ID 44 Clone 2D4-1 Heavy Chain CDR1 Amino Acid Sequence (AbM) Sequence ID 45 Clone 2D4-1 Heavy Chain CDR1 Amino Acid Sequence (Kabat) Sequence ID No. 46 Clone 2D4-1 Heavy Chain CDR1 Amino Acid Sequence (Contact) Sequence ID 47 Clone 2D4-1 Heavy Chain CDR1 Amino Acid Sequence (IMGT) Sequence ID 48 Clone 2D4-1 Heavy Chain CDR2 Amino Acid Sequence (Chothia) Sequence ID 49 Clone 2D4-1 Heavy Chain CDR2 Amino Acid Sequence (AbM) Sequence ID 50 Clone 2D4-1 Heavy Chain CDR2 Amino Acid Sequence (Kabat) Sequence ID 51 Clone 2D4-1 Heavy Chain CDR2 Amino Acid Sequence (Contact) Sequence ID 52 Clone 2D4-1 Heavy Chain CDR2 Amino Acid Sequence (IMGT) Sequence ID 53 Clone 2D4-1 Heavy Chain CDR3 Amino Acid Sequence (Chothia) Sequence ID 54 Clone 2D4-1 Heavy Chain CDR3 Amino Acid Sequence (AbM) Sequence ID 55 Clone 2D4-1 Heavy Chain CDR3 Amino Acid Sequence (Kabat) Sequence ID 56 Clone 2D4-1 Heavy Chain CDR3 Amino Acid Sequence (Contact) Sequence ID 57 Clone 2D4-1 Heavy Chain CDR3 Amino Acid Sequence (IMGT) Sequence ID 58 Clone 2D4-1 Light Chain CDR1 Amino Acid Sequence (Chothia) Sequence ID 59 Clone 2D4-1 Light Chain CDR1 Amino Acid Sequence (AbM) Sequence ID 60 Clone 2D4-1 Light Chain CDR1 Amino Acid Sequence (Kabat) Sequence ID 61 Clone 2D4-1 Light Chain CDR1 Amino Acid Sequence (Contact) Sequence ID 62 Clone 2D4-1 Light Chain CDR1 Amino Acid Sequence (IMGT) Sequence ID 63 Clone 2D4-1 Light Chain CDR2 Amino Acid Sequence (Chothia) Sequence ID 64 Clone 2D4-1 Light Chain CDR2 Amino Acid Sequence (AbM) Sequence ID 65 Clone 2D4-1 Light Chain CDR2 Amino Acid Sequence (Kabat) Sequence ID 66 Clone 2D4-1 Light Chain CDR2 Amino Acid Sequence (Contact) Sequence ID 67 Clone 2D4-1 Light Chain CDR2 Amino Acid Sequence (IMGT) Sequence ID 68 Clone 2D4-1 Light chain CDR3 amino acid sequence (Chothia) Sequence ID 69 Clone 2D4-1 Light Chain CDR3 Amino Acid Sequence (AbM) Sequence ID 70 Clone 2D4-1 Light Chain CDR3 Amino Acid Sequence (Kabat) Sequence ID 71 Clone 2D4-1 Light Chain CDR3 Amino Acid Sequence (Contact) Sequence ID 72, clone 2D4-1, light chain CDR3 amino acid sequence (IMGT) Sequence ID 73 Clone 3C3-1 HFR1 amino acid sequence (Chothia) Sequence ID 74 Clone 3C3-1 HFR1 amino acid sequence (AbM) Sequence ID 75 Clone 3C3-1 HFR1 amino acid sequence (Kabat) Sequence ID 76 Clone 3C3-1 HFR1 amino acid sequence (Contact) Sequence ID 77 Clone 3C3-1 HFR1 amino acid sequence (IMGT) Sequence ID 78 Clone 3C3-1 HFR2 amino acid sequence (Chothia) Sequence ID 79 Clone 3C3-1 HFR2 amino acid sequence (AbM) Sequence ID 80 Clone 3C3-1 HFR2 amino acid sequence (Kabat) Sequence ID 81 Clone 3C3-1 HFR2 amino acid sequence (Contact) Sequence ID 82 Clone 3C3-1 HFR2 amino acid sequence (IMGT) Sequence ID 83 Clone 3C3-1 HFR3 amino acid sequence (Chothia) Sequence ID 84 Clone 3C3-1 HFR3 amino acid sequence (AbM) Sequence ID 85 Clone 3C3-1 HFR3 amino acid sequence (Kabat) Sequence ID 86 Clone 3C3-1 HFR3 amino acid sequence (Contact) Sequence ID 87 Clone 3C3-1 HFR3 amino acid sequence (IMGT) Sequence ID 88 Clone 3C3-1 HFR4 amino acid sequence (Chothia) Sequence ID 89 Clone 3C3-1 HFR4 amino acid sequence (AbM) Sequence ID 90 Clone 3C3-1 HFR4 amino acid sequence (Kabat) Sequence ID 91 Clone 3C3-1 HFR4 amino acid sequence (Contact) Sequence ID 92 Clone 3C3-1 HFR4 amino acid sequence (IMGT) Sequence ID 93 Clone 3C3-1 LFR1 amino acid sequence (Chothia) Sequence ID 94 Clone 3C3-1 LFR1 amino acid sequence (AbM) Sequence ID 95 Clone 3C3-1 LFR1 amino acid sequence (Kabat) Sequence ID 96 Clone 3C3-1 LFR1 amino acid sequence (Contact) Sequence ID 97 Clone 3C3-1 LFR1 amino acid sequence (IMGT) Sequence ID 98 Clone 3C3-1 LFR2 amino acid sequence (Chothia) Sequence ID 99 Clone 3C3-1 LFR2 amino acid sequence (AbM) Sequence ID No. 100 Clone 3C3-1 LFR2 amino acid sequence (Kabat) Sequence ID No. 101 Clone 3C3-1 LFR2 amino acid sequence (Contact) Sequence ID 102 Clone 3C3-1 LFR2 amino acid sequence (IMGT) Sequence ID 103 Clone 3C3-1 LFR3 amino acid sequence (Chothia) Sequence ID No. 104 Clone 3C3-1 LFR3 amino acid sequence (AbM) Sequence ID No. 105 Clone 3C3-1 LFR3 amino acid sequence (Kabat) Sequence ID No. 106 Clone 3C3-1 LFR3 amino acid sequence (Contact) Sequence ID No. 107 Clone 3C3-1 LFR3 amino acid sequence (IMGT) Sequence ID 108 Clone 3C3-1 LFR4 amino acid sequence (Chothia) Sequence ID 109 Clone 3C3-1 LFR4 amino acid sequence (AbM) Sequence ID No. 110 Clone 3C3-1 LFR4 amino acid sequence (Kabat) Sequence ID 111 Clone 3C3-1 LFR4 amino acid sequence (Contact) Sequence ID 112 Clone 3C3-1 LFR4 amino acid sequence (IMGT) Sequence ID 113 Clone 2D4-1 HFR1 amino acid sequence (Chothia) Sequence ID 114 Clone 2D4-1 HFR1 amino acid sequence (AbM) Sequence ID 115 Clone 2D4-1 HFR1 amino acid sequence (Kabat) Sequence ID 116 Clone 2D4-1 HFR1 amino acid sequence (Contact) Sequence ID 117 Clone 2D4-1 HFR1 amino acid sequence (IMGT) Sequence ID 118 Clone 2D4-1 HFR2 amino acid sequence (Chothia) Sequence ID 119 Clone 2D4-1 HFR2 amino acid sequence (AbM) Sequence ID 120 Clone 2D4-1 HFR2 amino acid sequence (Kabat) Sequence ID 121 Clone 2D4-1 HFR2 amino acid sequence (Contact) Sequence ID 122 Clone 2D4-1 HFR2 amino acid sequence (IMGT) Sequence ID 123 Clone 2D4-1 HFR3 amino acid sequence (Chothia) Sequence ID 124 Clone 2D4-1 HFR3 amino acid sequence (AbM) Sequence ID 125 Clone 2D4-1 HFR3 amino acid sequence (Kabat) Sequence ID 126 Clone 2D4-1 HFR3 amino acid sequence (Contact) Sequence ID 127 Clone 2D4-1 HFR3 amino acid sequence (IMGT) Sequence ID 128 Clone 2D4-1 HFR4 amino acid sequence (Chothia) Sequence ID 129 Clone 2D4-1 HFR4 amino acid sequence (AbM) Sequence ID 130 Clone 2D4-1 HFR4 amino acid sequence (Kabat) Sequence ID 131 Clone 2D4-1 HFR4 amino acid sequence (Contact) Sequence ID 132 Clone 2D4-1 HFR4 amino acid sequence (IMGT) Sequence ID 133 Clone 2D4-1 LFR1 amino acid sequence (Chothia) Sequence ID 134 Clone 2D4-1 LFR1 amino acid sequence (AbM) Sequence ID 135 Clone 2D4-1 LFR1 amino acid sequence (Kabat) Sequence ID 136 Clone 2D4-1 LFR1 amino acid sequence (Contact) Sequence ID 137 Clone 2D4-1 LFR1 amino acid sequence (IMGT) Sequence ID 138 Clone 2D4-1 LFR2 amino acid sequence (Chothia) Sequence ID 139 Clone 2D4-1 LFR2 amino acid sequence (AbM) Sequence ID 140 Clone 2D4-1 LFR2 amino acid sequence (Kabat) Sequence ID 141 Clone 2D4-1 LFR2 amino acid sequence (Contact) Sequence ID 142 Clone 2D4-1 LFR2 amino acid sequence (IMGT) Sequence ID 143 Clone 2D4-1 LFR3 amino acid sequence (Chothia) Sequence ID 144 Clone 2D4-1 LFR3 amino acid sequence (AbM) Sequence ID 145 Clone 2D4-1 LFR3 amino acid sequence (Kabat) Sequence ID 146 Clone 2D4-1 LFR3 amino acid sequence (Contact) Sequence ID 147 Clone 2D4-1 LFR3 amino acid sequence (IMGT) Sequence ID 148 Clone 2D4-1 LFR4 amino acid sequence (Chothia) Sequence ID 149 Clone 2D4-1 LFR4 amino acid sequence (AbM) Sequence ID 150 Clone 2D4-1 LFR4 amino acid sequence (Kabat) Sequence ID 151 Clone 2D4-1 LFR4 amino acid sequence (Contact) Sequence ID 152 Clone 2D4-1 LFR4 amino acid sequence (IMGT) Sequence ID 153 Clone 3C3-1 Heavy Chain Amino Acid Sequence Sequence ID 154 Clone 3C3-1 Light Chain Amino Acid Sequence Sequence ID 155 Clone 2D4-1 Heavy Chain Amino Acid Sequence Sequence ID 156 Clone 2D4-1 Light Chain Amino Acid Sequence Sequence ID 157 CD8 signal peptide sequence Sequence ID 158 Spacer / Linker Amino Acid Sequence Sequence ID 159: CD8 hinge and transmembrane amino acid sequence Sequence ID 160 4-1BB / CD137 Co-stimulatory Domain Sequence ID 161 CD28 co-stimulatory domain Sequence ID 162: CD3-ζ intracellular signal transduction domain Sequence ID 163 IRES nucleic acid sequence Sequence ID 164 M_Cayenne RFP amino acid sequence Sequence ID No. 165: Human EphA3 amino acid sequence (precursor) Sequence ID 166: Human EphA3 mature amino acid sequence Sequence ID No. 167: Human EphA3 extracellular domain amino acid sequence Sequence ID No. 168: Human EphA3 transmembrane domain amino acid sequence Sequence ID No. 169: Human EphA3 cytoplasmic domain amino acid sequence Sequence ID No. 170: Human EphA3 Eph ligand-binding domain amino acid sequence Sequence ID No. 171: Human EphA3 fibronectin type III domain amino acid sequence Sequence ID No. 172: Human EphA3 fibronectin type III domain amino acid sequence Sequence ID No. 173: Human EphA3 protein kinase domain amino acid sequence Sequence ID 174: Human EphA3 steril α-motif amino acid sequence

[0048] [Detailed description of the invention] The present invention is, at least in part, based on the production of monoclonal antibodies targeting EphA3, and the subsequent creation of chimeric antigen receptors (CARs) based on the binding domains of these monoclonal antibodies. These monoclonal antibodies may be particularly suitable for the treatment and / or prevention of cancer, such as glioblastoma pleomorphoni. Furthermore, T cells expressing these CARs may also be suitable for adoptive immunotherapy in subjects with cancer.

[0049] The present invention relates to a novel EphA3-binding molecule having novel and / or improved properties compared to known anti-EphA3 antibodies. In one embodiment, the present invention provides a novel EphA3-binding molecule comprising at least one complementarity-determining region (CDR) having an amino acid sequence shown in any one of SEQ ID NOs. 13-72 and / or Tables 2-5 or an amino acid sequence that is at least 70% identical thereto.

[0050] EphA3 Ephrin type A receptor 3 (EphA3; also known as EPH receptor A3, EPH-like kinase 4, human embryo kinase, tyrosine-protein kinase TYRO4, and tyrosine-protein kinase receptor ETK1) includes all known and native EphA3 molecules, including the full-length EphA3 protein, as well as its fragments, variants, and derivatives. EphA3 includes, but is not limited to, mammalian EphA3, such as human EphA3 identified by UniProtKB accession number P29320 (as shown in SEQ ID NO: 165). In humans, EphA3 is encoded by the EPHA3 gene (also known as ETK, ETK1, HEK, and TYRO4). The function of EphA3 is described, for example, by Boyd et al., J Biol Chem, 267(5):3262-3267, which is incorporated herein by reference in its entirety. EphA3 is a 110 kDa single-pass type I transmembrane protein that functions as a receptor tyrosine kinase, binding in a chaotic manner to membrane-bound ephrin family ligands present on the surface of neighboring cells and initiating contact-dependent bidirectional signaling to adjacent cells.

[0051] The 20 amino acids at the N-terminus of SEQ ID NO: 165 constitute the signal peptide, and therefore the mature EphA3 (i.e., after processing to remove the signal peptide) has the amino acids shown in SEQ ID NO: 166. Positions 21-541 of SEQ ID NO: 165 form an extracellular domain (SEQ ID NO: 167), positions 542-565 form a transmembrane domain (SEQ ID NO: 168), and positions 566-983 form a cytoplasmic domain (SEQ ID NO: 169). The extracellular domain contains an Eph ligand-binding domain (shown in SEQ ID NO: 170, positions 29-207 in SEQ ID NO: 165) and two fibronectin type III domains (shown in SEQ ID NOs: 171 and 172, positions 325-435 and 436-531 in SEQ ID NO: 165, respectively). The cytoplasmic domain contains a protein kinase domain (shown in SEQ ID NO: 173, positions 621-882 in SEQ ID NO: 165). The cytoplasmic domain also contains a steril α motif (SAM) (as shown in SEQ ID NO: 174, at positions 911-975 in SEQ ID NO: 165).

[0052] EphA3 mature amino acid sequence: MDCQLSILLLLSCSVLDSFGELIPQPSNEVNLLDSKTIQGELGWYSYPSHGWEISGVDEHYTPIRTYQVCNVMDHSQNNWLRTNWVPRNSAQKIYVELKFTLRDCNSIPLVLGTCKETFNLYYMESDDDHGVKFREHQFTKIDTIAADESFTQMDLGDRILKLNTEIREVGPVNKKGFYLAFQDVGACVALVSVRVYFKKCPFTVKNLAMFPDTVPMDSQSLVEVRGSCVNNSKEEDPPRMYCS TEGEWLVPIGKCSCNAGYEERGFMCQACRPGFYKALDGNMKCAKCPPHSSTQEDGSMNCRCENNYFRADKDPPSMACTRPPSPRNVISNINETSVILDWSWPLDTGGRKDVTFNIICKKCGWNIKQCEPCSPNVRFLPRQFGLTNTTVTVTDLLAHTNYTFEIDAVNGVSELSSPPRQFAAVSITTNQAAPSPVLTIKKDRTSRNSISLSWQEPEHPNGIILDYEVKYEKQEQETSYTILRARG TNVTISSLKPDTIYVFQIRARTAAGYGTNSRKFEFETSPDSFSISGESSQVVMIAISAAVAIILLTVVIYVLIGRFCGYKSKHGADEKRLHFGNGHLKLPGLRTYVDPHTYEDPTQAVHEFAKELDATNISIDKVVGAGEFGEVCSGRLKLPSKKEISVAIKTLKVGYTEKQRRDFLGEASIMGQFDHPNIIRLEGVVTKSKPVMIVTEYMENGSLDSFLRKHDAQFTVIQLVGMLRGIASGMKYLSDMGYVHRDLAARNILINSNLVCKVSDFGLSRVLEDDPEAAYTTRGGKIPIRWTSPEAIAYRKFTSASDVWSYGIVLWEVMSYGERPYWEMSNQDVIKAVDEGYRLPPMDCPAALYQLMLDCWQKDRNNRPKFEQIVSILDKLIRNPGSLKIITSAAARPSNLLLDQSNVDITTFRTTGDWLNGVWTAHCKEIFTGVEYSSCDTIAKISTDDMKKVGVTVVGPQKKIISSIKALETQSKNGPVPV [Sequence ID 165]

[0053] In this specification, "EphA3" means EphA3 derived from any species, and includes isoforms, fragments, variants (including mutants), or homologs derived from any species of EphA3.

[0054] As used herein, a protein “fragment,” “variant,” or “homologous” may optionally be characterized as having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of a reference protein (e.g., a reference isoform). In some embodiments, a reference protein fragment, variant, isoform, or homologous may be characterized by its ability to perform the function exhibited by the reference protein.

[0055] A "fragment" generally refers to a segment, domain, part, or region of a reference protein that constitutes less than 100% of the amino acid sequence of the reference protein. A "variant" generally refers to a protein that has an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of the reference protein, but retains a fairly high degree of sequence identity (e.g., at least 60%) with respect to the amino acid sequence of the reference protein. An "isoform" generally refers to a variant of a reference protein expressed by the same species as the reference protein. A "homologous" generally refers to a variant of a reference protein produced by a different species compared to the species of the reference protein. Homologous compounds include orthologues.

[0056] The fragment may be of any length (by the number of amino acids), but may optionally be at least 20% of the length of the reference protein (i.e., the protein from which the fragment originates), and may have a maximum length of one of 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the length of the reference protein. The fragments of EphA3 can have a minimum length of one of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 550 amino acids or up to approximately 600 amino acids, and a maximum length of one of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450, 550 amino acids or up to approximately 600 amino acids.

[0057] In some embodiments, EphA3 is EphA3 derived from mammals (e.g., primate (rhesus macaque, cynomolgus macaque, non-human primate, or human) and / or rodent (e.g., rat or mouse) EphA3). An isoform, fragment, variant, or homolog of EPhA3 can be optionally characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of an immature or mature EphA3 isoform derived from a given species, e.g., human.

[0058] Isoforms, fragments, variants, or homologs may optionally be functional isoforms, fragments, variants, or homologs that possess the functional properties / activities of reference EphA3, for example, as determined by analysis using an assay suitable for their functional properties / activity. For example, isoforms, fragments, variants, or homologs of EphA3 may, for example, show association with EphA5 or retain kinase activity.

[0059] In some embodiments, EphA3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to sequence numbers 165 or 166. In some embodiments, a fragment of EphA3 comprises or consists of an amino acid sequence having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to one of sequence numbers 167, 170, 171, or a combination thereof.

[0060] EphA3 is a member of the ephrin receptor subfamily of the protein tyrosine kinase family and is known to be abnormally expressed in various human cancers, including malignant melanoma, glioblastoma, lung cancer, and breast cancer. Increased expression of EphA3 may promote tumor cell proliferation, angiogenesis, and invasion.

