T cell receptors for immunotherapy

Abstract

The present invention provides T cell receptors (TCRs) and TCR variable regions that are capable of selectively binding to SLC45A2. The TCRs may be used in various therapies, such as autologous cell transplantation, to treat cancer, such as skin melanoma, uveal melanoma, mucosal melanoma, or metastatic melanoma. Also provided are methods for expanding a population of T cells targeting SLC45A2.

Description

[0001] This application is a divisional application. The corresponding parent application has application number 201880075955.X, filing date October 12, 2018, and the name of the invention is “T cell receptor for immunotherapy”.

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 571,447, filed on October 12, 2017, the entire contents of which are hereby incorporated by reference.

[0003] Incorporation of Sequence Listing

[0004] The Sequence Listing contained in a file entitled "UTFCP1314WO.txt" of 44.7 KB (as measured in Microsoft Windows) created on October 12, 2018, is submitted herewith by electronic submission and is incorporated herein by reference. Technical Field

[0005] The present invention relates generally to the fields of immunology and medicine. More specifically, it relates to T cell receptors (TCRs). In certain embodiments, the TCRs can be used to treat cancer. Background Art

[0006] T cell-based therapies have shown significant promise as a method for treating many cancers; unfortunately, this approach has also been hampered by the lack of immunogenic antigenic targets for common cancers and potential toxicity to non-cancerous tissues. These T cell-based therapies can include adoptive cell transfer (ACT) and vaccination regimens. ACT typically involves infusing a large number of autologous activated tumor-specific T cells into a patient, for example to treat cancer. ACT has induced therapeutic clinical responses in melanoma patients (Yee, 2002; Dudley, 2002; Yee, 2014). In general, in order to produce an effective anti-tumor T cell response, the following three steps are generally required: sensitizing and activating antigen-specific T cells, migrating activated T cells to the tumor site, and recognizing and killing the tumor by antigen-specific T cells. The selection of target antigens is important for the induction of effective antigen-specific T cells.

[0007] Although several tumor-associated antigens have been identified for melanoma and a few other solid tumor malignancies, there are few immunogenic targets for pancreatic, ovarian, gastric, lung, cervical, breast, and head and neck cancers. There is a need to identify and validate new epitopes and target antigens for these common and difficult-to-treat malignancies. Summary of the invention

[0008] In certain embodiments, the disclosure provides an engineered T cell receptor (TCR) comprising an alpha chain CDR3 and / or a beta chain CDR3, wherein the alpha chain CDR3 is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 5, 15, 25, 35 or 45, and the beta chain CDR3 is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 10, 20, 30, 40 or 50. In specific aspects, the TCR has a CDR3 amino acid sequence of SEQ ID NO: 5 and 10, 15 and 20, 25 and 30, 35 and 40 or 45 and 50. In certain aspects, the TCR has the CDR1, CDR2 and CDR3 amino acid sequences of SEQ ID NO: 3-5 and 8-10, 13-15 and 18-20, 23-25 ​​and 28-30, 33-35 and 38-40 or 43-45 and 48-50. In certain aspects, the engineered TCR binds HLA-A2, HLA-A*0201, HLA-A24 and / or HLA-A*2402.

[0009] In certain aspects, the TCR comprises an alpha chain variable region that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32 or 42 and / or a beta chain variable region that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37 or 47. In specific aspects, the TCR comprises an alpha chain of SEQ ID NO: 2, 12, 22, 32 or 42 and / or a beta chain of SEQ ID NO: 7, 17, 27, 37 or 47. In certain aspects, the TCR may comprise the alpha and beta chains of SEQ ID NOs: 2 and 7, 12 and 17, 22 and 27, 32 and 37, or 42 and 47, respectively. In specific aspects, the TCR may have variations in the sequence of the variable regions of the alpha and / or beta chains while keeping the sequence of the CDR regions constant.

[0010] In certain aspects, the TCR comprises an alpha chain having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO: 1, 11, 21, 31 or 41 and / or a beta chain having at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO: 6, 16, 26, 36 or 46. In specific aspects, the TCR comprises an alpha chain comprising the nucleotide sequence of SEQ ID NO: 1, 11, 21, 31 or 41 and / or a beta chain comprising the nucleotide sequence of SEQ ID NO: 6, 16, 26, 36 or 46.

[0011] In certain aspects, the TCR is further defined as a soluble TCR, wherein the soluble TCR does not comprise a transmembrane domain.

[0012] In some aspects, the TCR further comprises a detectable label. In some aspects, the TCR covalently binds a therapeutic agent. In specific aspects, the therapeutic agent is an immunotoxin or a chemotherapeutic agent.

[0013] Further provided herein is a multivalent TCR complex comprising a plurality of the TCRs of the embodiments. In some aspects, the multivalent TCR comprises 2, 3, 4 or more TCRs that are bound to each other. In particular aspects, the multivalent TCR is present in a lipid bilayer, in a liposome or is connected to a nanoparticle. In some aspects, the TCR is bound to each other via a linker molecule.

[0014] In another embodiment, a polypeptide encoding the TCR of the embodiment is provided. Also provided herein is a polynucleotide encoding the polypeptide of the embodiment.

[0015] Other embodiments provide expression vectors encoding the TCR of the embodiments. In some aspects, the sequence encoding the TCR is under the control of a promoter. In specific aspects, the expression vector is a viral vector. In a specific aspect, the viral vector is a retroviral vector. In some aspects, the vector further encodes a linker domain. In some aspects, the linker domain is located between the α chain and the β chain. In some aspects, the linker domain comprises one or more cleavage sites. In some aspects, the one or more cleavage sites are furin cleavage sites and / or P2A cleavage sites. In some aspects, the furin cleavage site is RAKR. In other aspects, the furin cleavage site is ATNFSLLKQAGDVEENPG (SEQ ID NO: 51). In some aspects, the one or more cleavage sites are separated by a spacer. In specific aspects, the spacer is SGSG or GSG.

[0016] In another embodiment, a host cell is provided that is engineered to express the TCR of the embodiment. In some aspects, the cell is a T cell, a NK cell, a constant NK cell, a NKT cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell. In some aspects, the host cell is an immune cell. In a specific aspect, the host cell is isolated from an umbilical cord. In some aspects, the T cell is a CD8 + In certain aspects, the T cells are T cells, CD4+ T cells or γδ T cells. In certain aspects, the T cells are regulatory T cells (Treg). In certain aspects, the cells are autologous. In certain aspects, the cells are allogeneic.

[0017] Another embodiment provides a method for engineering a host cell of the embodiment, the method comprising contacting the immune cell with the TCR of the embodiment or the expression vector of the embodiment. In some aspects, the immune cell is a T cell or a peripheral blood lymphocyte. In some aspects, contact is further defined as transfection or transduction. In some aspects, transfection includes electroporating the RNA encoding the TCR of the embodiment into the immune cell.

[0018] In an additional aspect, the method further comprises, prior to transducing the immune cells, producing a viral supernatant from an expression vector encoding the TCR of the embodiments.

[0019] In some aspects, the immune cell is a stimulated lymphocyte. In some aspects, the stimulated lymphocyte is a human lymphocyte. In some aspects, stimulation includes contacting the immune cell with OKT3 and / or IL-2 or incubating the immune cell in OKT3 and / or IL-2.

[0020] In some aspects, the method also includes sorting immune cells to separate T cells engineered by TCR. In some aspects, the method also includes performing T cell cloning by serial dilution. In some aspects, the method also includes amplifying T cell clones by a rapid amplification scheme.

[0021] In another embodiment, a method for treating cancer in a subject is provided, the method comprising administering an effective amount of a TCR engineered cell of the embodiment to the subject. In some aspects, the subject is identified as having an HLA-A*0201 allele or an HLA-A*2402 allele. In some aspects, the subject is a human.

[0022] In certain aspects, the TCR engineered cell is a T cell or a peripheral blood lymphocyte. In specific aspects, the T cell is a CD8 + T cells, CD4 + T cells or Tregs.

[0023] In some aspects, the cancer is melanoma. In specific aspects, the melanoma is cutaneous melanoma, uveal melanoma, mucosal melanoma or metastatic melanoma. In some aspects, the TCR engineered cells are autologous or allogeneic.

[0024] In further aspects, the method further comprises depleting lymphocytes of the subject prior to administering the SLC45A2-specific T cells. In certain aspects, the lymphocyte depletion comprises administering cyclophosphamide and / or fludarabine.

[0025] In some aspects, the method further comprises administering a second anti-cancer therapy. In some aspects, the therapy is chemotherapy, immunotherapy, surgery, radiation therapy, or biological therapy. In some aspects, the TCR engineered cells and / or the at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, topically, or by direct injection or infusion. In some aspects, the subject is determined to have cancer cells that overexpress SLC45A2.

[0026] In certain embodiments, the present disclosure provides a TCR that selectively binds to SLC45A2. In certain embodiments, the α and β portions of the TCR sequences provided herein may be included in a chimeric antigen receptor (CAR), which can be used in adoptive T cell therapy. In certain embodiments, the α and β portions of the TCR may be encoded in DNA, which may be used, for example, to treat melanoma. Alternatively, the α and β variable regions of the TCR may be included in a protein (such as a TCR or a solubilized protein) and used in anticancer therapies such as adoptive immunotherapy. In certain preferred embodiments, the TCR, CAR or soluble peptide selectively binds to a specific epitope of SLC45A2, such as SLC45A2. 382-390 or SLC45A2 393-402 Immunogenic epitopes. It is foreseen that the TCR can lead to a decrease in toxicity to non-cancerous cells and can be particularly useful for the treatment of melanoma (e.g., skin melanoma, uveal melanoma, mucosal melanoma). In certain embodiments, the cloned T cell receptor can be included in a chimeric T cell receptor (CAR) and used in adoptive T cell transfer or immunotherapy.

[0027] In certain aspects, the present disclosure provides soluble TCRs that can be used directly to treat HLA-A2 positive cancer patients. Soluble bispecific T cell engaging molecules can be generated by linking the SLC45A2 TCR to a CD3-specific Fab fragment. The T cell engaging TCR can bind to the tumor cell surface by presenting the corresponding peptide / MHC complex, and then the Fab fragment cross-links to the CD8 + The TCR on the surface of T cells, thereby causing cell activation and elimination of target cells. Therefore, this soluble bispecific TCR construct can be directly used to treat cancer patients.

[0028] Finally, soluble TCR can be used as a probe for the diagnostic evaluation of peptides / MHC in tumor cells, or for guiding therapeutic molecules to the tumor site. The soluble TCR molecule can also be labeled with a tracer (such as a fluorescent probe or a radioactive probe) and then used for the diagnostic evaluation of the presentation of peptides / MHC in tumor cells. In addition, the soluble TCR molecule can be connected to a therapeutic molecule (such as a toxin) to guide these therapeutic molecules to the tumor site to treat cancer patients.

[0029] In certain aspects, SLC45A2-specific T cells may be administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, or by direct injection or infusion, optionally in combination with a second therapeutic agent.

[0030] Another aspect of the disclosure relates to a pharmaceutical composition comprising a peptide of the disclosure or a peptide as described above and an excipient. The pharmaceutical preparation can be formulated for parenteral administration, intravenous injection, intramuscular injection, inhalation or subcutaneous injection. In certain embodiments, the peptide is contained in a liposome, a lipid-containing nanoparticle or a lipid-based carrier.

[0031] Another aspect of the disclosure relates to specific T cell receptor variable regions (eg, SEQ ID NOs: 51-70).