[0061] Target region on the target molecule The antigen-binding molecule of the present invention was specifically designed to target a particular region of EphA3. Following analyses of expected antigenicity, function, and safety, the EphA3 region to be targeted was selected using a two-step approach. Subsequently, antibodies specific to the target region of EphA3 were prepared using a peptide corresponding to the target region as an immunogen to produce a specific monoclonal antibody, and a subsequent screening to identify antibodies capable of binding to native EphA3. This approach provides control over the antibody epitope.

[0062] The antigen-binding molecules of the present invention can be defined by referring to the region of EphA3 to which they bind. The antigen-binding molecules of the present invention can bind to a specific region of interest of EphA3. In some embodiments, the antigen-binding molecules can bind to a linear epitope of EphA3 consisting of a continuous sequence of amino acids (i.e., the primary sequence of amino acids). In some embodiments, the antigen-binding molecules can bind to a structural epitope of EphA3 consisting of a discontinuous sequence of amino acids within the amino acid sequence.

[0063] In some embodiments, the antigen-binding molecule binds to EphA3. In some embodiments, the antigen-binding molecule binds to the extracellular domain of EphA3 (e.g., the region shown in SEQ ID NO: 167). In some embodiments, the antigen-binding molecule binds to the domain of the Eph ligand-binding domain (e.g., the region shown in SEQ ID NO: 170). In some embodiments, the antigen-binding molecule binds to one or both of the fibronectin type III domains (e.g., the regions shown in SEQ ID NOs: 171 and 172).

[0064] The region of the peptide / polypeptide to which the antibody binds can be determined by those skilled in the art using a variety of methods well known in the art, including X-ray crystallography, any analysis of antibody-antigen complexes, peptide scanning, mutagenesis mapping, hydrogen-deuterium exchange analysis by mass spectrometry, phage display, competitive ELISA, and "protection" methods based on proteolysis. Such methods are described, for example, by Gershoni et al., BioDrugs, 2007, 21(3):145-156, which is incorporated herein by reference in its entirety.

[0065] In some embodiments, the antigen-binding molecule can bind to the same EphA3 region to which an antibody containing the VH and VL sequences of one of the antibody clones 3C3-1 or 2D4-1 described herein binds, or to an EphA3 region that overlaps therewith.

[0066] As used herein, “isolated” means a material that has been removed from its natural state or otherwise manipulated artificially, such as an EphA3-binding molecule. An isolated material may substantially or essentially lack components normally associated with it in its natural state, or it may be manipulated to become artificially composed with components normally associated with it in its natural state. An isolated material may be in a recombinant, chemically synthesized, enhanced, purified, or partially purified form.

[0067] As used herein, “protein” refers to an amino acid polymer, where amino acids include D-amino acids, L-amino acids, and natural and / or unnatural amino acids. As typically used herein, “peptide” refers to a protein containing 50 or fewer consecutive amino acids. As typically used herein, “polypeptide” refers to a protein containing more than 50 consecutive amino acids. It should be understood that the term “protein” also includes, but is not limited to, protein-containing molecules such as glycoproteins and lipoproteins.

[0068] In some embodiments, the antigen-binding molecule of the present invention can be bound to a polypeptide comprising or consisting of one of the amino acid sequences of SEQ ID NOs: 165, 166, 167, 170, 171, or 172.

[0069] The ability of an antigen-binding molecule to bind to a given peptide / polypeptide can be analyzed by methods well known to those skilled in the art, including analysis by ELISA, immunoblotting (e.g., Western blotting), immunoprecipitation, surface plasmon resonance (SPR; see, e.g., Hearty et al., Methods Mol. Biol. (2012) 907:411-442), or biolayer interferometry (see, e.g., Lad et al., (2015) J. Biomol. Screen 20(4):498-507).

[0070] In embodiments in which an antigen-binding molecule can bind to a peptide or polypeptide containing a reference amino acid sequence, the peptide or polypeptide may contain one or more additional amino acids at one or both ends of the reference amino acid sequence. In some embodiments, the peptide / polypeptide may contain, for example, 1-5, 1-10, 1-20, 1-30, 1-40, 1-50, 5-10, 5-20, 5-30, 5-40, 5-50, 10-20, 10-30, 10-40, 10-50, 20-30, 20-40, or 20-50 additional amino acids at one or both ends of the reference amino acid sequence.

[0071] In some embodiments, in the context of the amino acid sequence of EphA3, the additional amino acids (or more) added to one or both ends (i.e., the N-terminus and C-terminus) of the reference sequence correspond to positions at the ends of the reference sequence. For example, if an antigen-binding molecule can bind to the sequence of SEQ ID NO: 3 and a peptide or polypeptide containing two additional amino acids at the C-terminus of SEQ ID NO: 3, then both of the additional amino acids could be valine, corresponding to positions 542 and 543 of SEQ ID NO: 165.

[0072] In some embodiments, the antigen-binding molecule can bind to a peptide / polypeptide to which an antibody containing one of the VH and VL sequences from antibody clone 3C3-1 or 2D4-1 described herein is bound.

[0073] antigen binding molecule This invention provides an antigen-binding molecule capable of binding to EphA3.

[0074] An "antigen-binding molecule" refers to a molecule capable of binding to a target antigen, and includes monoclonal antibodies, polyclonal antibodies, monospecific antibodies, and multispecific antibodies (e.g., bispecific antibodies), as well as antibody fragments, insofar as they exhibit binding to the corresponding target molecule.

[0075] In certain embodiments, the EphA3-binding molecules described herein are antibodies or antibody fragments. As used herein, “antibody” is an immunoglobulin protein or contains an immunoglobulin protein. The term “immunoglobulin” includes any antigen-binding protein products of the mammalian immunoglobulin gene complex, including immunoglobulin isotypes IgA, IgD, IgM, IgG, and IgE, and their antigen-binding fragments. The term “immunoglobulin” includes immunoglobulins that are recombinant, chimeric, or humanized, or otherwise modified or variant amino acid residues, sequences, and / or glycosylated, whether naturally occurring or produced by artificial intervention (e.g., by recombinant DNA technology).

[0076] Generally, antibodies and antibody fragments can be polyclonal or monoclonal. In certain embodiments, the antibody or antibody fragment is one of the monoclonal antibodies (or a fragment thereof) shown in Figure 1, for example, a 3C3-1 or 2D4-1 monoclonal antibody, or a fragment thereof.

[0077] The present invention also includes, within its scope, antibody fragments of polyclonal or monoclonal antibodies described herein, such as Fv, Fc, Fab, or F(ab')2 fragments. Alternatively, the EphA3 conjugates of the present invention may include single-chain Fv(scFv) and / or scFab antibodies. Such scFv can be prepared according to the methods described, for example, in U.S. Patent No. 5,091,513, European Patent No. 239,400, or in the paper by Winter and Milstein, 1991, Nature 349:293, which are incorporated herein by reference. The present invention also envisions including polyvalent recombinant antibody fragments containing multiple scFv, so-called diabodies, triabodies, and / or tetrabodies, as well as dimerizable activated demibodies (e.g., International Publication No. 2007 / 062466). For example, such antibodies can be prepared according to the methods described in Holliger et al., 1993 Proc Natl Acad Sci USA 90:6444~6448; or Kipriyanov, 2009 Methods Mol Biol 562:177~93, which are incorporated herein by reference in their entirety.

[0078] Furthermore, it is understood that antibodies can be produced as recombinant synthetic antibodies or antibody fragments by expressing nucleic acids encoding antibodies or antibody fragments in appropriate host cells. Non-limiting examples of methods for the expression and selection of recombinant antibodies are presented in Chapter 17 of CURRENT PROTOCOLS IN IMMUNOLOGY by Coligan et al., and in Zuberbuhler et al., 2009, Protein Engineering, Design & Selection 22 169.

[0079] Typically, antibodies have their respective light chain variable regions (V) which contain complementarity-determining regions (CDRs) 1, 2, and 3 amino acid sequences. L or VL) and heavy chain variable region (V H (or VH), and each light chain steady region (C L) and the heavy chain constant region (CH1, CH2, CH3) are included. Therefore, antibodies generally contain six CDRs (three in the heavy chain variable region and three in the light chain variable region). These six CDRs together define the antibody paratope, which is the part of the antibody that binds to the target antigen.

[0080] The antigen-binding molecule of the present invention can be designed and prepared using a monoclonal antibody (mAb) sequence capable of binding to EphA3. Alternatively, the antigen-binding region of an antibody, such as a single-chain variable fragment (scFv), Fab, and F(ab')2 fragment, can also be used / prepared. The "antigen-binding region" is any fragment of an antibody capable of binding to a target to which a given antibody is specific.

[0081] V H Region and V L Each region includes a framework region (FR) on both sides of the CDR, which serves as a scaffold for the CDR. From the N-terminus to the C-terminus, V H The region contains the following structure: N-terminus-[HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C-terminus, and V L The region contains the following structure: N-terminus-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]-[LC-FR4]-C-terminus.

[0082] CDR identification and numbering can be carried out according to any known CDR numbering scheme, including those by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)), Chothia (Chothia et al., J. Mol. Biol. 196:901~917 (1987)), AbM, and Contact.

[0083] In some embodiments, the antigen-binding molecule comprises the CDRs of an antigen-binding molecule capable of binding to EphA3. In some embodiments, the antigen-binding molecule comprises the FRs of an antigen-binding molecule capable of binding to EphA3. In some embodiments, the antigen-binding molecule comprises the CDRs and FRs of an antigen-binding molecule capable of binding to EphA3. That is, in some embodiments, the antigen-binding molecule is the V H region and V L region of an antigen-binding molecule capable of binding to EphA3.

[0084] In some embodiments, the antigen-binding molecule is the VH / VL region of an EphA3-binding antibody (i.e., anti-EphA3 antibody clone 3C3-1 or 2D4-1) as described in detail herein, or a V H region and V L region derived therefrom.

[0085] Non-limiting examples of CDR amino acid sequences are shown in SEQ ID NOs: 13-72 and / or Tables 2-5. Identification and numbering of the CDRs were performed using abYsis version 3.4.1 and IMGT / V-QUEST. Antibodies according to the invention may comprise one, two or three V L CDR amino acid sequences (e.g., CDR1, CDR2 and / or CDR3) and / or one, two, or three V H CDR amino acid sequences (e.g., CDR1, CDR2 and / or CDR3), such as those shown in SEQ ID NOs: 13-72 and / or Tables 2-5.

[0086] In some embodiments, the EphA3 binder is (a) a heavy chain immunoglobulin variable region (VH) polypeptide comprising a CDR1 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 13-17, a CDR2 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 18-22, and a CDR3 having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 23-27, and / or (b) A light chain immunoglobulin variable region (VL) polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 28-32, CDR2 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 33-37, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 38-42. Includes.

[0087] In such embodiments, the VH polypeptide preferably comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and / or the VL polypeptide preferably comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0088] In an alternative embodiment, the EphA3 binder is (a) A VH polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 43-47, CDR2 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 48-52, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 53-57, and / or (b) A VL polypeptide comprising CDR1 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 58-62, CDR2 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 63-67, and CDR3 having an amino acid sequence at least 70% identical to any one of SEQ ID NOs. 68-72. Includes.

[0089] In this regard, the VH polypeptide may contain the amino acid sequence shown in SEQ ID NO: 155 or an amino acid sequence that is at least 70% identical thereto, and / or the VL polypeptide may contain the amino acid sequence shown in SEQ ID NO: 156 or an amino acid sequence that is at least 70% identical thereto.

[0090] The CDR and FR of the VH and VL regions of the antibodies described in detail herein are defined below according to the International IMGT (ImMunoGeneTics) Information System (LeFranc et al., Nucleic Acids Res., (2015) 43 (Database issue): D413~22), as described in LeFranc et al., Dev.Comp.Immunol. (2003) 27:55~77. In some embodiments, the antigen-binding molecule includes a VH region according to (1) or (2) below: (1)(3C3-1)The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 16, HC-CDR2 having the amino acid sequence of SEQ ID NO: 22, HC-CDR3 having the amino acid sequence of SEQ ID NO: 27, Or a variant in which one or more of HC-CDR2, HC-CDR2, or HC-CDR3 have one, two, or three amino acids substituted with other amino acids. A VH region incorporating this. (2)(2D4-1)The following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 47, HC-CDR2 having the amino acid sequence of SEQ ID NO: 52, HC-CDR3 having the amino acid sequence of SEQ ID NO: 57, Or a variant in which one or more of HC-CDR2, HC-CDR2, or HC-CDR3 have one, two, or three amino acids substituted with other amino acids. A VH region incorporating this.

[0091] In some embodiments, the antigen-binding molecule includes a VH region according to (3) or (4) below: (3)(3C3-1)FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 97, HC-FR2 having the amino acid sequence of SEQ ID NO: 102, HC-FR3 having the amino acid sequence of SEQ ID NO: 107, HC-FR4 having the amino acid sequence of SEQ ID NO: 112, Or a variant in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have one, two, or three amino acids substituted with other amino acids. A VH region incorporating this. (4)(2D4-1)FR below: HC-FR1 having the amino acid sequence of SEQ ID NO: 137, HC-FR2 having the amino acid sequence of SEQ ID NO: 142, HC-FR3 having the amino acid sequence of SEQ ID NO: 147, HC-FR4 having the amino acid sequence of SEQ ID NO: 152, Or a variant in which one or more of HC-FR1, HC-FR2, HC-FR3, or HC-FR4 have one, two, or three amino acids substituted with other amino acids. A VH region incorporating this.

[0092] In some embodiments, the antigen-binding molecule includes a VH region comprising a CDR according to one of (1) and (2) above, and an FR according to (3) or (4) above.

[0093] In some embodiments, the antigen-binding molecule includes a VH region by one of the following (5) or (6): (5) The VH region including the CDR according to (1) and the FR according to (3). (6) A VH region including CDR by (2) and FR by (4).

[0094] In some embodiments, the antigen-binding molecule includes a VL region according to (7) or (8) below: (7)(3C3-1) The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 32, LC-CDR2 having the amino acid sequence of SEQ ID NO: 37, LC-CDR3 having the amino acid sequence of SEQ ID NO: 42, Or a variant in which one or more of LC-CDR2, LC-CDR2, or LC-CDR3 have one, two, or three amino acids substituted with other amino acids. A VL domain incorporating this. (8)(2D4-1)The following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 62, LC-CDR2 having the amino acid sequence of SEQ ID NO: 67, LC-CDR3 having the amino acid sequence of SEQ ID NO: 72, Or a variant in which one or more of LC-CDR2, LC-CDR2, or LC-CDR3 have one, two, or three amino acids substituted with other amino acids. A VL domain incorporating this.

[0095] In some embodiments, the antigen-binding molecule includes a VL region according to (9) or (10) below: (9)(3C3-1)FR below: LC-FR1 having the amino acid sequence of SEQ ID NO: 97, LC-FR2 having the amino acid sequence of SEQ ID NO: 102, LC-FR3 having the amino acid sequence of SEQ ID NO: 107, LC-FR4 having the amino acid sequence of SEQ ID NO: 112, Or a variant in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one, two, or three amino acids substituted with other amino acids. A VL domain incorporating this. (10)(2D4-1) and below FR: LC-FR1 having the amino acid sequence of SEQ ID NO: 137, LC-FR2 having the amino acid sequence of SEQ ID NO: 142, LC-FR3 having the amino acid sequence of SEQ ID NO: 147, LC-FR4 having the amino acid sequence of SEQ ID NO: 152, Or a variant in which one or more of LC-FR1, LC-FR2, LC-FR3, or LC-FR4 have one, two, or three amino acids substituted with other amino acids. A VL domain incorporating this.

[0096] In some embodiments, the antigen-binding molecule includes a VL region comprising a CDR according to one of (1) and (2) above, and an FR according to (3) or (4) above.

[0097] In some embodiments, the antigen-binding molecule includes a VH region by one of the following (11) or (12): (11) A VH region including CDR by (7) and FR by (9). (12) A VH region including CDR by (8) and FR by (10).

[0098] The VH and VL regions of the antigen-binding domain of an antibody together constitute the Fv region. In some embodiments, the antigen-binding molecule according to the present invention includes or consists of an Fv region that binds to EphA3. In some embodiments, the VH and VL regions of Fv are provided as a single polypeptide, i.e., a single-chain Fv (scFv), joined by a linker region.

[0099] In some embodiments, the present invention provides isolated antibodies and CAR fragments of the present invention.

[0100] The fragments of the present invention can be prepared by the methods described herein. Alternatively, the fragments can be prepared by digestion of antibodies or CAR proteins using proteinases, such as endoLys-C, endoArg-C, endoGlu-C, and V8-protease. The digested fragments can be purified by chromatographic techniques well known in the art.

[0101] A particular embodiment of the present invention provides an immunogenic fragment of the EphA3 antigen-binding molecule of the present invention. "Immunogenic" means that it can induce an immune response after administration to animals such as humans, mice, or rabbits. The immune response may include, but is not limited to, the production, activation, or stimulation of the innate and / or adaptive parts of the immune system, including immune cells such as B lymphocytes and / or T lymphocytes, NK cells, granulocytes, macrophages, and dendritic cells, and / or molecules such as antibodies, cytokines, and chemokines.

[0102] Antibody fragments include, but are not limited to, Fab and Fab'2 fragments, diabodies, triabodies, bispecific antibodies, and single-chain antibody fragments (e.g., scFv). In some embodiments, the antibody fragment is the amino acid sequence of CDR1, CDR2 and / or CDR3, as shown in, for example, SEQ ID NOs. 13-72, or V, as shown in, for example, SEQ ID NOs. 153-156. H and / or V L The antibody fragment may contain at least a portion of the amino acid sequence. A preferred antibody fragment contains the entirety of at least one light chain variable region (CDR) and / or the entirety of at least one heavy chain variable region (CDR).

[0103] In some embodiments, the EphA3 conjugates provided herein are recombinant, human, or humanized antibodies, or antibody fragments. As used herein, “humanized” antibodies may include antibodies of fully or at least partially human origin, including modified antibodies or antibody fragments obtained from non-human “foreign” species. In some embodiments, antibodies and antibody fragments may be modified to be administerable to one species that originated in or is derived from the same or another “foreign” species without inducing an adverse immune response to the “foreign” antibody. Complementarity-determining region (CDR) or variable region (i.e., V H and V L Human antibodies or non-human antibody fragments containing domains can be "grafted" onto a human antibody scaffold or skeleton to produce "humanized" antibodies or antibody fragments. In some embodiments, human or non-human CDRs or V L and V L The domain can be recombinantly grafted with the constant region of a human antibody.

[0104] In some embodiments, the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin heavy chain constant sequence. In some embodiments, the immunoglobulin heavy chain constant sequence is or is derived from an IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM heavy chain constant sequence.

[0105] In some embodiments, the immunoglobulin heavy chain constant region sequence is the human immunoglobulin G1 constant region sequence (IGHG1: UniProt accession number P01857, v1, SEQ ID NO. 175). Positions 1-98 of SEQ ID NO. 175 form the CH1 region (SEQ ID NO. 176). Positions 99-110 of SEQ ID NO. 175 form the hinge region between the CH1 and CH2 regions (SEQ ID NO. 177). Positions 111-223 of SEQ ID NO. 175 form the CH2 region (SEQ ID NO. 178). Positions 222-330 of SEQ ID NO. 175 form the CH3 region (SEQ ID NO. 179).

[0106] Immunoglobulin heavy chain constant region gamma 1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[Sequence ID 175]

[0107] In some embodiments, the CH1 region includes or consists of the sequence of SEQ ID NO: 176, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 176.