[0032] The term "chimeric antigen receptor (CAR)" used herein may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and includes an engineered receptor that is transplanted artificially specifically to a specific immune effector cell. CAR can be used to confer the specificity of monoclonal antibodies on T cells, thereby allowing the production of a large number of specific T cells, for example, for use in adoptive cell therapy. In a specific embodiment, CAR instructs cells to be specific for, for example, tumor-associated antigens. In certain embodiments, CAR comprises an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising a tumor-associated antigen binding region. In a particular aspect, CAR comprises a fusion of a single-chain variable fragment (scFv) derived from a monoclonal antibody with a CD3-ζ transmembrane domain and an intracellular domain. The specificity of other CAR designs can be derived from a ligand of a receptor (eg, a peptide) or from a pattern recognition receptor, such as Dectin. In some cases, the interval of the antigen recognition domain can be modified to reduce activation-induced cell death. In some cases, CAR includes a domain for other costimulatory signal transductions, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10 and / or OX40. In some cases, molecules can be co-expressed with CAR, including costimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products, homing receptors, chemokines, chemokine receptors, cytokines and cytokine receptors for conditionally ablating T cells after adding prodrugs.

[0033] As used herein, "substantially free" with respect to a particular component is used herein to indicate that the particular component is not intentionally formulated in the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any unintended contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the particular component is undetectable using standard analytical methods.

[0034] HLA-A2 stands for human leukocyte antigen serotype A2 and is also known as HLA-A* 02. Several serotypes of gene products of many HLA-A*02 alleles are well known, including HLA-A*0201, *0202, *0203, *0206, *0207 and *0211 gene products.

[0035] HLA-A24 stands for human leukocyte antigen serotype A24 and is also referred to as HLA-A*24. Several serotypes of gene products of many HLA-A*24 alleles are well known, including HLA-A*2402 and *2403 gene products.

[0036] When used in the claims and / or specification, the terms "inhibit," "reduce," or "prevent" or any variation of these terms includes any measurable decrease or complete inhibition to achieve the desired result.

[0037] The term "effective," as the term is used in this specification and / or claims, means sufficient to achieve a desired, expected, or intended result.

[0038] In the claims and / or description, the term "a" or "an" when used in conjunction with the term "comprising" may mean "one", but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one".

[0039] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa.In addition, the compositions of the invention can be used to implement the methods of the invention.

[0040] The terms "about," "substantially," and "approximately" generally refer to plus or minus 5% of the stated value.

[0041] The term "or" as used in the claims is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports a definition referring to only alternatives and "and / or."

[0042] As used in this specification and claims, the words "comprising" (and any forms of comprising, such as "including" and "containing"), "having" (and any forms of having, such as "having" and "with"), "including" (and any forms of comprising, such as "including" and "containing"), or "containing" (and any forms of containing, such as "including" and "including") are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0043] Other objects, features and advantages of the present invention will be apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating preferred embodiments of the present invention, are given by way of example only, because those skilled in the art will appreciate various changes and modifications within the spirit and scope of the present invention from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The following drawings form part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0045] Figure 1 : Schematic diagram depicting the retroviral construct containing the TCRβ chain, peptide linker and TCRα chain.

[0046] Figure 2A -B: Specific lysis of target cells by TCR-transfected T cells. + ) and Mel888 (HLA A2 - ) cells, cytotoxic activity of TCR clone #Vb3. To test the cytotoxic activity of parental T cells, a standard chromium release assay was performed and compared between TCR-transfected T cells and parental T cell clones. Figure 2A , TCR-transfected T cells could cleave the HLA-A24-matched target Mel888, but not the HLA-A24-mismatched target Mel526, both of which expressed SLC45A2. Figure 2B , the cytotoxic activity of parental T cells showed similar lysis.

[0047] Figure 3A -B: Stable expression of TCR using TCR retrovirus. SLC45A2 tetramer and CD8 staining of TCR clone Vb3 and parental clone. Figure 3A , activated autologous PBMCs were transduced with retrovirus containing TCR genes. 8 days later, T cells were stained with SLC45A2-PE conjugated tetramers. SLC45A2 tetramer-positive T cells were sorted and REP was performed. Figure 3B , generating parental T cell clones from autologous PMBCs.

[0048] Figure 4A -B: Specific lysis of target cells by TCR-transfected T cells. + ) and Mel888 (HLA A2 -) cells to test the cytotoxic activity of TCR clone #24. To test the cytotoxic activity of parental T cells, a standard chromium release assay was performed and compared between TCR-transfected T cells and parental T cell clones. Figure 4A , TCR-transfected T cells could cleave the HLA-A24-matched target Mel888, but not the HLA-A24-mismatched target Mel526, both of which expressed SLC45A2. Figure 4B , the cytotoxic activity of parental T cells showed similar lysis.

[0049] Figure 5A -B: Stable expression of TCR using TCR retrovirus. SLC45A2 tetramer and CD8 staining of TCR clone #24 and parental clone. Figure 5A , activated autologous PBMCs were transduced with retrovirus containing TCR genes. 8 days later, T cells were stained with SLC45A2-PE conjugated tetramers. SLC45A2 tetramer-positive T cells were sorted and REP was performed. Figure 5B , parental T cell clones were generated from autologous PMBCs.

[0050] Fig. 6A -B: Specific lysis of target cells by TCR-transfected T cells. + ) and Mel888 (HLA A2 - ) cells were tested for cytotoxic activity of TCR clone #39. To test the cytotoxic activity of parental T cells, a standard chromium release assay was performed and a comparison was made between TCR-transfected T cells and parental T cell clones. Fig. 6A , TCR-transfected T cells could cleave the HLA-A24-matched target Mel888, but not the HLA-A24-mismatched target Mel526, both of which expressed SLC45A2. Figure 6B , the cytotoxic activity of parental T cells showed similar lysis.

[0051] Fig. 7A -B: Stable expression of TCR using TCR retrovirus. SLC45A2 tetramer and CD8 staining of TCR clone #39 and parental clone. Fig. 7A , activated autologous PBMCs were transduced with retrovirus containing TCR genes. 8 days later, T cells were stained with SLC45A2-PE conjugated tetramers. SLC45A2 tetramer-positive T cells were sorted and REP was performed. Figure 7B , parental T cell clones were generated from autologous PMBCs.

[0052] Figure 8 : Tetramer staining detection of TCR engineered T cells. The TCR from SLC45A2CTL (#39 clone) was cloned into the retroviral expression vector pMSGV1, and recombinant retrovirus was generated from the infection of PBMC. After infection, tetramers + populations of CD8 + and CD4 + T cells appeared. CD8 + tetramers + and CD4 + tetramers + populations were sorted and amplified using a rapid amplification protocol (REP), and their purity was tested thereafter.

[0053] Fig. 9 : Peptide binding titration assay of TCR engineered T cells. T2 cells were pulsed with different concentrations of SLC45A2 peptide (from 10 pg / mL to 10 μg / mL) and 51 Cr labeling. CD8+ or CD4+ TCR engineered T cells were used as effector cells and co-cultured with T2 cells (E:T=20:1). Detection after 4 hours of co-culture 51 Cr release.

[0054] Fig.10 : CD8+TCR engineered T cells recognize endogenously presented epitopes. CD8+TCR engineered T cells were able to kill Mel526 (HLA-A2+, SLC45A2+) and Mel888-A2 (forced expression of HLA-A2, SLC45A2+) tumor cell lines, but not A375 (HLA-A2+, SLC45A2-) or Mel624 (HLA-A2+, SLC45A2+) tumor cell lines ( Fig.10 ). However, T cells were able to kill A375 cells pulsed with SLC45A2 peptide.

[0055] Fig.11 : Recognition of endogenously presented epitopes by CD4+TCR engineered T cells. Although CD4+TCR engineered T cells did not significantly generate tetramer+ populations after REP, they still killed tumor cells after long-term co-culture (20 h).

[0056] Fig. 12A-E: TCR engineered T cells respond specifically when encountering target cells. An internal cytokine staining (ICS) assay was performed to detect the specific response of TCR engineered T cells when they encounter target cells. Mel526 (naturally presenting endogenous epitopes of SLC45A2), A375 (SLC45A2 negative), T2 pulsed with SLC45A2 peptide, and T2 pulsed with M26 peptide (negative control) were co-cultured with TCR engineered T cells (CD8+ or CD4+, E:T=10:1). After overnight incubation, TNF-α ( Fig. 12A )、CD107a( Fig. 12B ), IFN-γ( Fig. 12C )、CD137( Fig.12D ) and IL-2( Fig.12E ) expression level.

[0057] Figures 13A-13J :Includes Clone #24( Fig.13A -B), Clone #39( Fig. 13C -D), clone #76( Fig.13E -F), clone Vβ3 ( Figure 13G -H) and clone Vβ22 ( Fig.13I -J) Sequences of the α and β chains of each TCR clone. Underlined: signal peptide; Highlighted: variable region; Underlined: CDR1, CDR2, CDR3; Black: constant region. DETAILED DESCRIPTION

[0058] I. Engineered Antigen Receptors

[0059] In different aspects, provided herein is a T cell receptor (TCR) (e.g., SEQ ID NO: 1-50) specifically binding to SLC45A2 or SLC45A2 peptides of the present disclosure. The antigen binding region of TCR can be included in a chimeric antigen receptor (CAR) as an extracellular domain comprising an antigen binding region. TCR can be transfected into cells (e.g., autologous or allogeneic cells), which can be used in adoptive cell transfer therapy. In certain embodiments, CAR is humanized to reduce immunogenicity (hCAR).

[0060] In certain embodiments, host cells of the present disclosure, such as T cells (e.g., CD4 + T cells, CD8 +T cells, γδT cells and Treg), NK cells, constant NK cells, NKT cells, mesenchymal stem cells (MSC), induced pluripotent stem (iPS) cells, to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, autologous cells or allogeneic cells (e.g., isolated from umbilical cord) are modified to express T cell receptors (TCRs) with antigen specificity for cancer antigens. In certain embodiments, the antigen receptor is specific for SLC45A2 (such as the peptide SLC45A2 382-390 or SLC45A2 393-402 In certain embodiments, the engineered TCR has an alpha chain CDR3 and / or a beta chain CDR3, wherein the alpha chain CDR3 has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 5, 15, 25, 35, or 45, and the beta chain CDR3 has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity with SEQ ID NO: 10, 20, 30, 40, or 50. In certain embodiments, the TCR has an alpha chain and / or a beta chain, wherein the alpha chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 1, 2, 11, 12, 21, 22, 31, 32, 41, or 42, and the beta chain has at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 6, 7, 16, 17, 26, 27, 36, 37, 46, or 47. Suitable modification methods are known in the art. See, e.g., Sambrook and Ausubel, supra. For example, T cells can be transduced to express a T cell receptor (TCR) with antigen specificity for a cancer antigen using the transduction techniques described in Heemskerk et al. Hum Gene Ther. 19:496-510 (2008) and Johnson et al. Blood 114:535-46 (2009).

[0061] Electroporation of RNA encoding full-length TCR α and β (or γ and δ) chains can be used as an alternative to overcome the long-term problem of autoreactivity caused by pairing of retrovirally transduced TCR chains and endogenous TCR chains. Even if this alternative pairing occurs in a transient transfection strategy, the autoreactive T cells that may be generated will usually lose this autoreactivity after a period of time because the introduced TCR α and β chains are only transiently expressed. When the expression of the introduced TCR α and β chains decreases, only normal autologous T cells remain. This is not the case when full-length TCR chains are introduced by stable retroviral transduction, which does not lose the introduced TCR chains, thereby causing constant autoreactivity in patients.