[0108] In some embodiments, the antigen-binding molecule of the present invention comprises one or more regions of an immunoglobulin light chain constant region sequence. In some embodiments, the immunoglobulin light chain constant region sequence is a human immunoglobulin lambda constant region sequence (IGLA;CA), for example, IGLC1, IGLC2, IGLC3, IGLC6, or IGLC7. In some embodiments, the CL region comprises or consists of the sequence of SEQ ID NO: 180, or a sequence having at least 60%, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 180.

[0109] Immunoglobulin lambda constant region MRPGTGQGGLEAPGEPGPNLRQRWPLLLLGLAVVTHGLLRPTAASQSRALGPGAPGGSSRSSLRSRWGRFLLQRGSWTGPRCWPRGFQSKHNSVTHVFGSGTQLTVLSQPKATPSVTLFPPSSEELQANKATLVCLMNDFYPGILTVTWKADGTPITQGVEMTTPSKQSNNKYAASSYLSLTPEQWRSRRSYSCQVMHEGSTVEKTVAPAECS [Sequence ID 180]

[0110] Immunoglobulin kappa constant region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [Sequence ID 212]

[0111] The VL region and light chain constant (CL) region of the antibody's antigen-binding region, as well as the VH region and heavy chain constant region 1 (CH1), together constitute the Fab region. In some embodiments, the antigen-binding molecule consists of VH, CH1, VL, and CL (e.g., C κ or C λ The Fab region includes a Fab region containing VH and CH1 (e.g., a VH-CH1 fusion polypeptide). In some embodiments, the Fab region includes a polypeptide containing VH and CL (e.g., a VH-CL fusion polypeptide). In some embodiments, the Fab region includes a polypeptide containing VH and CL (e.g., a VH-CL fusion polypeptide), as well as a polypeptide containing VL and CH (e.g., a CL-CH1 fusion polypeptide), i.e., in some embodiments, the Fab region is a CrossFab region. In some embodiments, the VH, CH1, VL, and CL regions of the Fab or CrossFab are provided as a single polypeptide joined by a linker region, i.e., as a single-chain Fab (scFab) or single-chain CrossFab (scCrossFab).

[0112] In some embodiments, the antigen-binding molecule of the present invention includes, consists of, or is essentially composed of a Fab region that binds to EphA3.

[0113] In some embodiments, the antigen-binding molecules described herein include or consist of a whole antibody that binds to EphA3. As used herein, “whole antibody” means an antibody having a structure substantially similar to that of immunoglobulin (Ig). Various types of immunoglobulins and their structures are described, for example, in Schroeder and Cavacini, J Allergy Clin Immunol (2010) 125(202): S41-S52, which is incorporated herein by reference in its entirety.

[0114] G-type immunoglobulins (i.e., IgG) are glycoproteins of approximately 150 kDa, comprising two heavy chains and two light chains. From the N-terminus to the C-terminus, the heavy chain contains a constant region (CH1, CH3, and CH3) with three constant domains following a VH, and similarly the light chain contains a CL following a VL. Depending on the heavy chain, immunoglobulins can be classified as IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM. The light chain can be kappa (κ) or lambda (λ).

[0115] In some embodiments, the antigen-binding molecules described herein include, consist of, or are essentially composed of, IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgA (e.g., IgA1, IgA2), IgD, IgE, or IgM that bind to EphA3.

[0116] Preferably, the EphA3 conjugate binds to an epitope of the EphA3 protein. As commonly used herein, “epitope” is an antigenic protein fragment comprising a continuous or discontinuous sequence of amino acids of a protein, where the epitope is recognized or bound by an element of the immune system, such as an antibody or other antigen receptor.

[0117] The present invention also includes variants of the EphA3 conjugates disclosed herein. In one embodiment, the variant is an EphA3 conjugate having an amino acid sequence that is at least 70% identical to any one of SEQ ID NOs: 13-72, which is referred to herein as the CDR "variant". In another embodiment, the variant has an amino acid sequence that is at least 70% identical to the VH and / or VL amino acid sequence of any one of SEQ ID NOs: 153-156.

[0118] Preferably, the EphA3 binder comprises at least one or more variants of a CDR capable of binding to the EphA3 protein.

[0119] In certain embodiments, the variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with respect to the amino acid sequence of a reference protein (e.g., a reference isoform), for example, one of the sequence numbers 13-156. The protein "variants" disclosed herein may have one or more amino acids deleted, inserted, or substituted with different amino acids. It is well understood in the art that some amino acids can be substituted or deleted without altering the biological activity of the peptide (conservative substitution). In some embodiments, reference protein fragments, variants, isoforms, and homologs can be characterized by their ability to perform the functions exhibited by the reference protein.

[0120] Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced by another amino acid having the same or similar chemical or physical properties. For example, conservative amino acid substitutions may include substitution of an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), substitution of an amino acid with a nonpolar side chain with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Cys, Val, etc.), substitution of a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), substitution of an uncharged amino acid with a polar side chain with another uncharged amino acid having a polar side chain (e.g., Asn, Gin, Ser, Thr, Tyr, etc.), substitution of an amino acid with a β-branched side chain with another amino acid having a β-branched side chain (e.g., Ile, Thr, and Val), and substitution of an amino acid with an aromatic side chain with another amino acid having an aromatic side chain (e.g., His, Phe, Trp, and Tyr).

[0121] Terms commonly used herein to describe sequence relationships between individual proteins and nucleic acids include “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” Because each nucleic acid / protein contains (1) only one or more portions of the complete nucleic acid / protein sequence shared by the nucleic acid / protein, and (2) one or more portions that differ between the nucleic acids / proteins, sequence comparison is typically performed by comparing sequences across a “comparison window” to identify and compare regions of local sequence similarity. A “comparison window” refers to a conceptual segment, typically at least six, nine, or twelve consecutive residues, that is compared to a reference sequence. For optimal alignment of each sequence, the comparison window may contain approximately 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence. The optimal sequence alignment for performing comparison window alignment can be performed by computer execution of an algorithm (GAP, BESTFIT, FASTA, and TFASTA in Intelligenetics' Geneworks program, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference), or by examination by any of the various selected methods and the best alignment produced by any of them (i.e., the one that yields the highest homology percentage across the comparison window). For example, one can refer to a BLAST family program, such as the one disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389, incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, edited by Ausubel et al. (John Wiley & Sons Inc NY, 1995-2015).

[0122] The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches, taking into account proper alignment using standard algorithms and the extent to which sequences are identical across a comparison window. Therefore, the “percentage of sequence identity” is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, I) exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. For example, “sequence identity” would be understood to mean the “percentage of matches” calculated by the DNASIS computer program (version 2.5 for Windows, sold by Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA).

[0123] Derivatives of antibodies, antibody fragments, or variants thereof disclosed herein are also provided.

[0124] As used herein, “derivative” antibodies, antibody fragments, or variants thereof are modified, for example, by conjugation or complexation with other chemical moieties, by post-translational modifications (e.g., phosphorylation, ubiquitination, glycosylation), by chemical modifications (e.g., crosslinking, acetylation, biotinylation, oxidation or reduction, etc.), by conjugation with labels (e.g., fluorophores, enzymes, radioisotopes), and / or by including additional amino acid sequences as will be understood in the art.

[0125] In this regard, for more detailed methods concerning the chemical modification of proteins, those skilled in the art should refer to Chapter 15 of *CURRENT PROTOCOLS IN PROTEIN SCIENCE*, edited by Coligan et al. (John Wiley & Sons NY 1995-2015).

[0126] Additional amino acid sequences may include fusion partner amino acid sequences that create the fusion protein. For example, fusion partner amino acid sequences can aid in the detection and / or purification of isolated fusion proteins. Non-limiting examples include metal-binding (e.g., polyhistidine) fusion partners, maltose-binding proteins (MBPs), protein A, glutathione S-transferase (GST), fluorescent protein sequences (e.g., GFP, RFP), epitope tags (e.g., myc, FLAG, and hemagglutinin tags).

[0127] The isolated proteins (e.g., EphA3 antibody, antibody fragments, and CARs), variants, fragments, and / or derivatives of the present invention can be prepared by any means known in the art, including but not limited to chemical synthesis, recombinant DNA technology, and proteolytic cleavage for generating peptide fragments.

[0128] Chemical synthesis includes solid-phase and liquid-phase synthesis. While such methods are well known in the art, examples of chemical synthesis provided in Chapter 9 of *SYNTHETIC VACCINES*, edited by Nicholson (Blackwell Scientific Publications), and Chapter 15 of *CURRENT PROTOCOLS IN PROTEIN SCIENCE*, edited by Coligan et al. (John Wiley & Sons, Inc. NY USA 1995-2008) are also referred to. In this regard, see also International Publications 99 / 02550 and 97 / 45444.

[0129] In one preferred embodiment, the EphA3 antibody, antibody fragment, and / or CAR protein of the present invention are recombinant proteins.

[0130] Recombinant proteins can be conveniently prepared by those skilled in the art using standard protocols such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 1989), particularly sections 16 and 17; Current Protocols in Molecular Biology, edited by Ausubel et al. (John Wiley & Sons, Inc., NY USA 1995-2008), particularly chapters 10 and 16; and Current Protocols in Protein Science, edited by Coligan et al. (John Wiley & Sons, Inc., NY USA 1995-2008), particularly chapters 1, 5 and 6.

[0131] Chimeric antigen receptor (CAR) The present invention also provides a chimeric antigen receptor (CAR) comprising the antigen-binding molecule or polypeptide of the present invention.

[0132] Accordingly, a relevant aspect of the present invention provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain including at least one CDR having an amino acid sequence shown in SEQ ID NOs. 13 to 72 or an amino acid sequence that is at least 70% identical thereto, a transmembrane domain, and an intracellular T cell signaling domain.

[0133] CARs are artificially constructed hybrid proteins or polypeptides containing an antigen-binding domain (e.g., a single-chain variable fragment (scFv)) of an antibody linked to a T cell signaling domain. A key feature of CARs is their ability to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner by utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigens independently of antigen processing and thus bypass the main mechanisms of tumor evasion. Furthermore, when expressed on T cells, CARs advantageously do not dimerize with the endogenous T cell receptor (TCR) α and β chains. The structure and manipulation of CARs are reviewed, for example, in Dotti et al., Immunol Rev (2014) 257(1), the entire text of which is incorporated herein by reference. CARs include a cell membrane anchoring region (also known as a transmembrane domain) and an antigen-binding region linked to a signaling region. An optional hinge region can result in the separation of the antigen-binding region from the cell membrane anchoring region and can act as a mobile linker. The CAR of the present invention includes an antigen-binding region comprising, consisting of, or essentially comprising the polypeptide according to the present invention.

[0134] The cell membrane anchoring region is located between the antigen-binding region and the signaling region of the CAR, resulting in anchoring of the CAR to the cell membrane of the CAR-expressing cell, such that the antigen-binding region is in the extracellular lumen and the signaling region is intracellular. In some embodiments, the CAR contains or is derived from one of the transmembrane amino acid sequences of CD3-ζ, CD4, CD8, or CD28. Preferably, the transmembrane domain is derived from a membrane protein selected from CD8α, CD8β, 4-1BB / CD137, CD28, CD34, CD4, FcεRIγ, CD16, OX40 / CD134, CD3-ζ, CD3ε, CD3γ, CD3δ, TCRα, CD32, CD64, VEGFR2, FAS, FGFR2B, and any combination thereof. In some specific embodiments, the transmembrane domain may be derived from the CD8 and / or CD28 transmembrane domain, which generally results in superior receptor stability. As used herein, a region “derived” from a reference amino acid sequence includes an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the reference sequence. In some embodiments, the transmembrane domain includes the amino acid sequence shown in SEQ ID NO: 159 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to it.

[0135] The transmembrane domain (i.e., cell membrane anchoring region) of the chimeric receptor described herein may be any form known in the art. As used herein, “transmembrane domain” refers to any protein structure that is thermodynamically stable in the cell membrane, preferably the eukaryotic cell membrane. Transmembrane domains suitable for use in the chimeric receptor as used herein can be obtained from naturally occurring proteins. Alternatively, the transmembrane domain may be a synthetic, non-naturally occurring protein segment (e.g., a hydrophobic protein segment that is thermodynamically stable in the cell membrane). See, for example, U.S. Patent No. 7,052,906 and International Application PCT Publication No. 2000 / 032776, incorporated herein by reference. For this purpose, the transmembrane domain may include a hydrophobic α-helix.

[0136] Any intracellular or cytoplasmic T cell signaling domain (e.g., CD3-ζ or FcεR1γ), for example, one containing an immunoreceptor tyrosine-based activation motif (ITAM) for phosphorylation and activation of CAR-expressing T cells, can be used to construct the chimeric receptor described herein. “ITAM” as used herein refers to a conserved protein motif commonly present in the tail of signaling molecules expressed on many immune cells. After antigen recognition, the receptors cluster and the signal is transmitted to the cell. The most commonly used T cell signaling component is CD3-ζ containing three ITAMs. This transmits an activation signal to the T cell after antigen binding. However, a fully competent activation signal may not be obtained from the CD3-ζ cytoplasmic signaling domain alone, and it will be understood that additional co-stimulatory signaling domains, e.g., those described below herein, may be utilized. For example, chimeric CD28 and / or 4-1BB / CD137 can be used with CD3-ζ to transmit proliferation / survival signals, or all three can be used together. Therefore, the endodomain of the CAR of the present invention may include a CD28 costimulatory domain (e.g., SEQ ID NO: 161), a 4-1BB / CD137 costimulatory domain (e.g., SEQ ID NO: 160), and a CD3-ζ intracellular signaling domain (e.g., SEQ ID NO: 162).

[0137] Furthermore, the signaling domain of the CAR may include a co-stimulatory sequence derived from the signaling domain of a co-stimulatory molecule to facilitate the activation of T cells expressing the CAR upon binding to a target protein. Activation of a co-stimulatory signaling domain in host cells (e.g., immune cells) can induce cells to increase or decrease cytokine production and secretion, phagocytic properties, proliferation, differentiation, survival, and / or cytotoxicity. Any co-stimulatory signaling domain of a co-stimulatory molecule may be suitable for use in the chimeric receptor described herein. The type(s) of the co-stimulatory signaling domain is selected based on factors such as the type of immune cell expressing the chimeric receptor (e.g., T cells, NK cells, macrophages, neutrophils, or eosinophils) and the desired immune effector function (e.g., ADCC effect). In other words, the term “co-stimulatory signaling domain,” as used herein, refers to at least a portion of a protein that mediates signaling within a cell to induce an immune response, such as effector function. The chimeric receptor costimulatory signaling domains described herein may be costimulatory protein-derived cytoplasmic signaling domains that transmit signals to modulate responses mediated by immune cells such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

[0138] Examples of co-stimulatory signaling domains for use in chimeric receptors may be cytoplasmic signaling domains of co-stimulatory proteins, and these may include, but are not limited to, members of the B7 / CD28 family (e.g., B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, and PDCD6), members of the TNF superfamily (e.g., 4-1BB / TNFSF9 / CD137, 4-1BB ligand / TNFSF9, BAFF / BLy S / TNFSF13B, BAFF-R / TNFRSF13C, CD27 / TNFRSF7, CD27 ligand / TNFSF7, CD30 / TNFRSF8, CD30 ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF1 8, GITR ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-alpha / TNF-β, OX40 / TNFRSF4, 0X40 ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNF, and TNFRII / TNFRSF1B), members of the SLAM family (e.g., 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, and SLAM / CD150), and any other co-stimulatory molecules, e.g., CD2, CD7, CD53, CD82 / The domains include Kai-1, CD90 / Thy1, CD96, CD160, CD200, CD300a / LMIR1, HLA class I, HLA-DR, Ikaros, integrin α4 / CD49d, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-1 / KIM-1 / HAVCR, TIM-4, TSLP, TSLP R, lymphocyte function-associated antigen-1 (LFA-1), and NKG2C. In some embodiments, the co-stimulatory signaling domain is 4-1BB, CD28, OX40, ICOS, CD27, GITR, HVEM, TIM1, LFA1 (CD11a) or CD2, or any variant thereof. In some embodiments, the co-stimulus signaling domains are derived from 4-1BB (e.g., SEQ ID NO: 160) and / or CD28 (e.g., SEQ ID NO: 161).

[0139] Furthermore, any variant of the co-stimulatory signaling domains described herein that can modulate the immune response of immune cells is also included in the scope of this disclosure. It is also envisioned that a chimeric receptor may contain multiple co-stimulatory signaling domains (e.g., two, three, four, or more). In some embodiments, a chimeric receptor contains two or more identical co-stimulatory signaling domains, e.g., two copies of the CD28 co-stimulatory signaling domain. In some embodiments, a chimeric receptor contains two or more co-stimulatory signaling domains derived from different co-stimulatory proteins, e.g., any two or more of the co-stimulatory proteins described herein. In some cases, a CAR is engineered to result in co-stimulation of different intracellular signaling pathways. For example, signaling associated with CD28 co-stimulation preferentially activates the phosphatidylinositol 3-kinase (P13K) pathway, while 4-1BB-mediated signaling circulates via the TNG receptor-associated factor (TRAF) adapter protein. Therefore, the signaling region of a CAR may sometimes contain co-stimulatory sequences derived from the signaling regions of multiple co-stimulatory molecules. In some embodiments, the CAR of the present invention comprises one or more costimulatory sequences comprising, consisting of, or derived from amino acid sequences of one or more intracellular domains among CD28, OX30, 4-1BB, ICOS, and CD27.

[0140] An optional hinge region can result in the separation of the antigen-binding domain from the transmembrane domain and can act as a mobile linker. The hinge region may be derived from IgG1. In some embodiments, the CAR of the present invention includes, comprises, or comprises an amino acid sequence of the hinge region of IgG1, or an amino acid sequence derived therefrom.

[0141] The CARs of the present invention may be considered, for example, first-generation, second-generation, third-generation, or fourth-generation CARs (i.e., related to T cells redirected for universal cytokine-mediated death (TRUCK)) that are known in the art. First-generation CARs typically involve ligating an antibody-derived scFv to the CD3-zeta (ζ or z) intracellular signaling domain of the T cell receptor via a hinge and transmembrane domain. Second-generation CARs incorporate additional domains (e.g., CD28, 4-1BB, or ICOS) to supply a co-stimulatory signal. Third-generation CARs typically contain two co-stimulatory domains fused to the TCR CD3-ζ chain. Third-generation co-stimulatory domains may include, for example, a combination of CD3-ζ, CD27, CD28, 4-1BB, ICOS, DAP-10, or 0X40. Therefore, the CAR of the present invention may contain an ectodomain, a hinge, a transmembrane domain, and an endodomain having one (first generation), two (second generation), or three (third generation) signaling domains derived from CD3-ζ and / or a co-stimulatory molecule.

[0142] In some embodiments, CARs are associated with T cells redirected for cytokine activity (e.g., TRUCK), also known as fourth-generation CARs. TRUCK is a CAR-redirected T cell used to induce effector activity in CAR T cells, and in addition, as a medium for producing and releasing transgenic cytokines (e.g., IL-12) that accumulate in targeted tissues (e.g., tumor tissue expressing EphA3). Transgenic cytokines are constitutively produced or released when CARs bind to a target. TRUCK cells can deliver various therapeutic cytokines to the target site. This can create therapeutic concentrations at the target site while avoiding the systemic toxicity of these same cytokines.

[0143] The CARs of the present invention preferably have antigen specificity for EphA3. The phrases “having antigen specificity” and “inducing an antigen-specific response,” as used herein, mean that the CAR can specifically bind to an antigen and immunologically recognize the antigen so that the binding of the CAR to the antigen induces an immune response. While not bound by any particular theory or mechanism, by inducing an antigen-specific response to EphA3, the CARs described herein are thought to provide one or more of the following: targeting and destruction of cancer cells expressing EphA3; reduction or elimination of cancer cells; promotion of immune cell infiltration into tumor sites; and enhancement / expansion of anti-cancer responses.

[0144] One embodiment of the present invention provides a CAR comprising an antigen-binding domain of one of the monoclonal antibodies described herein, for example, the one shown in Figure 1. In certain embodiments, the CAR comprises an antigen-binding domain of a 3C3 or 2D4 monoclonal antibody that specifically binds to EphA3. In this regard, preferred embodiments of the present invention provide a CAR comprising an antigen-binding domain comprising, consisting of, or essentially a single-chain variable fragment (scFv) of a 3C3 or 2D4 antigen-binding domain.

[0145] The antigen-binding domain may include a light chain variable region and / or a heavy chain variable region. In one embodiment of the present invention, the heavy chain variable region includes a CDR1 region, a CDR2 region, and a CDR3 region. In this regard, the antigen-binding domain includes a heavy chain CDR1 region containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 13-17, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92% identical to any one of SEQ ID NOs: 18-22. It may include a heavy chain CDR2 region containing an amino acid sequence that is 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and one or more heavy chain CDR3 regions containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs. In an alternative embodiment, the antigen-binding domain is a heavy chain CDR1 region containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs. 48–52 or at least 70%, 75%, 80%, 85%, 90%, 91%, It includes a heavy chain CDR2 region containing an amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and one or more heavy chain CDR3 regions containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of sequence numbers 53 to 57. Preferably, the heavy chain includes all of the CDR1, CDR2, and CDR3 regions selected from amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs.

[0146] In one embodiment of the present invention, the light chain variable region includes a light chain CDR1 region, a light chain CDR2 region, and a light chain CDR3 region. In this regard, the antigen-binding domain includes a light chain CDR1 region containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs. 33 to 37, or at least 70%, 75%, 80%, 85%, 90%, 91%, 92% identical to any one of SEQ ID NOs. It includes a light chain CDR2 region containing an amino acid sequence that is identical by %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, and one or more light chain CDR3 regions containing an amino acid sequence that is identical by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to any one of SEQ ID NOs. In an alternative embodiment, the antigen-binding domain is a light chain CDR1 region containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs. 63–67 or at least 70%, 75%, 80%, 85%, 90%, 91%, It includes a light chain CDR2 region containing an amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and one or more light chain CDR3 regions containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of sequence numbers 68-72. Preferably, the light chain includes all of the CDR1, CDR2, and CDR3 regions, selected from amino acid sequences that are at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0147] The heavy chain variable region of the antigen-binding domain may contain, consist of, or be essentially composed of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 153 or 155. The light chain variable region of the antigen-binding domain may contain, consist of, or be essentially composed of, an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 154 or 156. Accordingly, in one embodiment of the present invention, the antigen-binding domain includes a heavy chain variable region containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 153, and / or a light chain variable region containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 154. In an alternative embodiment, the antigen-binding domain includes a heavy chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 155, and / or a light chain variable region comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 156. Preferably, the antigen-binding domain includes an amino acid sequence that is identical to both SEQ ID NOs. 153 and 154, or both SEQ ID NOs. 155 and 156, or to them by at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0148] In one embodiment of the present invention, the light chain variable region and the heavy chain variable region may be joined by a spacer or linker sequence. The linker may include any preferred amino acid sequence. In one embodiment of the present invention, the linker may include, consist of, or be essentially the amino acid sequence shown in SEQ ID NO: 158 or an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0149] Furthermore, the CAR may include additional spacer or linker sequences that link the antigen-binding domain to the transmembrane domain and spatially separate the antigen-binding domain from its end domain. Movable spacer or hinge regions allow the antigen-binding domain to be oriented in various directions to enable EphA3 binding. For example, hinge domains of antibodies such as IgG, IgA, IgM, IgE, or IgD antibodies are also suitable for use in the chimeric receptor described herein. In some embodiments, the hinge domain is a hinge domain connecting the constant domains CH1 and CH2 of the antibody. Thus, additional spacer sequences may include, for example, an IgG1 Fc region, an IgG1 hinge, or a CD8 stalk or hinge, or a combination thereof.

[0150] The antigen-binding domain is expected to further include a leader sequence or a signal peptide sequence. The leader sequence can be a peptide sequence (e.g., about 5, 10, 15, 20, 25, or 30 amino acids in length) located at the N-terminus of the newly synthesized protein (e.g., adjacent to the heavy chain variable region), which directs the protein into the secretory pathway. The leader sequence may include any suitable leader sequence known in the art, such as those derived from CD8, granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, CD28, mouse kappa chain, and CD16. In one embodiment, the leader sequence is a CD8 leader sequence. In this regard, the antigen-binding domain may include, consist of, or be essentially a leader sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 157. In one embodiment of the present invention, the leader sequence promotes the expression of CAR on the cell surface, but the presence of the leader sequence in the expressed CAR is not necessary for the CAR to function. Therefore, once the CAR is expressed on the cell surface, the leader sequence may be cleaved from the CAR. For this reason, in one embodiment of the present invention, the CAR lacks a leader sequence.

[0151] The antigen-binding domain of a CAR is typically fused to an endodomain containing or associating an intracellular or cytoplasmic T cell signaling domain, via a spacer and / or hinge region and a transmembrane domain. When a CAR binds to a target antigen, this results in the transmission of an activation signal to the T cell expressing the target antigen. The endodomain is the part of the CAR involved in signal transduction and may thus contain one or more costimulatory domains and / or one or more intracellular T cell signaling domains.

[0152] The scope of the present invention includes the functional portions of CARs as described herein. The term “functional portion” as used in reference to a CAR means any portion or fragment of the CAR of the present invention that retains the biological activity of the CAR from which it is a part (parent CAR). A functional portion includes portions of a CAR that retain the ability to recognize target cells or to detect, treat, or prevent disease to a similar, the same, or higher degree as the parent CAR. Based on the parent CAR, a functional portion may consist of, for example, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more of the parent CAR.

[0153] The functional moiety may contain additional amino acids at its amino-terminus, carboxyl-terminus, or both, which are not found in the amino acid sequence of the parent CAR. Preferably, the additional amino acids do not interfere with the biological function of the functional moiety, such as recognizing target cells, detecting cancer, or treating or preventing cancer. More preferably, the additional amino acids enhance the biological activity compared to that of the parent CAR.

[0154] The scope of the present invention includes functional variants of CARs as described herein. The term "functional variant," as used herein, means a CAR, polypeptide, or protein having substantial or significant sequence identity or similarity to the parent CAR, wherein the functional variant retains the biological activity of the original CAR from which the variant originated. Functional variants include, for example, variants of the parent CAR (parent CAR) described herein that retain the ability to recognize target cells to a similar, equal, or greater degree than the parent CAR. With respect to the parent CAR, the functional portion may be, for example, identical to the parent CAR by at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99%, or more, with respect to the amino acid sequence.

[0155] A functional variant may include, for example, the amino acid sequence of the parent CAR having at least one conserved amino acid substitution. Alternatively or additionally, a functional variant may include the amino acid sequence of the parent CAR having at least one non-conserved amino acid substitution. In this case, it is preferable that the non-conserved amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution may enhance the biological activity of the functional variant such that its biological activity is increased compared to that of the parent CAR.

[0156] The CARs (including functional portions and functional variants) of embodiments of the present invention can be of any length, that is, the CAR (or its functional portion or functional variant) can contain any number of amino acids, provided that it retains its biological activity (e.g., the ability to specifically bind to an antigen, the ability to detect diseased cells in mammals, or the ability to treat or prevent a disease in mammals). For example, the CAR may be about 50 to about 5000 amino acid lengths, for example, 50, 70, 75, 100, 125, 150, 175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino acid lengths.

[0157] Furthermore, cells containing CAR according to the present invention are also provided. CAR according to the present invention can be used to generate immune cells that express CAR, such as CAR T cells or CAR NK cells. Manipulation of immune cells with CAR can be performed in vitro during culture.

[0158] The antigen-binding region of the CAR of the present invention can be provided in any preferred form, such as scFv, scFab, etc.

[0159] Nucleic acids and vectors The present invention provides one or more nucleic acids or nucleic acids encoding an antigen-binding molecule, polypeptide, or CAR according to the present invention.

[0160] In some embodiments, the nucleic acid is purified or isolated from, for example, other nucleic acids or biological materials of natural origin. In some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0161] [[ID=४]]Thus, in another aspect, the invention contemplates an isolated nucleic acid that encodes or is complementary to a nucleic acid sequence encoding an isolated protein (e.g., an antibody and a CAR protein, including fragments, variants, and derivatives thereof) disclosed herein.

[0162] The nucleotide sequences encoding the isolated proteins of the invention can be readily deduced from one or more of the complete nucleic acid sequences provided herein (e.g., see SEQ ID NOs: 1-12), but are not limited thereto.

[0163] [[ID=1२]]This aspect also includes fragments, variants, and derivatives of the isolated nucleic acid, such as those previously described herein.

[0164] As used herein, the term "nucleic acid" refers to single-stranded or double-stranded DNA and RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, rRNA, RNAi, siRNA, cRNA, and self-catalytic RNA. Additionally, the nucleic acid may be a DNA-RNA hybrid. Nucleic acids typically include nucleotide sequences containing nucleotides that include the A, G, C, T, or U bases. However, the nucleotide sequence may include other bases such as, but not limited to, inosine, methylcytosine, methylinosine, methyladenosine, and / or thiouridine.

[0165] [[ID=२०]]Thus, in certain embodiments, the isolated nucleic acid is cDNA.

[0166] [[ID=२४]]A "polynucleotide" is a nucleic acid having 80 or more contiguous nucleotides, while an "oligonucleotide" has less than 80 contiguous nucleotides.

[0167] The "probe" is preferably a single-stranded or double-stranded oligonucleotide or polynucleotide labeled for the purpose of detecting complementary sequences in Northern blotting or Southern blotting.

[0168] The "primer" is typically a single-stranded oligonucleotide having preferably 15 to 50 consecutive nucleotides, which can be annealed to a complementary nucleic acid "template" and extended in a template-dependent manner by the action of a DNA polymerase, such as Taq polymerase, RNA-dependent DNA polymerase, or Sequenase (trademark).

[0169] In one embodiment, the nucleic acid variant encodes a variant of the isolated protein of the present invention.

[0170] In another embodiment, the nucleic acid variant has at least 40%, 45%, 50%, 55%, 60%, or 65%, 66%, 67%, 68%, or 69% nucleotide sequence identity with the isolated nucleic acid of the present invention, preferably at least 70%, 71%, 72%, 73%, 74%, or 75%, more preferably at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity with respect to the isolated nucleic acid of the present invention.

[0171] In a particular embodiment, the isolated nucleic acid of this embodiment comprises (a) (i) a nucleic acid encoding a segment, domain, portion, or region (including variants or derivatives thereof) of an antibody and / or isolated CAR protein as described herein, such as SEQ ID NOs. 13-156 and Table 1, and (b) one or more optional additional nucleic acid sequences. In this regard, the additional nucleic acid sequences may, but are not limited to, heterologous nucleic acid sequences that may be located at the 5' and / or 3' ends of the isolated nucleic acid sequence.

[0172] The present invention also envisions modified nucleic acids, such as those that utilize codon sequence redundancy. In a more detailed example, the use of codons can be modified to optimize nucleic acid expression in a particular organism or cell species.

[0173] The present invention further provides the use of modified purines (e.g., inosine, methylinosine, and methyladenosine) and modified pyrimidines (e.g., thiouridine and methylcytosine) in nucleic acids of the present invention.

[0174] Those skilled in the art will understand that the isolated nucleic acids of the present invention can be conveniently prepared using standard protocols, such as those described in Chapters 2 and 3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel et al., John Wiley & Sons NY, 1995-2008).

[0175] In yet another embodiment, complementary nucleic acids hybridize with the nucleic acids of the present invention under high stringency conditions.

[0176] In this specification, "hybridize and hybridization" is used to refer to the pairing of at least partially complementary nucleotide sequences that generate DNA-DNA, RNA-RNA, or DNA-RNA hybrids. Hybrid sequences containing complementary nucleotide sequences arise through base pairing.

[0177] As used herein, "stringency" refers to the temperature, ionic strength conditions, and the presence or absence of certain organic solvents and / or surfactants during hybridization. Higher stringency indicates a higher level of complementarity required between the hybridizing nucleotide sequences.

[0178] "Stringent conditions" refer to conditions under which only nucleic acids that have complementary bases at a high frequency hybridize.

[0179] Stringent conditions are well known in the art and are incorporated herein by reference, such as those described in Chapters 2.9 and 2.10 of Ausubel et al., op. cit. Those skilled in the art will also recognize that various factors can be manipulated to optimize the specificity of hybridization. Optimizing the stringency of the final wash can help ensure a high degree of hybridization.

[0180] Complementary nucleotide sequences can be identified by blotting techniques, which typically include steps of immobilizing nucleotides on a matrix (preferably a synthetic membrane such as nitrocellulose), hybridization, and detection using a labeled probe or other complementary nucleic acid. Southern blotting is used to identify complementary DNA sequences, and Northern blotting is used to identify complementary RNA sequences. Dot blotting and slot blotting can be used to identify complementary DNA / DNA, DNA / RNA, or RNA / RNA polynucleotide sequences. Such techniques are well known to those skilled in the art and are described in Ausubel et al., op. cit., pp. 2.9.1-2.9.20. According to such methods, Southern blotting involves separating DNA molecules according to size by gel electrophoresis, transferring the size-separated DNA to a synthetic membrane, and hybridizing the membrane-bound DNA to a complementary nucleotide sequence. When identifying complementary nucleic acids in cDNA or genomic DNA libraries, alternative blotting steps, such as plaque or colony hybridization processes, are used. Other typical examples of this procedure are described in Chapters 8 through 12 of Sambrook et al., *MOLECULAR CLONING. A Laboratory Manual* (Cold Spring Harbor Press, 1989).

[0181] Methods for detecting labeled nucleic acids hybridized to immobilized nucleic acids are well known to those skilled in the art. Such methods include autoradiography, chemiluminescence, fluorescence, and colorimetric detection.

[0182] Nucleic acids can also be isolated, detected, and / or subjected to recombinant DNA techniques using nucleic acid sequence amplification methods.

[0183] Suitable nucleic acid amplification methods that cover both thermal and isothermal methods are well known to those skilled in the art and include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), rolling circle replication (RCR), nucleic acid sequence-based amplification (NASBA), Q-β replicase amplification, recombinase polymerase amplification (RPA), and helicase-dependent amplification.

[0184] As used herein, "amplification product" refers to a nucleic acid product generated by nucleic acid amplification.

[0185] Nucleic acid amplification methods can include certain quantitative and semi-quantitative methods such as qPCR, real-time PCR, and competitive PCR, as are well known in the art.

[0186] In some embodiments, the nucleic acid can be present in a gene construct that facilitates delivery and expression of the nucleic acid. In some embodiments, the present invention provides one or more vectors comprising one or more nucleic acids according to the present invention.

[0187] Thus, in yet another aspect, the present invention provides a gene construct comprising (i) an isolated nucleic acid described herein, or (ii) an isolated nucleic acid comprising a nucleotide sequence complementary thereto. In one embodiment, the isolated nucleic acid is operably linked or connected to one or more regulatory sequences in a vector (e.g., an expression vector).

[0188] Preferably, the gene construct may be in the form of a plasmid, bacteriophage, cosmid, yeast or bacterial artificial chromosome, or comprise its genetic components, as is well understood in the art. The gene construct may be suitable for the maintenance and proliferation of isolated nucleic acids in bacteria or other host cells, manipulation by recombinant DNA technology, and / or expression of the nucleic acids or encoded proteins of the present invention.

[0189] For the purpose of host cell expression, the gene construct may be an expression construct. Preferably, the expression construct comprises the nucleic acid of the present invention operably ligated to one or more additional sequences in an expression vector. "Vector," as used herein, is a nucleic acid molecule used as a medium for transferring exogenous nucleic acids into cells. The vector may be a vector for expressing nucleic acids in cells. The "expression vector" may be a self-replicating extrachromosomal vector, such as a plasmid, or a vector incorporated into the host genome. In this regard, the vector can introduce the nucleic acid of the present invention into host cells, such as T cells, so that the cells express an EphA3-specific CAR or EphA3 conjugate. For this purpose, the vector should ideally be able to sustain high levels of expression in T cells.

[0190] Such vectors may include a promoter sequence operably ligated to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer.

[0191] "Operatively linked" means that the additional nucleotide sequence(s) (e.g., regulatory nucleic acid sequence) is positioned relative to the nucleic acid of the present invention, preferably to initiate, regulate, or otherwise control transcription. Typically, the selected nucleic acid sequence and the regulatory nucleic acid sequence (e.g., promoter and / or enhancer) are covalently linked such that the expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette).

[0192] Regulatory nucleic acid sequences are generally considered appropriate for the host cells used for expression. Numerous types of suitable expression vectors and preferred regulatory sequences are known in the art for various host cells.

[0193] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, transcription start and termination sequences, translation start and termination sequences, and an enhancer or activator sequence.

[0194] Constitutive or inductive promoters, as known in the art, are envisioned in the present invention.

[0195] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors, transposon-based vectors, and artificial chromosomes.

[0196] In certain embodiments, the expression vector is or comprises one or more viral delivery systems, such as an adenovirus vector, an adeno-associated virus (AAV) vector, a herpesvirus vector, a retrovirus vector (e.g., a gamma-retrovirus vector, e.g., a mouse leukemia virus (MLV)-derived vector), a lentivirus vector, a vaccinia virus vector, and a baculovirus vector.

[0197] In some embodiments, the vector may be a eukaryotic vector, for example, a vector containing elements necessary for protein expression from the vector in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, for example, containing a cytomegalovirus (CMV) or SV40 promoter to promote protein expression.

[0198] In a further embodiment, the present invention provides host cells transformed with nucleic acid molecules or gene constructs described herein.

[0199] Suitable host cells for expression may be prokaryotic or eukaryotic. Suitable host cells include, but are not limited to, mammalian cells (e.g., HeLa, HEK293T, Jurkat cells), yeast cells (e.g., Saccharomyces cerevisiae), insect cells used with or without a baculovirus expression system (e.g., Sf9, Trichoplusia ni), plant cells (e.g., Chlamydomonas reinhardtii, Pheodactylum tricornutum), or bacterial cells, such as Escherichia coli. The introduction of gene constructs into host cells (whether prokaryotes or eukaryotes) is well known in this field, and is described, for example, in Chapters 9 and 16 of *CURRENT PROTOCOLS IN MOLECULAR BIOLOGY*, edited by Ausubel et al. (John Wiley & Sons, Inc. 1995-2009).

[0200] CAR-expressing cells The Disclosure also provides cells containing or expressing CARs according to the Disclosure. Furthermore, the Disclosure also provides cells containing or expressing nucleic acids encoding CARs. Manipulation of CARs into T cells may be performed during in vitro culture for transduction and expression, such as during the augmentation of T cells for adoptive T cell therapy. Methods for manipulating immune cells to express CARs are known to those skilled in the art, for example, described in Wang and Riviere, Mol Ther Oncolytics, (2016) 3:16015, which is incorporated herein by reference in its entirety. “At least one cell” will be understood to include multiple cells, for example, a population of such cells.