[0062] Exemplary antigen receptors (including CARs and recombinant TCRs) and methods for engineering receptors and introducing the receptors into cells include, for example, those described in the following documents: International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US20132 87748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application No. EP2537416, and / or Sadelain et al., Cancer Discov. April 2013; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., October 2012; 24(5): 633-39; Wu et al., Cancer, March 2012 18(2): 160-75. In certain aspects, the genetically engineered antigen receptor includes the CAR described in U.S. Pat. No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668A1.

[0063] AT cell receptor (TCR)

[0064] In certain embodiments, the genetically engineered antigen receptor includes a recombinant T cell receptor (TCR) and / or a TCR cloned from a naturally occurring T cell." T cell receptor" or "TCR" means such a molecule: it contains variable α and β chains (also referred to as TCRα and TCRβ, respectively) or variable γ and δ chains (also referred to as TCRy and TCRδ, respectively), and it is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In certain embodiments, the TCR is in the form of αβ. In certain embodiments, the engineered TCR has SEQ ID NO:5, 15, 25, 35 or 45 α chain CDR3 and / or SEQ ID NO:10, 20, 30, 40 or 50 β chain CDR3. In certain embodiments, the TCR has an alpha chain of SEQ ID NO: 1, 2, 11, 12, 21, 22, 31, 32, 41, or 42 and a beta chain of SEQ ID NO: 6, 7, 16, 17, 26, 27, 36, 37, 46, or 47, respectively.

[0065] In general, TCRs in the form of αβ and γδ are usually similar in structure, but the T cells expressing them may have different anatomical locations or functions. TCRs may appear on the cell surface or in a soluble form. Typically, TCRs appear on the surface of T cells (or T lymphocytes), where they are generally responsible for identifying antigens bound to major histocompatibility complex (MHC) molecules. In certain embodiments, TCRs may also contain constant domains, transmembrane domains and / or short cytoplasmic tails (see, e.g., Janeway et al., Immunobiology:The Immune System in Health and Disease, 3rd edition, Current Biology Publications, p. 433, 1997). For example, in some aspects, each chain of TCR may have an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region and a short cytoplasmic tail at the C-terminal end. In certain embodiments, TCRs are bound to the constant proteins of the CD3 complexes involved in mediating signal transduction. Unless otherwise indicated, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length TCRs, including TCRs in αβ form or γδ form.

[0066] Thus, for the purposes of this article, reference to a TCR includes any TCR or functional fragment, such as an antigen binding portion of a TCR that binds to a specific antigenic peptide bound in an MHC molecule (i.e., an MHC-peptide complex). "Antigen binding portion" or "antigen binding fragment" of a TCR are used interchangeably to represent a molecule that contains a portion of the structural domains of a TCR but binds to an antigen (e.g., an MHC-peptide complex) bound by a complete TCR. In some cases, the antigen binding portion contains the variable domains of a TCR, such as a variable α chain and a variable β chain of a TCR, which are sufficient to form a binding site for binding to a specific MHC-peptide complex, such as typically wherein each chain contains three complementarity determining regions.

[0067] In certain embodiments, the variable domains of the TCR chains combine to form a loop, or are similar to the complementarity determining region (CDR) of an immunoglobulin, which imparts antigen recognition and determines peptide specificity and determines peptide specificity by forming a binding site for a TCR molecule. In general, similar to immunoglobulins, CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., PNAS USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; See also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In certain embodiments, CDR3 is the main CDR responsible for identifying processed antigens, although the CDR1 of the α chain has also been shown to interact with the N-terminal portion of the antigen peptide, and the CDR1 of the β chain interacts with the C-terminal portion of the peptide. It is believed that CDR2 recognizes MHC molecules. In certain embodiments, the variable region of the β-chain may contain another high variability (HV4) region.

[0068] In certain embodiments, the TCR chain contains a constant domain. For example, similar to immunoglobulins, the extracellular portion of a TCR chain (e.g., an α-chain, a β-chain) can contain two immunoglobulin domains, a variable domain at the N-terminus (e.g., a V a or Vp; usually amino acids 1-116 based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest, U.S. Pat. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th edition), and a constant domain adjacent to the cell membrane (e.g., the α chain constant domain or C a, typically amino acids 117-259 based on Kabat; the β-chain constant domain or Cp, typically amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing the CDRs. The constant domain of the TCR domain contains a short linker sequence in which cysteine ​​residues form a disulfide bond to form a connection between the two chains. In certain embodiments, the TCR may have additional cysteine ​​residues in each of the α chain and the β chain such that the TCR contains two disulfide bonds in the constant domain.

[0069] In certain embodiments, the TCR chain may contain a transmembrane domain. In certain embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows TCR to bind to other molecules (such as CD3). For example, a TCR containing a constant domain with a transmembrane region can anchor the protein in the cell membrane and bind to the constant subunit of a CD3 signal transducer (apparatus) or a complex.

[0070] In general, CD3 is a multiprotein complex that can have three different chains (γ, δ and ε) (in mammals) and a ζ chain. For example, in mammals, the complex can contain a homodimer of a CD3γ chain, a CD3δ chain, two CD3ε chains and a CD3ζ chain. CD3γ, CD3δ and CD3ε chains are highly correlated cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane region of CD3γ, CD3δ and CD3ε chains is negatively charged, which is a feature that allows these chains to be combined with positively charged T cell receptor chains. The intracellular tails of CD3γ, CD3δ and CD3ε chains each contain a single conserved motif (called an activation motif or ITAM based on immunoreceptor tyrosine), and each CD3ζ chain has three conserved motifs. Generally, ITAM participates in the signal transduction ability of TCR complexes. These auxiliary molecules have a negatively charged transmembrane region and play a role in transmitting signals from TCR to cells. CD3 chains and ζ chains form so-called T cell receptor complexes together with TCR.