[0201] Cells containing or expressing CARs according to this disclosure may be eukaryotic cells, for example, mammalian cells. Mammals may be humans or non-human mammals (for example, rabbits, guinea pigs, rats, mice, or other rodents (including any animals of the order Rodentia), cats, dogs, pigs, sheep, goats, domestic cattle (cattle, for example, dairy cows, or any animals of the order Artiodactyla), horses (including any animals of the order Equidae), donkeys, and non-human primates).

[0202] In some embodiments, the cells may be from or obtained from a human subject. When CAR-expressing cells are to be used to treat a subject, the cells may be from the subject to be treated with the CAR-expressing cells (i.e., the cells may be autologous), or the cells may be obtained from a different subject (i.e., the cells may be homogeneous).

[0203] In certain embodiments, the cells are immune cells or include immune cells. The cells may be hematopoietic cells, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. The lymphocytes may be, for example, T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs), or their precursors. The cells may express, for example, CD3 polypeptide (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), TCR polypeptide (TCRα or TCRβ), CD27, CD28, CD4, or CD8.

[0204] Preferably, the immune cells are T cells including CD4+ helper T cells and / or CD8+ cytotoxic T cells (e.g., cytotoxic T lymphocytes (CTLs)). In this regard, the T cells of this embodiment may be a mixed population of CD4+ helper T cells / CD8+ cytotoxic T cells.

[0205] The use of CAR T cells has the advantage that they can be administered systemically and are automatically induced in both primary and metastatic tumors (see Manzo et al., Human Mol Genetics (2015) R67-73).

[0206] In some embodiments, the cells are antigen-specific T cells. In these embodiments, "antigen-specific" T cells are cells that exhibit specific functional properties of a T cell in response to an antigen for which the T cell is specific, or cells that express the said antigen. In some embodiments, the properties are functional properties associated with effector T cells, such as cytotoxic T cells.

[0207] In some embodiments, antigen-specific T cells may exhibit one or more of the following characteristics: for example, cytotoxicity, proliferation, IFN-γ expression, CD107a expression, IL-2 expression, TNF expression, perforin expression, granzyme expression, granulysin expression, and / or FAS ligand (FASL) expression in response to an antigen for which the T cell is specific or to cells containing / expressing that antigen. Antigen-specific T cells include a TCR capable of recognizing peptides of the antigen for which the T cell is specific when presented by an appropriate MHC molecule. Antigen-specific T cells may also be CD4+ T cells and / or CD8+ T cells.

[0208] In some embodiments, the T cell-specific antigen may be a peptide or polypeptide of a virus, such as cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriolarinitis virus (LCMV), or herpes simplex virus (HSV).

[0209] Advantageously, the isolated CARs of the present invention have specificity for EphA3 and can stimulate a large number of T cells (cell 10) regardless of the patient's existing immune repertoire. 8~10 pieces 10 It can be used for CAR gene transfer, a rapid and reliable approach that can generate more than 10 cells per patient. For example, in retroviral or lentiviral transduction, it may be necessary to culture pre-activation T cells for only 48 hours. Furthermore, a large number of autologous T cells can be obtained by leukocyte apheresis or isolation of peripheral blood mononuclear cells (PBMCs) from a blood sample derived from the target. In this way, 10 cells for injection can be obtained. 8 ~10 9 It is possible to manipulate individual transformed or transfected T cells within a few days.

[0210] Therefore, the host cells (e.g., T cells) of the present invention can be used by adoptive transfer to treat EphA3-related diseases, disorders, or conditions, such as cancer. For this purpose, T cells are typically isolated from a biological sample taken from a subject including a donor subject for use in adoptive transfer of recombinant cells.

[0211] Preferably, T cells transduced or transformed by the CAR of the present invention (for example, CARs as shown in Figures 4-1 to 4-3) contain a mixture of naive cells, central memory cells, and effector memory cells.

[0212] In alternative embodiments, the host cell is a stem cell, such as a hematopoietic stem cell (HSC), or a cell derived therefrom. For this purpose, the host cell may be a genetically modified stem cell that, upon differentiation, produces T cells expressing the CAR of the present invention.

[0213] In some embodiments, host cells, such as T cells, are genetically engineered to express cytokines, chemokines, and / or their receptors.

[0214] For this purpose, CAR T cells can be designed in several ways to enhance tumor cytotoxicity and specificity, evade tumor immunosuppression, avoid host rejection, and extend their therapeutic half-life. For example, TRUCK (T-cells Redirected for Universal Cytokine Killing) T cells are designed to express and release cytokines such as IL-12, which promote tumor death, while also possessing CARs. These CAR T cells are sometimes called "armoured CARs" because they are designed to release molecular payloads upon activation of CARs after localizing to the tumor environment. Exemplary cytokines include IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-α, IFN-γ, TNF, TRAIL, FLT3 ligand, lymphotactin, and TGF-β.

[0215] "Automated" or "homing" CART cells are engineered to express chemokine receptors in addition to their CAR. Since certain chemokines are upregulated in tumors, incorporating chemokine receptors can aid in tumor migration and infiltration by adoptive T cells, thereby enhancing both the specificity and functionality of CAR T cells. Universal CAR T cells also possess a CAR but are engineered not to express endogenous TCR (T cell receptor) and MHC (major histocompatibility complex) proteins. Removing these two proteins from the signaling repertoire of adoptive T cell therapy can prevent graft-versus-host disease and rejection, respectively. Furthermore, armored CART cells are so named because they possess the ability to avoid tumor immunosuppression and tumor-induced CAR T cell dysfunction. These specific CAR T cells can also be engineered to possess a CAR but not express checkpoint inhibitors. Alternatively, these CAR T cells can be co-administered with monoclonal antibodies (mAbs) that block checkpoint signaling. Administration of anti-PDL1 antibodies significantly restored the ability of CAR TILs (tumor-infiltrating lymphocytes) to kill them. While the PD1-PDL1 and CTLA-4-CD80 / CD86 signaling pathways have been studied, it is also possible to design armored CAR-T cells that target other immune checkpoint signaling molecules, including LAG-3, Tim-3, IDO-1, 2B4, and KIR. Other intracellular inhibitors of TILs include phosphatases (SHP1), ubiquitin ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinases). Furthermore, CAR T cells can be engineered to express proteins or receptors that protect them from or make them resistant to the effects of tumor-secreted cytokines. For example, transduced CTLs (cytotoxic T lymphocytes) with double-negative TGF-β receptors are resistant to TGF-β-mediated immunosuppression from lymphomas. These transduced cells showed significantly increased antitumor activity in vivo compared to their corresponding controls.

[0216] In another embodiment, the present invention provides a method for producing an isolated protein described herein (for example, an isolated EphA3 binder or CAR), comprising the steps of (i) culturing a pre-transformed host cell as previously described herein, and (ii) isolating the protein from the host cell cultured in step (i).

[0217] Recombinant proteins can be conveniently prepared by those skilled in the art using, for example, the standard protocols described in Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, 1989), particularly sections 16 and 17; Current Protocols in Molecular Biology, edited by Ausubel et al. (John Wiley & Sons, Inc. 1995-2009), particularly chapters 10 and 16; and Current Protocols in Protein Science, edited by Coligan et al. (John Wiley & Sons, Inc. 1995-2009), particularly chapters 1, 5, and 6.

[0218] In one related aspect, the present invention provides an isolated EphA3 binder or a CAR produced by the method described above.

[0219] In a further embodiment, the present invention is (i) EphA3 binders of the first embodiment described above, and / or (ii) The CAR in the second embodiment described above (Including its fragments, variants, and derivatives) It is present in antibodies or antibody fragments that bind to and / or are produced against.

[0220] Preferably, the antibody or antibody fragment specifically binds to the isolated EphA3 binder or CAR. Preferably, the antibody or antibody fragment is a CDR, V as described herein. H Domain and / or V LThe antibody or antibody fragment of this embodiment specifically or selectively binds to or recognizes the complete or partial amino acid sequence of the domain (e.g., SEQ ID NOs. 13-156). In this regard, the antibody or antibody fragment of this embodiment binds to or recognizes its specific CDR, V in the sample. H Domain or V L These may be suitable for use in methods for detecting or isolating T cells expressing CARs having a domain. For this purpose, the antibodies and antibody fragments of the present invention may be particularly suitable for affinity chromatography purification of isolated EphA3 conjugates and CARs as described herein. For example, refer to the affinity chromatography procedure described in Chapter 9.5 of the aforementioned book by Coligan et al.

[0221] Antibodies may be polyclonal or monoclonal, native or recombinant. Well-known protocols applicable to the production, purification, and use of antibodies are described, for example, in Chapter 2 of the aforementioned book by Coligan et al., and in Harlow, E. & Lane, D. Antibodies. A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratory, 1988, both of which are incorporated herein by reference.

[0222] Generally, the antibodies of the present invention bind to or conjugate with the isolated proteins, fragments, variants, or derivatives of the present invention. For example, the antibodies may be polyclonal antibodies. Such antibodies can be prepared, for example, by injecting the isolated proteins, fragments, variants, or derivatives of the present invention into a production species, which may include mice or rabbits, to obtain polyclonal antiserum. Methods for producing polyclonal antibodies are well known to those skilled in the art. Exemplary protocols that can be used are described, for example, in Coligan et al., op. cit. and Harlow & Lane, 1988, op. cit.

[0223] Monoclonal antibodies can be produced, for example, by immortalizing spleen or other antibody-producing cells derived from a production species inoculated with one or more isolated proteins, fragments, variants, or derivatives of the present invention, using the standard method described in the paper by Koehler & Milstein, 1975, Nature 256, 495, which is incorporated herein by reference, or by the recent modification thereof described in the aforementioned book by Coligan et al.

[0224] CMV-specific T cells In certain embodiments, CMV-specific T cells (e.g., CD4 T cells and / or CD8 T cells) expressing a TCR (e.g., αβTCR or γδTCR) that recognizes a peptide containing an MV epitope (e.g., a CMV epitope listed in Table 1) are provided herein. Thus, in some preferred embodiments, the T cells of the present invention are T cells that recognize a peptide containing a CMV epitope listed in Table 1.

[0225] [Table 1] JPEG0007846002000002.jpg77149

[0226] In some preferred embodiments of this type, the T cells further comprise an antigen-binding molecule that binds to EphA3, as described above and / or elsewhere in this specification. In some embodiments, the T cells provided herein can be manipulated to express a CAR, as described above and elsewhere in this specification. For example, CMV-specific T cells further comprise an EphA3-binding CAR.

[0227] In some embodiments, methods for generating, activating, and / or inducing the proliferation of T cells (e.g., CTLs) that recognize one or more CMV epitopes described herein are provided herein. In some embodiments, a sample containing CTLs (e.g., a PBMC sample) is isolated and exposed to a pool of immunogenic peptides disclosed herein to collect stimulated CTLs. Preferably, the pool of immunogenic peptides consists essentially of each of the CMV peptide epitope amino acid sequences shown in Table 1. In certain embodiments, the exposed sample is incubated for at least 14 days. In some such embodiments, the exposed sample is incubated with IL-21 on day 0. Preferably, the exposed sample is incubated with IL-2 on day 2. In more preferred embodiments, the incubation of the exposed sample includes the addition of IL-2 every 3 days.

[0228] In some embodiments, the PBMC sample is derived from a healthy donor. In certain embodiments, the PBMC is derived from an immunocompromised donor. In some such embodiments, the donor is undergoing immunosuppressive therapy. In some embodiments, the donor is a recipient of a parenchymal organ transplant. In further embodiments, the donor is undergoing antiviral therapy.

[0229] In some embodiments, a sample containing CTLs (e.g., a PBMC sample) is incubated in a culture medium with an APC that presents a peptide containing the CMV epitope described herein on a class I MHC complex. A suitable preparation of this type of APC is described, for example, in International Patent Application Publication 2019 / 220209, which is incorporated herein by reference in its entirety. The APC may be autologous to the subject from which the T cells were obtained. In some embodiments, a sample containing T cells is incubated two or more times with the APC provided herein. In some embodiments, T cells are incubated with the APC in the presence of at least one cytokine, e.g., IL-2, IL-4, IL-7, IL-15, and / or IL-21. An exemplary method for inducing T cell proliferation using an APC is described, for example, in U.S. Patent Application Publication 2015 / 0017723, which is incorporated herein by reference.

[0230] The expression of biomarkers by CMV peptide-specific T cells can also be evaluated by any suitable method, such as flow cytometry. In some embodiments, CMV peptide-specific T cells are stimulated with a CMV-specific peptide and then sorted by flow cytometry. Preferably, CMV peptide-specific T cells are stimulated and / or surface-stained according to the protocol described in international application PCT Publication 2019 / 220209, which is incorporated herein by reference. In some embodiments, CMV peptide-specific T cells are incubated with one or more antibodies specific to CD107a and then sorted by flow cytometry. In some embodiments, CMV peptide-specific T cells are incubated with one or more antibodies that bind to intracellular cytokines, e.g., antibodies specific to IFN-γ, IL-2, and / or TNF. In some embodiments, CMV peptide-specific T cells are incubated with antibodies against intracellular cytokines and then sorted by flow cytometry.

[0231] In some embodiments, the method further includes obtaining a sample containing T cells from a donor (e.g., obtaining a PBMC sample from a donor). In some embodiments, autologous T cells (e.g., CD4+ T cells or CD8+ T cells) are isolated from the sample. In some embodiments, the sample consists mostly or entirely of allogeneic T cells.

[0232] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express CD107a.

[0233] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express IFN-γ.

[0234] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express TNF.

[0235] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the T cells (e.g., CTLs) in the sample express IL-2.

[0236] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% of T cells (e.g., CTLs) in the sample. 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a and IFN-γ.

[0237] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45% T cells (e.g., CTLs) %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a and TNF.

[0238] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45% of T cells (e.g., CTLs) in the sample. 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a and IL-2.

[0239] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45% T cells (e.g., CTLs) %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFN-γ and TNF.

[0240] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45% T cells (e.g., CTLs) %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFN-γ and IL-2.

[0241] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 4% of T cells (e.g., CTLs) in the sample. 5%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express TNF and IL-2.

[0242] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, and 4% T cells (e.g., CTLs) 6%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express IFN-γ, TNF, and IL-2.

[0243] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, and 4% T cells (e.g., CTLs) 6%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, TNF, and IL-2.

[0244] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, and 46% T cells (e.g., CTLs) %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IFN-γ, and IL-2.

[0245] In some embodiments, the sample contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, and 46% T cells (e.g., CTLs) %, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IFN-γ, and TNF.

[0246] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46% of T cells (e.g., CTLs) in the sample. 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% express CD107a, IFN-γ, TNF, and IL-2.

[0247] In some embodiments of the methods disclosed herein, T cells (e.g., CTLs) exhibit reactivity to multiple peptide epitopes derived from multiple CMV antigens. In this regard, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47% of T cells (e.g., CTLs). %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% are responsive to multiple CMV epitopes. In certain embodiments, T cells (e.g., CTLs) are responsive to any one or a combination of the CMV peptide epitope amino acid sequences shown in Table 1. In some embodiments, T cells (e.g., CTLs) are responsive to one or a combination of pp50, pp65, IE-I, gB, and gH.

[0248] The expression of T cell biomarkers and / or CMV reactivity can be measured and / or analyzed either before or after T cell (e.g., CTL) proliferation by any of the methods disclosed herein, for example, by exposure to a pool of immunogenic CMV peptide epitopes.

[0249] In some embodiments, CMV reactivity and biomarker expression are quantified before stimulation of T cells (e.g., CTLs). Alternatively or additionally, CMV reactivity and biomarker expression may be quantified after stimulation of T cells (e.g., CTLs). In some embodiments, CMV reactivity is measured by quantifying the percentage of T cells in a sample expressing CD107a. In some embodiments, CMV reactivity is measured by quantifying the percentage of T cells in a sample expressing IFN-γ. In some embodiments, CMV reactivity is measured by quantifying the percentage of T cells in a sample expressing TNF. In some embodiments, CMV reactivity is measured by quantifying the percentage of T cells in a sample expressing IL-2. In some embodiments, CMV reactivity is measured as the percentage of T cells expressing multiple biomarkers (e.g., two or more, preferably all four, of CD107a, IFN-γ, TNF, and IL-2). In some embodiments, CMV reactivity is calculated by quantifying the percentage of T cells in a sample expressing CD107a, IFN-γ, TNF, and IL-2. T cells may be isolated from the sample (e.g., a PBMC sample or a sample containing T cells) either before or after quantifying the percentage of CMV reactivity. Thus, in some embodiments, CMV reactivity is the percentage of T cells having desired characteristics(s) in a sample that is predominantly composed of T cells.

[0250] In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample expressing CD107a. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample expressing IFN-γ. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample expressing TNF. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD8+ lymphocytes in a sample expressing IL-2. In some embodiments, CMV reactivity is measured as the percentage of CD8+ lymphocytes expressing multiple biomarkers (e.g., two or more, preferably all four, of CD107a, IFN-γ, TNF, and IL-2). CD8+ lymphocytes may be isolated from the sample (e.g., a PBMC sample or a CD8+ lymphocyte sample) either before or after the quantification of the CMV reactivity percentage. Therefore, in some embodiments, CMV reactivity is the percentage of CD8+ lymphocytes having the desired characteristics(s) in a sample that is predominantly composed of CD8+ lymphocytes.

[0251] In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample expressing CD107a. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample expressing IFN-γ. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample expressing TNF. In some embodiments, CMV reactivity is measured by quantifying the percentage of CD3+ lymphocytes in a sample expressing IL-2. In some embodiments, CMV reactivity is measured as the percentage of CD3+ lymphocytes expressing multiple biomarkers (e.g., two or more, preferably all four, of CD107a, IFN-γ, TNF, and IL-2). CD3+ lymphocytes may be isolated from the sample (e.g., a PBMC sample or a CD3+ lymphocyte sample) either before or after the quantification of the CMV reactivity percentage. Therefore, in some embodiments, CMV reactivity is the percentage of CD3+ lymphocytes having the desired characteristics(s) in a sample that is predominantly composed of CD3+ lymphocytes.

[0252] In some of the most preferred embodiments of the present invention, T cells present an EphA3 antigen-binding molecule on their surface. For example, T cells may present an EphA3-binding CAR on their surface.

[0253] The T cells may or may not be autologous to the subject. In some embodiments, the T cells are stored in a cell bank before being administered to the subject. In some preferred embodiments, the T cells are homogeneous to the subject.

[0254] Pharmaceutical composition In yet another embodiment, the present invention provides a composition comprising an EphA3 binder as described herein, a CAR as described herein, an isolated nucleic acid as described herein, a gene construct as described herein, and / or a host cell as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0255] In some embodiments, compositions (e.g., pharmaceutical compositions) comprising CMV-specific CTLs expressing or displaying EphA3 CARs, or preparations thereof formulated with a pharmaceutical carrier, and methods for administering such pharmaceutical compositions are provided herein.

[0256] "Pharmacologically acceptable carrier, diluent, or excipient" means a solid or liquid filler, diluent, or encapsulating material that can be safely used in systemic administration.

[0257] In some embodiments, the composition may further contain an adjuvant. As used herein, the term “adjuvant” broadly refers to an immunological or pharmaceutical agent that modulates or enhances the immunological response to a composition in vitro or in vivo. For example, an adjuvant may increase the presence of an antigen over time, assist in the absorption of antigens by antigen-presenting cells, activate macrophages and lymphocytes, and support cytokine production. By altering the immune response, an adjuvant may enhance the efficacy or safety of a drug by allowing for a reduction in the dose of an immuno-interacting agent or preparation. For example, an adjuvant may prevent T-cell depletion and enhance the efficacy or safety of certain immuno-interacting agents or preparations. Examples of adjuvants include, but are not limited to, immunomodulatory proteins, adjuvant 65, α-GalCer, aluminum phosphate, aluminum hydroxide, calcium phosphate, β-glucan peptide, CpG DNA, GPI-0100, lipid A and its modifications (e.g., monophosphorylated lipid A), lipopolysaccharides, lipovant, montanide, N-acetylmuramyl-L-alanyl-D-isoglutamine, Pam3CSK4, Quil-A, and trehalose dimicholate.

[0258] Methods for preparing these formulations or compositions include the step of associating the agents described herein with a carrier and optionally one or more accessory components. Generally, formulations are prepared by uniformly and closely associating the agents described herein with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0259] The pharmaceutical compositions of the present invention, suitable for parenteral administration, comprise one or more of the agents described herein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injection solutions or dispersions immediately before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostatic agents, solutes that make the blood and preparation of the intended recipient isotonic, or suspending agents or thickeners.

[0260] Depending on the specific administration route, various carriers known in the art can be used. These carriers can be selected from the group including sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils (such as olive oil), synthetic oils, polyols (such as glycerol, propylene glycol, polyethylene glycol), alginic acid, phosphate buffer, emulsifiers, isotonic salines, and salts such as mineral salts including hydrochlorides, bromides, and sulfates, organic acids such as acetates, propionates, and malons, and pyrogen-free water. Further examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, ethanol, and suitable mixtures thereof, as well as injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0261] Regardless of the selected route of administration, the agents and / or pharmaceutical compositions of the present invention, which can be used in a preferred hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0262] A useful reference describing pharmaceutically acceptable carriers, diluents, and excipients is Remington's Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991), which is incorporated herein by reference.

[0263] therapeutic use Aspects of this disclosure relate particularly to the use of the antigen-binding agents and / or cells described herein in the treatment of cancer in the subject.

[0264] Accordingly, this disclosure provides a method for treating or preventing cancer in a subject, comprising the step of administering to the subject a therapeutically effective amount of an EphA3 conjugate described herein, or at least one T cell comprising an EphA3-specific chimeric antigen receptor (CAR) described herein, or a composition described herein, thereby treating or preventing cancer in the subject.

[0265] As used generally herein, the terms “cancer,” “tumor,” “malignant,” and “malignant tumor” refer to a disease or condition, or cells or tissues associated with such disease, characterized by abnormal or abnormal cell proliferation, differentiation, and / or migration, often accompanied by an abnormal or abnormal molecular phenotype, including carcinogenesis, expression of tumor markers, expression or loss of activity of tumor suppressor factors, and / or expression of abnormal or abnormal cell surface markers, one or more gene mutations or other gene changes.

[0266] Cancer may include, but is not limited to, any invasive or potentially invasive cancer, tumor, or other malignant tumor, as listed in the NCI Cancer Index at http: / / www.cancer.gov / cancertopics / alphalist, including all major cancer forms such as sarcomas, carcinomas, lymphomas, leukemias, and blastomas. These may include, but are not limited to, cancers of the reproductive system including breast cancer, lung cancer including lung adenocarcinoma, ovarian cancer, cervical cancer, uterine cancer and prostate cancer, cancers of the brain and nervous system, cancers of the gastrointestinal system including head and neck cancer, colon cancer, colorectal cancer and stomach cancer, cancers of the skin including liver cancer, kidney cancer, melanoma and skin cancer, cancers of the hematopoietic system including lymphoid cancer and myelomonocytic cancer, cancers of the endocrine system including pancreatic cancer and pituitary cancer, and cancers of the musculoskeletal system including bone cancer and soft tissue cancer. In certain embodiments, cancer is a solid tumor such as glioblastoma multiforme. Preferably, cancer expresses, for example, EphA3, or overexpresses it.

[0267] Methods of treating cancer can be prophylactic, preventive, or therapeutic, and are suitable for treating cancer in mammals, particularly humans. As used herein, “treating,” “treatment,” or “therapy” means a therapeutic intervention, a set of procedures, or a protocol that at least improves the symptoms of cancer after cancer and / or its symptoms have at least begun to develop. Cancer treatment or palliative care may be effective in preventing the progression of cancer, for example, in preventing the worsening of the condition, or in slowing the rate at which a more serious disease state develops. As used herein, “preventing,” “preventing,” or “prevention” means a therapeutic intervention, a set of procedures, or a protocol initiated before the onset of cancer and / or its symptoms in order to prevent, suppress, or delay the onset or progression of cancer or its symptoms.

[0268] In some embodiments, approximately 1 × 10⁶ T cells per single dose. 5 ~Approx. 1×10 8 A number of T cells are administered to the target. In some embodiments, approximately 1 × 10⁶ T cells are administered per single dose. 6 ~Approx. 1×10 7A number of T cells is administered to the target. In some embodiments, 1 × 10⁶ T cells are administered. 6 pieces, 1×10 7 pieces, 1.5×10 7 pieces, or 2 × 10 7 A number of T cells (e.g., CTLs) are administered to the subject. Multiple doses may be administered to the subject. In some embodiments, an initial dose of T cells (e.g., autologous CTLs) is administered, followed by one or more additional doses of T cells (e.g., autologous CTLs), for example, increasing in dose over time as treatment progresses. In some embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more doses are administered. The subject may receive additional doses that are the same as or different from the initial dose. For example, a lower dose may be administered followed by a higher dose. Doses may be administered daily, twice a week, weekly, every other week, once a month, once every two months, once every three months, or once every six months. In some embodiments, the subject does not experience any adverse effects as a result of T cell (e.g., allogeneic CTL) administration.

[0269] The term “therapeutic dose” refers to a sufficient amount of a particular agent, such as an EphA3 conjugate or CAR, to achieve the desired effect in the subject being treated with that agent. For example, this could be the amount of a composition comprising one or more EphA3 conjugates and / or CARs described herein that is necessary to reduce, alleviate and / or prevent cancer or cancer-related diseases, disorders, or conditions, including cancer metastasis and recurrence. In some embodiments, the “therapeutic dose” is a sufficient amount to reduce or eliminate the symptoms of cancer. In other embodiments, the “therapeutic dose” is a sufficient amount to achieve the desired biological effect, such as an effective amount to reduce or prevent cancer growth, recurrence, and / or metastasis.

[0270] Ideally, the therapeutically effective dose of a drug is sufficient to induce the desired outcome without causing significant cytotoxicity in the subject. The effective dose of a drug useful for reducing, mitigating, and / or preventing cancer is thought to depend on the subject being treated, the type and severity of any associated disease, disorder, and / or condition (e.g., the number and location of any associated metastases), and the mode of administration of the therapeutic composition.

[0271] It will be understood that the methods of this embodiment may include one or more further cancer treatments in addition to those listed above. Such cancer treatments may include, but are not limited to, drug therapy, chemotherapy, antibody, nucleic acid and other biomolecular therapies, radiotherapy, surgery, nutritional therapy, relaxation or meditation therapy and other natural or holistic therapies. Generally, drugs, biomolecules (e.g., antibodies, inhibitory nucleic acids, e.g., siRNA) or chemotherapeutic agents are referred to herein as “anti-cancer agents” or “anti-cancer agents.”

[0272] In some embodiments, the subjects are also administered anticancer compounds. Exemplary anticancer compounds include alemtuzumab (Campath®), alitretinoin (Panretin®), anastrozole (Arimidex®), bevacizumab (Avastin®), bexarotene (Targretin®), bortezomib (Velcade®), bosutinib (Bosulif®), and brentuximab vedocitine. (Adcetris (registered trademark)), cabozantinib (Cometriq (trademark)), carfilzomib (Kyprolis (trademark)), cetuximab (Erbitux (registered trademark)), crizotinib (Xalkori (registered trademark)), dasatinib (Sprycel (registered trademark)), deniroikin difutitox (Ontak (registered trademark)), erlotinib hydrochloride (Tarceva (registered trademark)), everonimus (Afumi Afmitor (registered trademark), exemestane (Aromasin (registered trademark)), fulvestrant (Faslodex (registered trademark)), gefitinib (Iressa (registered trademark)), ibritumomab tiuxetan (Zevalin (registered trademark)), imatinib mesylate (Gleevec (registered trademark)), ipilimumab (Yervoy (trademark)), lapatinib disitrate (Tykerb (registered trademark)), Trozole (Femara®), Nilotinib (Tasigna®), Ofatumumab (Arzerra®), Panitumumab (Vectibix®), Pazopanib Hydrochloride (Votrient®), Pertuzumab (Peijeta®), Pralatrexate (Folotyn®), Regorafenib (Stivarga®),Rituximab (Rituxan®), Romidepsin (Istodax®), Sorafenib tosylate (Nexavar®), Sunitinib malate (Sutent®), Tamoxifen, Temsirolimus (Torisel®), Toremifene (Fareston®), Tositumomab and 131I-Tositumomab (Bexal®) Examples include, but are not limited to, exxar (registered trademark), trastuzumab (Herceptin (registered trademark)), tretinoin (Vesanoid (registered trademark)), vandetanib (Caprelsa (registered trademark)), vemurafenib (Zelboraf (registered trademark)), vorinostat (Zolinza (registered trademark)), and Ziv-aflibercept (Zaltrap (registered trademark)).

[0273] In some embodiments, the subject is also administered a chemotherapeutic agent. Examples of such chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carbocone, metsuredopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (especially bratacin and bratacinone); and camptothecin (synthetic analogues). (including topotecan), bryostatin, callistatin, CC-1065 (including its synthetic analogues adzelesin, karzelesin and beizelesin), cryptophycin (especially cryptophycin 1 and cryptophycin 8), dorastatin, duocalmycin (including synthetic analogues KW-2189 and CB1-TM1), eryuterobin, pancratistatin, sarcodictiin, spongstatin, nitrogen mustard, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloret Min, mechloretamine oxide hydrochloride, melphalan, nobenbicin, fenestrine, prednimustine, trophosphamide, uracil mustard, etc., nitrosourea, such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine and ranimustine, antibiotics, such as engine antibiotics (e.g., calicheamicin, especially calicheamicin gamma(1,1) and calicheamicin omega(1,1)), dinemycin including dinemycin A, bisphosphonates, such as clodronate, esperamicin , as well as neocartinostatin chromophores and related pigment proteins, enediin antibiotic chromophores, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin),Epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, queramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin, antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU), folic acid analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate, pr Pyrimidine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine, etc.; androgens, such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.; anti-adrenal agents, such as aminoglutethimide, mitotane, trilostane, etc.; folic acid supplements, such as folinic acid, etc.; acegraton, aldofos Famidoglycoside, aminolevulinic acid, enyluracil, amsacrin, bestrabusil, bisanthren, edatraxate, defofamine, demecolsin, diaziquan, elfomitin, eriptinium acetate, epotilon, etoglucide, gallium nitrate, hydroxyurea, lentinan, ronidaminin, mytansinoids such as mytansin and anthamitosin, mitoglucon, mitoxantrone, mopidammole, nitramine, pentostatin, fenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydramine Zide, procarbazine, PSK polysaccharide complex, razoxane, lyzoxin, schizofuran, spirogermanium, tenuazonic acid, triadicone, 2,2',2"-trichlorotriethylamine, trichothecin (especially T-2 toxin, veraculin A, loridine A and anguidin), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitractol, pipobromane, gasitosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxoids, e.g., paclitaxel and docetaxel, chlorambucil, gemcitabine,Examples of pharmaceutically acceptable substances include, but are not limited to, 6-thioguanine, mercaptopurine, methotrexate, platinum-coordinated complexes such as cisplatin, oxaliplatin, and carboplatin, vinblastine, platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, xeloda, ibandronate, irinotecan (e.g., CPT-11), the topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, capecitabine, and any of the above pharmaceutically acceptable salts, acids, or derivatives.

[0274] In some embodiments, the subject is also administered an immunotherapy agent. Immunotherapy refers to treatments that utilize the subject's immune system to treat or prevent a condition, such as the use of cancer vaccines, cytokines, target-specific antibodies, T-cell therapy, and dendritic cell therapy.

[0275] In some embodiments, immunomodulatory proteins are also administered to the subjects. Examples of immunomodulatory proteins include B lymphocyte chemoattractant ("BLC"), CC motif chemokine 11 ("eotaxin-1"), eosinophil chemotactic protein 2 ("eotaxin-2"), granulocyte colony-stimulating factor ("G-CSF"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), 1-309, intercellular adhesion molecule 1 ("ICAM-1"), interferon-γ ("IFN-γ"), interleukin-1α ("IL-1α"), interleukin-1β ("IL-1β"), interleukin-1 receptor blocker ("IL-1ra"), interleukin-2 ("IL-2"), interleukin-4 ("IL-4"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), and interleukin-6 soluble receptor ("IL-6") Interleukin-7 ("IL-7"), Interleukin-8 ("IL-8"), Interleukin-10 ("IL-10"), Interleukin-11 ("IL-11"), Interleukin-12 subunit beta ("IL-12 p40" or "IL-12") p70"), interleukin-13 ("IL-13"), interleukin-15 ("IL-15"), interleukin-16 ("IL-16"), interleukin-17 ("IL-17"), chemokine (CC motif) ligand 2 ("MCP-1"), macrophage colony-stimulating factor ("M-CSF"), γ-interferon-induced monokine ("MIG"), chemokine (CC motif) ligand 2 ("MIP-1α"), chemokine (CC motif) ligand 4 ("MIP-1β"), macrophage inflammatory protein-1-delta ("MIP-1δ"), platelet-derived growth factor subunit B ("PDGF-BB"), chemokine (CC motif) ligand 5, "RANTES" (Regulated on Activation, Normal T-cell Expressed andSecreted), TEMP metallopeptidase inhibitor 1 ("TIMP-1"), TIMP metallopeptidase inhibitor 2 ("TIMP-2"), tumor necrosis factor ("TNF"), tumor necrosis factor lymphotoxin-beta ("TNF-β"), soluble TNF receptor type 1 ("sTNFRI"), sTNFRIIAR, brain-derived neurotrophic factor ("BDNF"), basic fibroblast growth factor ("bFGF"), osteomorphogenetic protein 4 ("BMP-4"), osteomorphogenetic protein 5 ("BMP-5"), osteomorphogenetic protein 7 ("BMP-7"), nerve growth factor (" β-NGF), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), endocrine gland-derived vascular endothelial growth factor (EG-VEGF), fibroblast growth factor 4 (FGF-4), keratinocyte growth factor (FGF-7), growth and differentiation factor 15 (GDF-15), glial cell-derived neurotrophic factor (GDNF), growth hormone, heparin-binding EGF-like growth factor (HB-EGF), hepatocyte growth factor (HGF), insulin-like growth factor-binding protein 1 (IGFBP-1), insulin-like growth factor-binding protein 2 ( IGFBP-2, insulin-like growth factor-binding protein 3 ("IGFBP-3"), insulin-like growth factor-binding protein 4 ("IGFBP-4"), insulin-like growth factor-binding protein 6 ("IGFBP-6"), insulin-like growth factor 1 ("IGF-1"), insulin, macrophage colony-stimulating factor ("M-CSFR"), nerve growth factor receptor ("NGFR"), neurotrophin 3 ("NT-3"), neurotrophin 4 ("NT-4"), osteoclast inhibitor ("osteoprotegerin"), platelet-derived proliferation Factor receptor ("PDGF-AA"), phosphatidylinositol-glycan biosynthesis ("PIGF"), Skp, quince, F-box-containing complex ("SCF"), stem cell factor receptor ("SCFR"), transforming growth factor alpha ("TGFα"), transforming growth factor beta-1 ("TGFβ1"), transforming growth factor beta-3 ("TGFβ3"), vascular endothelial growth factor ("VEGF"), vascular endothelial growth factor receptor 2 ("VEGFR2"), vascular endothelial growth factor receptor 3 ("VEGFR3"), VEGF-D6Ckine, tyrosine-protein kinase receptor FIFO ("Axl"), beta-cellulin ("BTC"), mucosa-associated epithelial chemokine ("CCL28"), chemokine (CC motif) ligand 27 ("CTACK"), chemokine (CXC motif) ligand 16 ("CXCL16"), CXC motif chemokine 5 ("ENA-78"), chemokine (CC motif) ligand 26 ("Eotaxin 3"), granulocyte chemotransduction protein 2 ("GCP-2"), GRO, chemokine (CC motif) ligand 14 ("HCC-1"), chemokine (CC motif) ligand 16 ("HCC-4"), interleukin 9 ("IL-9"), interleukin 17F ("IL-17F"), interleukin 18 binding protein ("IL-18") BPa), interleukin 28A ("IL-28A"), interleukin 29 ("IL-29"), interleukin 31 ("IL-31"), CXC motif chemokine 10 ("IP-10"), chemokine receptor CXCR3 ("I-TAC"), leukemia suppressor ("LIF"), Light, chemokine (C motif) ligand ("lymphotactin"), monocyte chemotactic protein 2 ("MCP-2"), monocyte chemotactic protein 3 ("MCP-3"), monocyte chemotactic protein 4 ("MCP-4"), macrophage-derived chemokine ("MDC"), macrophage migration inhibitor ("MIF"), chemokine (CC motif) ligand 20 ("MIP-3α"). CC motif chemokine 19 ("MIP-3β"), chemokine (CC motif) ligand 23 ("MPIF-1"), macrophage-stimulating protein alpha chain ("MSPα"), nucleolar assembly protein 1-like 4 ("NAP-2"), secreted phosphorylated protein 1 ("osteopontin"), lung activation regulatory cytokine ("PARC"), platelet factor 4 ("PF4"), stromal cell-derived factor-1 alpha ("SDF-1α"), chemokine (CC motif) ligand 17 ("TRC"), thymic-expressed chemokine ("TECK"), thymic interstitial lymphapoietin ("TSLP")4-1BB), CD166 antigen ("ALCAM"), cluster differentiation 80 ("B7-1"), tumor necrosis factor receptor superfamily member 17 ("BCMA"), surface antigen classification 14 ("CD14"), surface antigen classification 30 ("CD30"), surface antigen classification 40 ("CD40 ligand"), cancer embryo antigen-associated cell adhesion molecule 1 (bile glycoprotein) ("CEACAM-1"), Death receptor 6 ("DR6"), deoxythymidine kinase ("Dtk") ), type 1 membrane glycoprotein ("endoglin"), receptor tyrosine protein kinase erbB-3 ("ErbB3"), endothelial-leukocyte adhesion molecule 1 ("E-selectin"), apoptosis antigen 1 ("Fas"), Fms-like tyrosine kinase 3 ("Flt-3L"), tumor necrosis factor receptor superfamily member 1 ("GITR"), tumor necrosis factor receptor superfamily member 14 ("HVEM"), intercellular adhesion molecule 3 (ICAM-3), IL-1 R4, IL-1 Rl, IL-10Rβ, IL-17R, IL-2Rγ, IL-21R, Lysosomal membrane protein 2 ("LIMPII"), Neutrophil gelatinase-associated lipocalin ("Lipocalin-2"), CD62L ("L-selectin"), Lymphatic endothelium ("LYVE-1"), MHC class I polypeptide-associated sequence A ("MICA"), MHC class I polypeptide-associated sequence B ("MICB"), NRGI-βI, Beta-type platelet-derived growth factor receptor ("PDGF Rβ"), Platelet endothelial cell adhesion molecule ("PECAM-1"), RAGE, Hepatitis A virus cell receptor 1 ("TIM-1"), Tumor necrosis factor receptor superfamily member IOC ("TRAIL") R3), trapin protein transglutaminase binding domain ("trapin-2"), urokinase receptor ("uPAR"), vascular cell adhesion protein 1 ("VCAM-1"), XEDAR, activin A, agouti-related protein ("AgRP"), ribonuclease 5 ("angiogenin"), angiopoietin 1, angiostatin, cathepsin S, CD40, latent family protein IB ("Cripto-1"), DAN, Dickkopf-related protein 1 ("DKK-1"), E-cadherin, epithelial cell adhesion molecule ("EpCAM"), Fas ligand (FasL or CD95L), FcgRIIB / C, follistatin, galectin-7, intercellular adhesion molecule 2 ("ICAM-2"), IL-13 RI, IL-13R2, IL-17B, IL-2 Ra, IL-2 Rb, IL-23, LAP, nerve cell adhesion molecule ("NrCAM"), plasminogen activator inhibitor-1 ("PAI-1"), platelet-derived growth factor receptor ("PDGF-AB"), resistin, stromal cell-derived factor 1 ("SDF-1β"), sgpl30, secreted frizzled-related protein 2 ("ShhN"), sialic acid-binding immunoglobulin lectin ("Siglec-5"), ST2, transforming growth factor beta-2 ("TGFβ2"), Tie-2, thrombopoietin ("TPO"), tumor necrosis factor receptor superfamily member 10D ("TRAIL") "R4"), induction receptor 1 expressed on bone marrow cells ("TREM-1"), vascular endothelial growth factor C ("VEGF-C"), VEGFR1, adiponectin, adypsin ("AND"), alpha-fetoprotein ("AFP"), angiopoietin-like 4 ("ANGPTL4"), beta-2-microglobulin ("B2M"), basal cell adhesion molecule ("BCAM"), glycosylation antigen 125 ("CA125"), cancer antigen 15-3 ("CA15-3"), carcinoembryonic antigen ("CEA"), cAMP receptor protein ("CRP"), human epidermal growth factor receptor 2 ("ErbB2"), follistatin, follicle-stimulating hormone ("FSH"), chemokine (CXC motif) ligand 1 ("GROα"), human chorionic gonadotropin ("βHCG"), insulin-like growth factor 1 receptor ("IGF-1") sR”), IL-1 sRII, IL-3, IL-18Rβ, IL-21, leptin, matrix metalloproteinase-1 ("MMP-1"), matrix metalloproteinase-2 ("MMP-2"), matrix metalloproteinase-3 ("MMP-3"), matrix metalloproteinase-8 ("MMP-8"), matrix metalloproteinase-9 ("MMP-9"), matrix metalloproteinase-10 ("MMP-10"), matrix metalloproteinase-13 ("MMP-13"), neuronal cell adhesion molecule ("NCAM-I"), entactin ("Nidogen-1"), neuron-specific enolase ("NSE"), oncostatin M ("OSM"), procalcitonin, prolactin, prostate-specific antigen ("PSA"), cytoplasmic acid Auricular methyl glycerin-like lectin 9 ("Siglec-9"), ADAM17 endopeptidase ("TACE"), thyroglobulin, metalloproteinase inhibitor 4 ("TIMP-4"), TSH2B4, disintegrin and metalloproteinase domain-containing protein 9 ("ADAM-9"), angiopoietin 2, tumor necrosis factor ligand superfamily member 13 / acid leucine-rich nuclear phosphoprotein 32 family member B ("APRIL"), osteomorphogenetic protein 2 ("BMP-2"), osteomorphogenetic protein 9 ("BMP-9"), complement component 5a ("C5a"), cathepsin L, CD200, CD97, kemarin, tumor necrosis factor receptor superfamily member 6B ("DcR3"), Fatty acid-binding protein 2 ("FABP2"), fibroblast-activating protein alpha ("FAP"), fibroblast growth factor 19 ("FGF-19"), galectin-3, hepatocyte growth factor receptor ("HGFR"), IFN-α / βR2, insulin-like growth factor 2 ("IGF-2"), insulin-like growth factor 2 receptor ("IGF-2R"), interleukin-1 receptor 6 ("IL-1R6"), interleukin-24 ("IL-24"), interleukin-33 ("IL-33"), kallikrein-14, asparagine endopeptidase ("Regmain"), oxidized low-density lipoprotein receptor 1 ("LOX-1"), mannose-binding lectin ("MBL"), neprilysin ("NEP"), Notch homolog 1, translocation-related (Drosophila) ("Notch-1"), nephroblastoma overexpression ("NOV"), osteoactivin, programmed cell death protein 1 ("PD-1"), N-acetylmuramoyl-L-alanine amidase ("PGRP-5"), serpin A4, secreted frizzled-related protein 3 ("sFRP-3"), thrombomodulin, Toll-like receptor 2 ("TLR2"), tumor necrosis factor receptor superfamily member 10A ("TRAIL"). RI), transferrin ("TRF"), WIF-IACE-2, albumin, AMICA, angiopoietin 4, B cell activator ("BAFF"), glycosylated antigen 19-9 ("CA19-9"), CD163, clatherin, CRTAM, chemokine (CXC motif) ligand 14 ("CXCL14"), cystatin C, decorin ("DCN"), Dickkopf-related protein 3 ("Dkk-3"), delta-like protein Protein 1 ("DLL1"), fetine A, heparin-binding growth factor 1 ("aFGF"), folate receptor alpha ("FOLR1"), furin, GPCR-associated sorting protein 1 ("GASP-1"), GPCR-associated sorting protein 2 ("GASP-2"), granulocyte colony-stimulating factor receptor ("GCSFR"), serine protease hepsin ("HAI-2"), interleukin-17B receptor ("IL-17B R"), interleukin-27 ("IL-27"), lymphocyte activator gene 3 ("LAG-3"), apolipoprotein AV ("LDL")Examples include, but are not limited to, pepsinogen I, retinol-binding protein 4 ("RBP4"), SOST, heparan sulfate proteoglycan ("Syndecan-1"), tumor necrosis factor receptor superfamily member 13B ("TACI"), tissue factor pathway inhibitor ("TFPI"), TSP-I, tumor necrosis factor receptor superfamily member 10b ("TRAIL R2"), TRANCE, troponin I, urokinase plasminogen activator ("uPA"), VE-cadherin (vascular endothelium), also known as cadherin 5, type 2 or CD144 ("VE-cadherin"), WNTl-inducible signaling pathway protein 1 ("WISP-1"), and nuclear factor κB receptor activator ("RANK").