[0071] In certain embodiments, the TCR can be a heterodimer of two chains α and β (or optionally γ and δ), or it can be a single-chain TCR construct. In certain embodiments, the TCR is a heterodimer containing two independent chains (α and β chains or γ and δ chains) connected, for example, by one or more disulfide bonds. In certain embodiments, the TCR for a target antigen (such as a cancer antigen) is identified and introduced into a cell. In certain embodiments, the nucleic acid encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of publicly available TCR DNA sequences. In certain embodiments, the TCR is obtained from a biological source, such as from a cell, such as from a T cell (such as a cytotoxic T cell), a T cell hybridoma, or other publicly available sources. In certain embodiments, T cells can be obtained from cells separated in vivo. In certain embodiments, high-affinity T cell clones can be separated from patients, and TCR can be separated. In certain embodiments, T cells can be cultured T cell hybridomas or clones. In certain embodiments, TCR clones for target antigens have been generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system or HLA). See, for example, tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15: 169-180 and Cohen et al. (2005) J Immunol. 175: 5799-5808). In certain embodiments, phage display is used to isolate TCRs for target antigens (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14: 1390-1395 and Li (2005) Nat Biotechnol. 23: 349-354). In certain embodiments, TCRs or their antigen-binding portions can be produced synthetically based on knowledge of TCR sequences.

[0072] B. Chimeric T cell receptor

[0073] In certain embodiments, the engineered antigen receptor comprises a chimeric antigen receptor (CAR), including an activating or stimulatory CAR, a co-stimulatory CAR (see WO2014 / 055668) and / or an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013). CARs typically include an extracellular antigen (or ligand) binding domain connected to one or more intracellular signaling components via a linker and / or a transmembrane domain in some aspects. Such molecules typically mimic or approximate signals through natural antigen receptors, signals through such receptors in combination with co-stimulatory receptors, and / or signals through co-stimulatory receptors alone. In certain embodiments, the CAR includes one or more antigen binding portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from a variable heavy (VH) chain and a variable light (VL) chain of a monoclonal antibody (mAb).

[0074] The arrangement of the antigen binding domain of CAR can be a multimer, such as a dibody or a multimer. By cross-pairing the variable parts of light chain and heavy chain into so-called dibodies, a multimer can be formed. In certain embodiments, the hinge portion of CAR can be shortened or excluded (i.e., a CAR containing only antigen binding domain, transmembrane region and intracellular signaling domain is produced). A variety of hinges can be used with the present invention, for example, as shown in Table 1. In certain embodiments, the hinge region may have a first cysteine ​​that is maintained or a first cysteine ​​that is mutated by proline or serine replacement, or truncated until the first cysteine. The Fc portion can be deleted from the scFv used as an antigen binding region to produce a CAR according to the present invention. In certain embodiments, the antigen binding region may encode only one of the Fc domains, such as CH2 or CH3 domains from human immunoglobulin. One can also include hinges, CH2 and CH3 regions of human immunoglobulin, which have been modified to improve dimerization and oligomerization. In certain embodiments, the hinge portion may comprise or consist of a peptide of 8-14 amino acids (e.g., a 12 amino acid peptide), a portion of CD8α, or IgG4 Fc. In certain embodiments, the antigen binding domain may be suspended from the cell surface using a domain that promotes oligomerization, such as CD8α. In certain embodiments, the antigen binding domain may be suspended from the cell surface using a domain recognized by monoclonal antibody (mAb) clone 2D3 (e.g., mAb clone 2D3 described in Singh et al., 2008).

[0075] The intracellular domain or intracellular signaling domain of CAR can generally cause or promote the activation of at least one normal effector function of an immune cell comprising CAR. For example, the intracellular domain can promote the effector function of T cells, for example, cell lysis activity or auxiliary activity, including the secretion of cytokines. The effector function in the initial, memory or memory T cells can include antigen-dependent proliferation. The term "intracellular signaling domain" or "intracellular domain" means a portion of CAR that can transduce effector function signals and / or instruct cells to perform specialized functions. Although generally the entire intracellular signaling domain can be included in CAR, in some cases, a truncated portion of the intracellular domain may be included. Typically, the intracellular domain includes a truncated intracellular domain, wherein the truncated intracellular domain retains the ability to transduce effector function signals in the cell.

[0076] In certain embodiments, the intracellular domain comprises a ζ chain of a T cell receptor or any homologue thereof (e.g., η, δ, γ or ε), an MB1 chain, B29, Fc RIII, Fc RI, and a combination of signaling molecules such as CD3ζ and CD28, CD27, 4-1BB, DAP-10, OX40, and combinations thereof, as well as other similar molecules and fragments. Intracellular signaling portions of other members of the activating protein family, such as FcγRIII and FcεRI, can be used. Examples of these optional transmembrane and intracellular domains can be found in: for example, Gross et al. (1992), Stancovski et al. (1993), Moritz et al. (1994), Hwu et al. (1995), Weijtens et al. (1996) and Hekele et al. (1996), all of which are incorporated herein by reference. In certain embodiments, the intracellular domain may comprise a human CD3ζ intracellular domain.

[0077] The antigen-specific extracellular domain and intracellular signaling domain are preferably connected by a transmembrane domain. The transmembrane domain that can be included in CAR includes, for example, human IgG4 Fc hinge and Fc region, human CD4 transmembrane domain, human CD28 transmembrane domain, transmembrane human CD3 ζ domain or cysteine ​​mutated human CD3 ζ domain or transmembrane domain from human transmembrane signaling protein, for example, CD16 and CD8 and erythropoietin receptor. Examples of transmembrane domains are provided in, for example, Table 1.

[0078] In certain embodiments, the intracellular domain comprises a sequence encoding a co-stimulatory receptor, such as a modified CD28 intracellular signaling domain or a CD28, CD27, OX-40 (CD134), DAP10 or 4-1BB (CD137) co-stimulatory receptor. In certain embodiments, the main signal triggered by CD3ζ, the additional signal provided by the human co-stimulatory receptor can be included in the CAR to more effectively activate the transformed T cells, which can help improve the in vivo persistence and treatment success of adoptive immunotherapy. As shown in Table 1, the intracellular domain or intracellular receptor signaling domain can include a single or FcγRIII co-stimulatory signaling domain (e.g., CD28, CD27, DAP10, CD137, OX40, CD2, 4-1BB) CD3 ζ chain in combination. In certain embodiments, the intracellular domain comprises part or all of one or more of TCR ζ chain, CD28, CD27, OX40 / CD134, 4-1BB / CD137, FcεRIγ, ICOS / CD278, IL-2Rβ / CD122, IL-2Rα / CD132, DAP10, DAP12 and CD40. In certain embodiments, the intracellular domain may comprise 1, 2, 3, 4 or more cytoplasmic domains. For example, it has been observed in certain CARs that at least two or three signaling domains fused together can produce an additive or synergistic effect.

[0079] In some aspects, an isolated nucleic acid fragment and an expression cassette can be produced, which include a DNA sequence encoding CAR. A variety of vectors can be used. In certain preferred embodiments, the vector can allow the DNA encoding CAR to be delivered to immune cells such as T cells. CAR expression can be under the control of a regulated eukaryotic promoter (e.g., MNDU3 promoter, CMV promoter, EF1α promoter, or ubiquitin promoter). And, if there is no other reason, the vector may include a selection marker to facilitate its in vitro operation. In certain embodiments, CAR can be expressed from mRNA transcribed from a DNA template in vitro.

[0080] Chimeric antigen receptor molecules are recombinant and distinguished by the ability to bind antigens and transduce activation signals via the immunoreceptor activation motifs (ITAMs) present in their cytoplasmic tails. Receptor constructs utilizing antigen binding moieties (e.g., produced from single-chain antibodies (scFv)) provide the additional advantage of being "universal" because they can bind to natural antigens on the target cell surface in a manner that is independent of HLA. For example, scFv constructs can be fused with sequences encoding the intracellular portion of the ζ chain (ζ) of the CD3 complex, the Fc receptor γ chain, and the sky tyrosine kinase (Eshhar et al., 1993; Fitzer-Attas et al., 1998). Redirected T cell effector mechanisms have been recorded in several mouse and human antigen-scFv:ζ systems, including tumor recognition and dissolution by CTL (Eshhar et al., 1997; Altenschmidt et al., 1997; Brocker et al., 1998).

[0081] In certain embodiments, TCR is included in CAR as an antigen binding domain (e.g., as a scFv region), and CAR further includes a hinge region, a transmembrane region, and an intracellular domain. For example, TCR (e.g., SEQ ID NO: 51-70) can be included in CAR together with a hinge region, a transmembrane region, and an intracellular domain, and the hinge region, the transmembrane region, and the intracellular domain are as described in Table 1 below.

[0082] Table 1. Regions that can be included in anti-SLC45A2 targeting CARs

[0083]

[0084]

[0085] Transmembrane domain CD28 CD137(4-1BB) CD8α CD3ζ

[0086]

[0087]

[0088] ζ-zeta; Δ-mutant; Note = 4-1BB is also known as CD137; "+" indicates a fusion of a different region.

[0089] II. Soluble TCR

[0090] In certain embodiments, the present disclosure provides soluble TCRs, such as the SLC45A2 TCRs provided herein. Soluble TCRs are useful not only for the purpose of studying specific TCR-pMHC interactions, but also potentially useful as diagnostic tools for detecting infections or detecting autoimmune disease markers. Soluble TCRs can also be used for staining, for example, for staining cells for the presence of specific peptide antigens presented in the context of MHC. Similarly, soluble TCRs can be used to deliver therapeutic agents (e.g., cytotoxic compounds or immunostimulatory compounds) to cells presenting specific antigens. Soluble TCRs can also be used to inhibit T cells, for example, those that react with autoimmune peptide antigens.

[0091] In the context of this application, "solubility" is defined as the solubility of a compound at a concentration of 1 mg / ml in phosphate buffered saline (PBS) (KCl 2.7 mM, KH 2 PO 4 1.5mM, NaCl 137mM and Na 2 PO4 8mM, pH 7.1-7.5. Life Technologies, Gibco BRL) TCRs were purified as monodisperse heterodimers and more than 90% of the TCRs retained the ability to be monodisperse heterodimers after incubation at 25°C for 1 hour.

[0092] In certain aspects, the present disclosure provides a soluble T cell receptor (sTCR) comprising: (i) all or part of a TCR alpha chain (e.g., SEQ ID NO: 1, 2, 11, 12, 21, 22, 31, 32, 41 or 42), excluding its transmembrane domain, and (ii) all or part of a TCR beta chain (e.g., SEQ ID NO: 6, 7, 16, 17, 26, 27, 36, 37, 46 or 47), excluding its transmembrane domain, wherein (i) and (ii) each comprise a functional variable domain and at least a portion of a constant domain of a TCR chain, and are connected by disulfide bonds between constant domain residues that are not present in a native TCR.

[0093] In certain aspects, the soluble TCR comprises a TCR alpha or gamma chain extracellular domain dimerized to a TCR beta or delta chain extracellular domain, respectively, with the aid of a pair of C-terminal dimerization peptides such as leucine zippers (International Patent Publication No. WO 99 / 60120; U.S. Pat. No. 7,666,604).