[0276] In some embodiments, the subjects are also administered immune checkpoint inhibitors. Immune checkpoint inhibition broadly refers to inhibiting checkpoints that cancer cells can produce to block or downregulate the immune response. Examples of immune checkpoint proteins include, but are not limited to, CTLA4, PD-1, PD-L1, PD-L2, A2AR, B7-H3, B7-H4, BTLA, KIR, LAG3, TIM-3, or VISTA. Immune checkpoint inhibitors may be antibodies or antigen-binding fragments thereof that bind to and inhibit immune checkpoint proteins. Examples of immune checkpoint inhibitors include, but are not limited to, nivolumab, pembrolizumab, pizilizumab, AMP-224, AMP-514, STI-A1110, TSR-042, RG-7446, BMS-936559, MEDI-4736, MSB-0020718C, AUR-012, and STI-A1010.

[0277] In some embodiments, the compositions provided herein (e.g., vaccine compositions provided herein) are administered prophylactically to prevent cancer and / or CMV infection. In some embodiments, the vaccine is administered to inhibit the growth of tumor cells. The vaccine may be administered before or after the detection of cancer cells or CMV-infected cells in a patient. Inhibition of tumor cell growth is understood to refer to the prevention, cessation, slowing, or death of tumor cell proliferation. In some embodiments, a pro-inflammatory response is induced after administration of a vaccine comprising the peptides, nucleic acids, antibodies, or APCs described herein. The pro-inflammatory immune response includes the production of pro-inflammatory cytokines and / or chemokines, such as IFN-γ and / or IL-2. Pro-inflammatory cytokines and chemokines are well known in the art.

[0278] Combination therapy involves administering active compounds sequentially, simultaneously, separately, and / or co-administering them so that the therapeutic effect of the administered first drug is not completely lost when subsequent therapies are administered. In some embodiments, the second drug may be formulated together with the first drug or as a separate pharmaceutical composition.

[0279] "Administering" or "administering" means introducing an isolated EphA3 conjugate, CAR, encoding nucleic acid, gene construct, cell, or composition disclosed herein into an animal subject via a specific selected route.

[0280] Administration of EphA3 conjugates, CARs or their variants, or encoding nucleic acids, gene constructs, or cells, or compositions containing them, may be by any known parenteral, topical, or enteral route, including but not limited to intravenous, intramuscular, intraperitoneal, intracranial, transdermal, oral, intranasal, anal, and intraocular routes.

[0281] Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, lozenges, capsules, suppositories, aerosols, and transdermal patches. These dosage forms may also include injection or implantation of controlled-release devices specifically designed for this purpose, or other forms of implants modified to act additionally in this manner. Controlled release of therapeutic agents can be achieved by coating them with hydrophobic polymers, for example, acrylic resins, waxes, higher aliphatic alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives such as hydroxypropyl methylcellulose. In addition, controlled release can also be achieved by using other polymer matrices, liposomes and / or microspheres.

[0282] The compositions of the present invention, suitable for oral or parenteral administration, can be presented as individual units such as capsules, sachets, or tablets, each containing a predetermined amount of one or more therapeutic agents of the present invention, as powders or granules, or as solutions or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. Such compositions can be prepared by any pharmaceutical method, all of which involve the step of associating one or more of the above-mentioned agents with a carrier comprising one or more required components. Generally, compositions are prepared by homogeneously and closely mixing the agents of the present invention with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into a desired presentation.

[0283] In another related aspect, the present invention lies in the use of the EphA3 conjugate described herein, the CAR described herein, the isolated nucleic acid described herein, the gene construct described herein and / or the host cell described herein in the manufacture of a pharmaceutical product for the prevention and / or treatment of cancer in a subject.

[0284] In one embodiment, the cancer is or includes glioblastoma pleomorphis.

[0285] Signs and conjugates In yet another aspect, the present invention provides a method for detecting EphA3 or cells expressing EphA3, comprising the step of forming a complex of the aforementioned EphA3-binding molecule or CAR with EphA3, thereby detecting EphA3 or cells expressing EphA3.

[0286] In one embodiment, the method includes a first step of contacting EphA3 or cells expressing EphA3 with an EphA3 antigen-binding molecule or CAR as described above or elsewhere in this specification.

[0287] Therefore, in some embodiments, the antigen-binding molecule of the present invention further includes a detectable site.

[0288] In certain embodiments, the cells are cancer cells or include cancer cells.

[0289] Therefore, it will be understood that the EphA3 conjugates or CARs disclosed herein may be used to assist in the medical diagnosis of cancer. Preferably, the method includes detecting EphA3, for example, when it is present in a biological sample or expressed by cancer cells obtained from a biological sample. In certain embodiments, the biological sample may be a pathological sample comprising one or more liquid, cell, tissue, organ or tissue samples obtained from a human. Non-limiting examples include, but are not limited to, blood, plasma, saliva, serum, lymphocytes, urine, feces, amniotic fluid, cervical samples, cerebrospinal fluid, tissue biopsies, bone marrow, and skin.

[0290] In some embodiments, the antigen-binding molecule includes a detectable moiety. For example, the EphA3 antigen-binding molecule and / or CAR may include fluorescent labeling, phosphorescent labeling, luminescence labeling, immunodetectable labeling (e.g., epitope tagging), radioactive labeling, chemical labeling, nucleic acid labeling, or enzymatic labeling. The antigen-binding molecule may be covalently or noncovalently labeled by the detectable moiety.

[0291] Examples of fluorescent labels include fluorescein, rhodamine, allophycocyanin, eosin, and NDB, green fluorescent protein (GFP), rare earth chelating agents (such as europium (Eu), terbium (Tb), and samarium (Sm)), tetramethylrhodamine, Texas Red, 4-methylumbelliferone, 7-amino-4-methylcoumarin, Cy3, and Cy5.

[0292] As a radioactive label, iodine 123 iodine 125 iodine 126 iodine 131 iodine 133 ,bromine 77 ,technetium 99m ,indium 111 ,indium 113m ,gallium 67 ,gallium 68 ,ruthenium 95 ,ruthenium 103 ,ruthenium 105 ,mercury 207 ,mercury 203 ,rhenium 99m ,rhenium 101 ,rhenium 105 ,scandium 47 , Tellurium 121m , Tellurium 122m , Tellurium 125 ,thulium 165 ,thulium 167 ,thulium 16 ,copper 67 fluorine 18 ,yttrium 90 ,palladium 100 bismuth 217 , and antimony 211 Examples of radioactive isotopes include the following.

[0293] Examples of luminescent labels include radioluminescent labels, chemiluminescent labels (e.g., acridinium esters, luminols, isoluminols), and bioluminescent labels. Examples of immunodetectable labels include haptens, peptides / polypeptides, antibodies, receptors, and ligands such as biotin, avidin, streptavidin, or digoxigenin. Examples of nucleic acid labels include aptamers. Examples of enzymatic labels include peroxidases, alkaline phosphatases, glucose oxidases, beta-galactosidases, and luciferases.

[0294] In some embodiments, the antigen-binding molecule of the present invention is conjugated with a chemical moiety. The chemical moiety may be a moiety that provides a therapeutic effect. Antibody-drug conjugates are reviewed, for example, in Parslow et al., Biomedicines. 2016 Sep;4(3):14. In some embodiments, the chemical moiety may be a drug moiety (e.g., a cytotoxic agent). In some embodiments, the drug moiety may be a chemotherapeutic agent.

[0295] The labels include biotin, avidin, digoxigenin, enzymes (e.g., alkaline phosphatase or horseradish peroxidase), fluorophores (e.g., FITC, Texas Red, coumarin), and radioisotopes (e.g., 125 I, 131 I, 67 Ga, 111 The group may be selected from, but is not limited to, those including (In) and / or directly visible labels (e.g., gold particles).

[0296] Preferably, the detection of EphA3 includes the step of forming a detectable complex between an EphA3 conjugate or CAR and EphA3 or cells expressing EphA3. The complex thus formed can be detected by any technique, assay or means known in the art, including but not limited to immunoblotting, immunohistochemistry, immunocytochemistry, immunoprecipitation, ELISA, flow cytometry, magnetic bead separation, biosensor-based detection systems such as surface plasmon resonance, and imaging such as PET imaging.

[0297] To facilitate detection, the EphA3 conjugate or CAR may be directly labeled as described herein, or a labeled secondary antibody may be used. The labeling may be as described herein.

[0298] In some embodiments, a detection kit may be provided that includes the antibody or antibody fragment disclosed herein together with one or more detection reagents, for example, but not limited to, enzymes, enzyme substrates (e.g., luminol, AMPPD, NBT), secondary antibodies and / or magnetic beads.

[0299] In another embodiment, the present invention provides an isolated protein comprising, essentially, or consisting of an amino acid sequence described in any one of SEQ ID NOs: 13-156 and / or Tables 4-7, or an amino acid sequence that is at least 70% identical thereto.

[0300] In a final aspect, the present invention provides an isolated nucleic acid comprising, consisting of, or essentially comprising a nucleic acid sequence that is at least 70% identical to any of the nucleic acid sequences described in Sequence IDs 1 to 12 and / or Table 3.

[0301] With respect to the embodiments described above, the term “subject” includes, but is not limited to, mammals, including humans, performance animals (such as horses, camels, and greyhounds), livestock (such as cattle, sheep, and horses), and companion animals (such as cats and dogs). In some embodiments, the subject is human.

[0302] To describe preferred embodiments in detail and to obtain practical effects, the following non-limiting embodiments are referenced. [Examples]

[0303] Example 1 Theoretical basis Adoptive immunotherapy using genetically modified T cells expressing chimeric antigen receptors (CARs) has shown considerable success in the treatment of hematological cancers. 4 Despite these breakthroughs, the success of CAR T-cells in treating solid tumors remains limited.

[0304] CARs redirect the cytolytic ability of T cells by utilizing the tumor targeting specificity of any antibody or receptor ligand. Their therapeutic value lies in the ability to target specific cancer biomarkers or combinations of markers by manipulating the binding region to achieve tumor activity while minimizing off-target activity. EphA3 has been identified as a therapeutic target in GBM and several other cancers. 6 EphA3 is overexpressed in cancer and is associated with tumor growth, invasiveness, and metastasis. 6~9 EphA3 appears to be crucial in maintaining tumor cells in a poorly differentiated state and promotes the self-regeneration of cancer stem cells (CSCs). Therefore, targeted inhibition of EphA3 is a promising therapeutic approach for treating solid tumors, and by targeting CSCs, it may also be effective against heterogeneous cancers, metastatic cancers, or cancers deemed resistant to treatment.

[0305] Therapeutic antibodies targeting EphA3 are currently undergoing clinical evaluation in patients with recurrent glioblastoma and have been well-tolerated and have demonstrated promising clinical activity in certain cancer cohorts (10). However, given the challenges of achieving and maintaining the pharmacological level of inhibitors, particularly in the brain (11), this invention explores a CAR T cell-based approach that may provide a targeted antitumor response in the brain, surpassing conventional strategies.

[0306] design EphA3 monoclonal antibody The extracellular domain sequence (P29320, 21-541aa) of human EphA3 was designed, optimized, synthesized, and then subcloned into a pcDNA3.4 vector. A transfection-grade plasmid was prepared maxi for expression in Expi293 cells. The cloning strategy is shown in Figure 1.

[0307] Expi293F cells were grown on an orbital shaker in an Erlenmeyer flask containing serum-free Expi293® expression medium at 8% CO2 and 37°C. On the day of transfection, DNA and transfection reagents were mixed in an optimal ratio and then added to the flask. The cell culture supernatant was collected on day 6 and loaded onto an affinity purification column for purification. After washing and elution with appropriate buffers, the eluted fraction was pooled and the buffer was replaced with the final formulation buffer. The purified protein was analyzed by SDS-PAGE and Western blotting to determine molecular weight and purity (Figure 2). Concentration was determined by a BCA® assay using BSA as a standard, yielding 1.77 mg / mL of protein with a purity of approximately 95%. To avoid multiple freeze-thaw cycles, the protein was divided into multiple aliquots and stored at -80°C.

[0308] Three BALB / c mice and three C57 mice were immunized with recombinant human EphA3 protein according to the immunization schedule shown in the table below.

[0309] [Table 2]

[0310] Cell fusion and clonal plating were performed by electrofusion for each group of mice. All fused cells from each fusion were plated into 96-well plates, and the conditioned medium was screened by ELISA using the EphA3 protein. The positive supernatant was confirmed by ELISA to be negative for unrelated his-tagged proteins. Based on EphA3 specificity, five parental hybridoma clones were selected for subcloning. The supernatants of the 10 monoclonal subclonal cells were evaluated for EphA3 binding efficiency with recombinant EphA3 in ELISA, or by flow cytometry, to identify the EphA3-expressing leukemia cell line LK63. 9 We screened for EphA3 binding efficiency with each other (Figure 3 and Figures 4-1 to 4-3). Hybridomas 3C3-1 and 2D4-1 were selected for sequencing, and clones with lower binding efficiency (such as 6C9-1) were excluded.

[0311] For sequencing, total RNA was isolated from 3C3-1 and 2D4-1 hybridoma cells using TRIzol® reagent. Subsequently, total RNA was reverse transcribed into cDNA using either isotype-specific antisense primers or general-purpose primers with the PrimeScript® 1st Strand cDNA synthesis kit. Heavy and light chain antibody fragments were amplified by rapid amplification of cDNA ends (RACE). The amplified antibody fragments were separately cloned into standard cloning vectors. Colony PCR was performed to screen clones with correctly sized inserts and consensus sequences listed in Table 3.

[0312] [Table 3]

[0313] The complementarity determination regions (CDRs) for 3C3-1 and 2D4-1 are listed in Tables 4-7.

[0314] The inventors have created various EphA3-specific high-affinity complementarity-determining regions (CDRs). These unique sequences form EphA3-specific binding domains and can be used to create single-chain variable fragments (scFv) against targeted EphA3 using CAR T cell technology, or for other applications where EphA3 is the target.

[0315] [Table 4]

[0316] [Table 5]

[0317] [Table 6]

[0318] [Table 7]

[0319] result EphA3 on glioma cell lines Clone 3C3-1 was used to screen glioma cell lines for EphA3 expression (Figure 5). U87 cells were negative, while D270 cells showed a proportion of EphA3-positive and negative tumor cells. U251 cells were predominantly EphA3-positive. These tumor cell lines are considered useful for testing immunotherapy approaches for heterogeneous tumors (D270) and for evaluating particularly high-grade GBM (U251) with upregulated EphA3 expression.

[0320] EphA3-CAR T cells A single-chain variable fragment (scFv) is joined together by a mobile peptide linker to a heavy-chain variable region (V H ) and light chain variable region (V L ) consist of. When the scFv sequences of clones 3C3-1 and 2D4-1 were compared, the alignment identity was less than 48%, which means that these are distinct sequences. Using these scFv sequences, we constructed lentiviral expression plasmids for producing our second-generation CAR constructs. Briefly, we ligated the individual coding sequences of anti-EphA3 scFv to the cytoplasmic regions of human 4-1BB or CD28 having a hinge, CD8 transmembrane region, and CD3-ζ (Figure 6). These sequences were subcloned into pD2109 (lentiviral skeleton plasmid-ATUM) to construct lentiviral expression plasmids. Lentiviral particles were produced via transfection into HEK293T human embryonic kidney cells. Using lipofectamine 2000, cells were transfected with expression plasmids (FA301 or FA302) and pMDL, pREV, and pVSV-G plasmids. pD2109 was used as a control. CAR sequence expression in 293T cells was confirmed by RT-PCR (Figure 7). Viral supernatant was collected 48 and 72 hours after transfection.

[0321] [Table 8] JPEG0007846002000010.jpg105149

[0322] EphA3-CAR expression in Jurkat cell lines Jurkat cells are an immortalized human T cell line, and were used to determine the titer of lentivirus-containing supernatant. Since the CAR construct is tandem with Ires_RFP, surface expression of RFP was used as a reporter for transduction. Therefore, transduction efficiency was determined by transducing Jurkat cells and quantifying RFP expression. Transduction efficiency was 32-58% (1 × 10⁻⁶).6 This range (in individual cells) results in a titer range of 3.2–5.8 × 10⁻⁶. 5 The titer was in the range of IU / mL. The titer of the control pD2109_GFP lentivirus was 8 × 10⁻⁶. 4 The level was IU / mL (Figure 8).

[0323] Both the CAR and RFP sequences have a CD8 reader sequence in their head for surface membrane expression. Nevertheless, to confirm CAR surface expression and binding to its target, cells were incubated with EphA3-His protein and stained with αHis-tagged Ab. FACS results showed that EphA3-CAR was expressed on the surface and primarily bound to EphA3 in cells with high RFP expression (Figure 9). CD69 is an early activation marker in T cells and is involved in proliferation and signaling. We used CD69 as a marker for EphA3-specific activation in Jurkat-CAR cells. Despite high levels of CD69 expression in RFP-negative Jurkat cells, the results showed moderate activation in cells expressing the CAR construct (RFP-positive cells) through interaction with membrane-bound EphA (Figure 10) or through incubation with Lk63 cells (EphA3-positive tumor cell line) (Figure 11). Although only a slight increase in CD69 expression was observed, the expression of activation markers in Jurkat-CAR is a promising indicator of CAR function. Therefore, we hypothesize that the low level of activation in these cells was due to a low MOI (metamorphosis of infection) used for transduction, resulting in a small number of integrations per cell (sometimes only one). We plan to address this issue in the future by enriching the lentivirus to increase the MOI for transduction.

[0324] PBMC-derived T cells for EphA3-CAR production PBMCs were collected from peripheral blood by density gradient centrifugation within 24 hours of venotomy. The PBMC fraction was extracted, washed, and counted. Polyclonal T cells were generated by activation and augmentation via CD3 and CD28 stimulation using T cell TransAct®. CMV-specific T cells were augmented from PBMCs using a previously described protocol. 10、11 In short, one-third of the PBMCs are incubated for one hour with a special pool of 26 T-cell peptide epitopes derived from multiple CMV antigens, washed, and then mixed with the remaining PBMCs. Subsequently, 2-5 × 10⁶ cells are placed in a flask. 6 pieces / cm 2 The seeds were sown at this density.

[0325] On the second day after stimulation, cells were transduced using pD2109 (GFP reporter) and FA301 (RFP reporter) lentiviruses. The cells were cultured in a medium containing recombinant IL-2, which was added every 2-3 days. FACS on the third day after transduction revealed that the transduction efficiency of both lentiviruses was low (Figure 12).

[0326] conclusion The inventors successfully produced an EphA3-specific monoclonal antibody and used it as the scFv in the CAR lentivirus construct. Jurkat-EphA3-CAR expresses a chimeric protein on its surface and upregulates the initial activation marker in response to EphA3 recognition. The inventors further demonstrate that transduction by EphA3-CAR is possible in both Jurkat cells and CMV-specific T cells.

[0327] Example 2 To improve viral titer and T cell transduction efficiency, the IRES and RFP reporter sequences were removed from the constructs to reduce insert size. Lentiviruses were constructed as described above using these smaller constructs, FA3-05-BBζ and FA3-06-28ζ, including a 4-hour ultracentrifugation step at 4°C and 10,500 rpm (SW 32 Ti centrifuge). Polyclonal T cells were cultured as described above and transduced on day 2. T cells expressing CAR were detected by surface staining with anti-mouse IgG AF546, and the cells were analyzed by flow cytometry. CAR transduction efficiency remained low even on day 12. Cells were selected for CAR+ expression and cultured for up to day 20 (Figure 13A).

[0328] The inventors then determined the in vitro function of these FA3-CARs. Transduced T cells were stimulated overnight with LK63, one of the EphA3+ tumor cell lines. Using a standard intracellular staining protocol, the inventors demonstrated that EphA3-CAR T cells induce equivalent target-induced cytokine secretion of TNF, a T cell-activating and immunomodulatory molecule, under either 4-1BB(FA305) or CD28(FA306) co-stimulation (Figure 13B).

[0329] The size of the FA3-06-28ζ construct was further reduced using a specially formulated pLV-Ef1a expression plasmid skeleton from Biosettia. Subsequent studies were conducted using lentiviral transduction T cells produced using this plasmid, which will be referred to as CAR EpHA3 T cells.

[0330] Using CAR EpHA3 lentivirus, polyclonal T cells (anti-CD3 / 28) +Transduction was performed on stimulated T cells and CMV-specific T cells. CAR expression was determined as described above, and CMV-CAR specificity was determined by FACS analysis using a peptide tetramer against HLA complex-CMV (Figure 14). The in vitro function of EphA3-CAR was determined as described above. Transduced T cells were stimulated overnight with LK63 cells. Using a standard intracellular staining protocol, the inventors demonstrated that EphA3-CAR T cells induce TNF-induced cytokine secretion. After stimulation, CAR T cells produced from CMV-pepmix expressed multiple effector molecules, including TNF, IFNγ, and CD107a, suggesting that these cells may have greater cell death capacity (Figure 15). To determine both the specificity and cell death capacity of EphA3-CAR, the inventors performed a real-time cytotoxicity assay (RTCA) using xCELLigence. This assay measures target cell death over a 100-hour period. The glioma cell line U251, which expresses endogenous EphA3, was used as a positive target, along with EphA3-negative glioma cells U87, which served as a negative control. Previous research by Day et al. demonstrated that the U251 EphA3+ glial cell line responds to anti-EphA3 (clone IIIA4) antibody in an orthotopic GBM model to validate the use of these cells as targets. 16 In the RTCA assay, incubation of target cells with EphA3-CAR T cells induced 80% cell lysis within 100 hours of treatment, and no death of EphA3-negative glioma cells was observed (Figure 16A). EphA3-CAR-induced death of target cells was observed by RTCA at effector-target ratios of 1:1, 5:1, and 10:1 (Figure 16B). To compare the killing ability of EphA3-CMV CAR T cells with that of EphA3 CAR, we performed RTCA using T cell:target U251 ratios of 1:1, 5:1, and 10:1, and observed efficient killing of target cells, particularly at 10:1, which was more evident in EphA3-CMV CAR T cells (Figure 16C).

[0331] Example 3 EphA3-CAR T cells exhibit a potent anti-tumor effect in vivo After showing that CAR EphA3 T cells have significant in vitro cytotoxicity against glioma cell lines, the inventors next evaluated their therapeutic ability in vivo.

[0332] For immunodeficient NOD.Rag1KO.IL2RγcKO (NRG) mice, a glioma cell line U251 (EphA3+) or U87 (EphA3-) expressing luciferase was subcutaneously transplanted into the flank (ectopic model) (A in Figure 17-1). Tumor size was measured or determined by bioluminescence. By the 10th day, the tumor reached approximately 25 mm 2 and thus the mice received the first of two intravenous injections of cells with EphA3-CAR T cells, NT (non-transduced) T cells, or CAR19 (non-specific CAR T cells) T cells. hCD45 was detected in the bloodstream on the 17th day, and most of this was CD4 + CAR T cells (B in Figure 17-1). Moreover, an increase in Ki67 expression was observed in U251-bearing mice that received EphA3-CAR T cell administration, suggesting target-induced proliferation of these CAR T cells in this treatment group (C in Figure 17-1).

[0333] Notably, treatment with CAR EphA3 T cells induced a complete response in mice transplanted with U251 (EphA3+) tumors, and the tumors completely disappeared by day 30 (D and F in Figure 17-2). Mice that received non-transduced (NT) T cells or non-specific T cells (CAR19 T cells), and U87 (EphA3 - )-bearing mice were unable to suppress tumor growth (D - G in Figure 17-2).

[0334] conclusion These data demonstrate that these CAR T cells target EphA3 and mediate potent antitumor activity. Treatment with EphA3 CAR T cells induces tumor regression in an ectopic xenograft GBM tumor model. These data support the use of EphA3 CAR T cells as a novel therapy for cancers such as GBM.

[0335] [References] 1. Doubrovina, E. et al.Adoptive immunotherapy with unselected or EBV-specific T cells forbiopsy-proven EBV+ lymphomas after allogeneic hematopoietic celltransplantation. Blood 119, 2644-2656 (2012). 2. Barrett, DM, Grupp,SA & June, CH Chimeric Antigen Receptor- and TCR-Modified T CellsEnter Main Street and Wall Street. J. Immunol. 195, 755-61 (2015). 3. Day, BW et al.EphA3 Maintains Tumorigenicity and Is a Therapeutic Target in Glioblastoma Multiforme. Cancer Cell 23, 238-248 (2013). 4. Tang, XX et al.Implications of EPHB6, EFNB2, and EFNB3 expressions in human neuroblastoma.Proc. Natl. Acad. Sci. (2000) doi:10.1073 / pnas.190123297. 5. Xi, H. Q., Wu, X. S., Wei, B. & Chen, L. Eph receptors and ephrins as targets for cancer therapy. J. Cell. Mol. Med. 16, 2894 - 2909 (2012). 6. Wykosky, J., Gibo, D.M. & Debinski, W. A novel, potent, and specific ephrinA1 - based cytotoxin against EphA2 receptor expressing tumor cells. Mol. Cancer Ther. (2007) doi:10.1158 / 1535 - 7163.mct - 07 - 0200. 7. Swords, R. T. et al. KB004, a Novel Non - Fucosylated Humaneered(registered trademark) Antibody, Targeting EphA3, Is Active and Well Tolerated in a Phase I / II Study of Advanced Hematologic Malignancies. Blood 124, (2014). 8. Razpotnik, R., Novak, N., Curin Serbec, V. & Rajcevic, U. Targeting Malignant Brain Tumors with Antibodies. Front. Immunol. 8, 1181 (2017). 9. Charmsaz, S. et al. EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia. Leukemia 31, 1779 - 1787 (2017). 10. Smith, C. et al. AutologousAdoptive T-cell Therapy for Recurrent or Drug-resistant CytomegalovirusComplications in Solid Organ Transplant Recipients: A Single-arm Open-labelPhase I Clinical Trial. Clin. Infect. Dis. 68, 632-640 (2019). 11. Smith, C. et al. AutologousCMV‐specific T cells are a safe adjuvant immunotherapyfor primary glioblastoma multiforme. J. Clin. Invest. (2020)doi:10.1172 / JCI138649. 12. Day, BW et al. EphA3Maintains Tumorigenicity and Is a Therapeutic Target in Glioblastoma Multiforme. Cancer Cell 23, 238-248 (2013).

[0336] This application claims priority to Australian Patent Application No. 2019903802, filed on 9 October 2019, the contents and elements thereof being incorporated herein by reference for all purposes.

Claims

1. An optionally isolated EphA3 binder comprising six complementarity-determining regions (CDRs), (a) A heavy chain immunoglobulin variable region (VH) polypeptide comprising CDR-H1 having the amino acid sequence shown in SEQ ID NO: 13, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 18, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 23, and A light chain immunoglobulin variable region (VL) polypeptide comprising CDR-L1 having the amino acid sequence shown in SEQ ID NO: 28, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 33, and CDR-L3 having the amino acid sequence shown in SEQ ID NO: 38, or (b) A heavy chain immunoglobulin variable region (VH) polypeptide comprising CDR-H1 having the amino acid sequence shown in SEQ ID NO: 43, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 48, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 53, and A light chain immunoglobulin variable region (VL) polypeptide comprising CDR-L1 having the amino acid sequence shown in SEQ ID NO: 58, CDR-L2 having the amino acid sequence shown in SEQ ID NO: 63, and CDR-L3 having the amino acid sequence shown in SEQ ID NO:

68. An EphA3 binder containing [the necessary components].

2. (a) The VH polypeptide comprises the amino acid sequence shown in SEQ ID NO: 153, and The VL polypeptide contains the amino acid sequence shown in SEQ ID NO: 154, or (b) The VH polypeptide comprises the amino acid sequence shown in SEQ ID NO: 155, and The VL polypeptide contains the amino acid sequence shown in SEQ ID NO:

156. The EphA3 binder according to claim 1.

3. The EphA3 conjugate according to claim 1 or 2, wherein the EphA3 conjugate is an antibody or an antibody fragment.

4. The EphA3 conjugate according to claim 3, which is a recombinant, human, or humanized antibody, or an antibody fragment.

5. An antigen-binding molecule capable of binding to EphA3, (i) The following CD-Rs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 13, HC-CDR2 having the amino acid sequence of SEQ ID NO: 18, HC-CDR3 having the amino acid sequence of SEQ ID NO: 23 A heavy-chain variable (VH) region incorporating, and (ii) The following CD-Rs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 28, LC-CDR2 having the amino acid sequence of SEQ ID NO: 33, LC-CDR3 having the amino acid sequence of SEQ ID NO: 38 Light chain variable (VL) region incorporating Includes or (i) The following CD-Rs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 43, HC-CDR2 having the amino acid sequence of SEQ ID NO: 48, HC-CDR3 having the amino acid sequence of SEQ ID NO: 53 A heavy-chain variable (VH) region incorporating, and (ii) The following CD-Rs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 58, LC-CDR2 having the amino acid sequence of SEQ ID NO: 63, LC-CDR3 having the amino acid sequence of SEQ ID NO: 68 Light chain variable (VL) region incorporating Antigen-binding molecules, including those mentioned above.

6. (i) an antigen-binding molecule according to claim 5, and (ii) an antigen-binding molecule capable of binding to an antigen other than EphA3.

7. A chimeric antigen receptor (CAR) comprising the antigen-binding molecule described in claim 5 or 6.

8. A chimeric antigen receptor (CAR) comprising an antigen-binding domain containing six CDRs encoded by the nucleic acid sequences shown in SEQ ID NOs: 1-6 or SEQ ID NOs: 7-12, a transmembrane domain, and an intracellular T cell signaling domain.

9. The antigen-binding domain, (a) A heavy chain immunoglobulin variable region (VH) polypeptide comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 13, CDR2 having the amino acid sequence shown in SEQ ID NO: 18, and CDR3 having the amino acid sequence shown in SEQ ID NO: 23, and A light chain immunoglobulin variable region (VL) polypeptide comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 28, CDR2 having the amino acid sequence shown in SEQ ID NO: 33, and CDR3 having the amino acid sequence shown in SEQ ID NO: 38, or (b) A heavy chain immunoglobulin variable region (VH) polypeptide comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 43, CDR2 having the amino acid sequence shown in SEQ ID NO: 48, and CDR3 having the amino acid sequence shown in SEQ ID NO: 53, and A light chain immunoglobulin variable region (VL) polypeptide comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 58, CDR2 having the amino acid sequence shown in SEQ ID NO: 63, and CDR3 having the amino acid sequence shown in SEQ ID NO:

68. The CAR according to claim 8, which includes, consists of, or is essentially made of.

10. (a) The VH polypeptide comprises the amino acid sequence shown in SEQ ID NO: 153, and The VL polypeptide contains the amino acid sequence shown in SEQ ID NO: 154, or (b) The VH polypeptide comprises the amino acid sequence shown in SEQ ID NO: 155, and The VL polypeptide contains the amino acid sequence shown in SEQ ID NO:

156. The CAR according to claim 9.

11. (a) The antigen-binding domain includes a linker such as a linker having the amino acid sequence shown in SEQ ID NO: 158, and / or (b) further comprising a leader sequence, preferably the leader sequence comprising, consisting of, or essentially comprising the amino acid sequence shown in SEQ ID NO: 157 The CAR according to any one of claims 7 to 10.

12. A CAR according to any one of claims 7 to 11, wherein the transmembrane domain comprises a CD8 transmembrane domain, and optionally the CD8 transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

159.

13. The CAR according to any one of claims 7 to 12, wherein the intracellular signaling domain includes a CD3 zeta intracellular signaling domain.

14. The CAR according to claim 13, wherein the intracellular signaling domain includes the CD3 zeta-amino acid sequence shown in SEQ ID NO:

162.

15. The CAR according to any one of claims 7 to 14, further comprising one or more costimulatory domains, such as a CD28 costimulatory domain having the amino acid sequence shown in SEQ ID NO: 161, and / or a CD137 costimulatory domain having the amino acid sequence shown in SEQ ID NO:

160.

16. An EphA3 binder according to any one of claims 1 to 4, or an isolated nucleic acid encoding a CAR according to any one of claims 7 to 15.

17. A gene construct comprising isolated nucleic acid according to claim 16.

18. comprising the nucleic acid described in claim 16 and / or the gene construct described in claim 17, A host cell that is either a T cell or contains T cells.

19. A method for preparing an isolated EphA3 binder or CAR, (i) the step of culturing the host cells according to claim 18, and (ii) A step of isolating the EphA3 binder or CAR from the host cells cultured in step (i). A method that includes this.

20. A composition comprising an EphA3 binder according to any one of claims 1 to 4, a CAR according to any one of claims 7 to 15, an isolated nucleic acid according to claim 16, a gene construct according to claim 17, and / or a host cell according to claim 18, and a pharmaceutically acceptable carrier, diluent, or excipient.

21. An in vitro method for detecting EphA3 or cells expressing EphA3, comprising the step of forming a complex of an EphA3 binder according to any one of claims 1 to 4 or a CAR and EphA3 according to any one of claims 7 to 15, thereby detecting EphA3 or cells expressing EphA3.

22. The method according to claim 21, wherein the cells are cancer cells or include cancer cells.

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