[0094] The soluble TCR of the present disclosure, which is preferably human, can be provided in a substantially pure form or as a purified or isolated preparation. For example, it can be provided in a form that is substantially free of other proteins.

[0095] A variety of soluble TCRs of the present disclosure can be provided as multivalent complexes. Therefore, in one aspect, the present disclosure provides a multivalent T cell receptor (TCR) complex comprising a plurality of soluble T cell receptors as described herein. Each of the plurality of soluble TCRs is preferably identical.

[0096] In its simplest form, the multivalent TCR complex according to the present invention comprises a polymer of two or three or four or more T cell receptor molecules that are preferably bound to each other (e.g., covalently or otherwise connected) by a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules, such as avidin, streptavidin, neutravidin and extravidin, each of which has four binding sites for biotin. Thus, biotinylated TCR molecules can be formed into a polymer of a T cell receptor with multiple TCR binding sites. The number of TCR molecules in the polymer will depend on the amount of TCR related to the amount of the linker molecule used to make the polymer, and also depends on whether there are any other biotinylated molecules. Preferred polymers are dimers, trimers or tetramers TCR complexes.

[0097] Suitable structures for use in the methods of the invention include membrane structures such as liposomes, and solid structures, which are preferably particles, such as beads, for example latex beads. Other structures that can be coated with T cell receptor molecules on the outside are also suitable. Preferably, the structure is coated with T cell receptor polymers rather than with individual T cell receptor molecules.

[0098] In the case of liposomes, the T cell receptor molecules or multimers thereof may be attached to or otherwise associated with the membrane. The techniques for this are well known to those skilled in the art.

[0099] A label or other part (e.g., toxic or therapeutic part) may be included in the multivalent TCR complex of the present invention. For example, a label or other part may be included in a mixed molecule polymer. An example of such a polymer molecule is a tetramer, which contains three TCR molecules and one peroxidase molecule. This can be achieved as follows: TCR and enzyme are mixed in a molar ratio of about 3:1 to produce a tetramer complex, and the desired complex is separated from any complex that does not contain the correct molecular ratio. These mixed molecules can contain any combination of molecules, provided that steric hindrance does not impair or significantly impair the desired function of the molecule. Due to the unlikely occurrence of steric hindrance, the positioning of the binding site on the streptavidin molecule is suitable for mixed tetramers.

[0100] Alternatively or additionally, the TCR (or its multivalent complex) of the present disclosure can be combined with a therapeutic agent (e.g., covalently or otherwise connected thereto), which can be, for example, a toxic moiety useful in cell killing, or an immunostimulant (such as an interleukin or cytokine). Compared to non-polymeric T cell receptor heterodimers, the multivalent TCR complex of the present invention can have an enhanced binding ability to TCR ligands. Therefore, the multivalent TCR complex according to the present invention is particularly useful for tracking or targeting cells presenting specific antigens in vitro or in vivo, and can also be used as an intermediate for producing another multivalent TCR complex with such uses. Therefore, the TCR or multivalent TCR complex can be provided in a pharmaceutically acceptable formulation for in vivo application.

[0101] The present disclosure also provides a method for delivering a therapeutic agent to a target cell, the method comprising contacting a potential target cell with a TCR or a multivalent TCR complex according to the present disclosure, under conditions that allow the TCR or multivalent TCR complex to attach to the target cell, wherein the TCR or multivalent TCR complex is specific for a TCR ligand and has a therapeutic agent bound thereto.

[0102] Specifically, soluble TCR or multivalent TCR complexes can be used to deliver therapeutic agents to the location of cells presenting specific antigens. This will be useful in many cases, especially for tumors. Therapeutic agents can be delivered so that they exert their effects locally but not only on the cells to which they bind. Therefore, a specific strategy envisions anti-tumor molecules connected to T cell receptors or multivalent TCR complexes specific for tumor antigens.

[0103] Many therapeutic agents can be used for this purpose, such as radioactive compounds, enzymes (e.g., perforin) or chemotherapeutic agents (e.g., cisplatin). In order to improve the limited toxic effects in the desired location, the toxin can be provided inside the liposomes connected to streptavidin so that the compound is released slowly. This can reduce the damaging effects during transport in vivo and help limit the toxin effects until after the TCR binds to the relevant antigen presenting cells.

[0104] Other suitable therapeutic agents include:

[0105] Small molecule cytotoxic agents, i.e. compounds with a molecular weight of less than 700 Daltons that have the ability to kill mammalian cells. Such compounds may also contain toxic metals that can have a cytotoxic effect. In addition, it should be understood that these small molecule cytotoxic agents also include prodrugs, i.e. compounds that decay or transform under physiological conditions to release the cytotoxic agent. Examples of such agents include cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetrexate glucuronate, auristatin E vincristine and doxorubicin;

[0106] ● Peptide cytotoxins, i.e. proteins or fragments thereof that have the ability to kill mammalian cells. Examples include ricin, diphtheria toxin, Pseudomonas exotoxin A, DNA enzymes, and RNA enzymes;

[0107] ● Radionuclides, which are unstable isotopes of elements that decay with the simultaneous emission of one or more of alpha or beta particles or gamma rays. Examples include iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and 213, actinium-225, and astatine-213;

[0108] ● Prodrugs, such as antibody-directed enzyme prodrugs; and

[0109] ● Immunostimulants, i.e., moieties that stimulate an immune response. Examples include: cytokines, such as IL-2; chemokines, such as IL-8; platelet factor 4; melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof; complement activators; foreign protein domains; allogeneic protein domains; viral / bacterial protein domains and viral / bacterial peptides.

[0110] The soluble TCR of the present disclosure can be used to regulate T cell activation by binding specific TCR ligands and thereby inhibiting T cell activation. Autoimmune diseases (e.g., type I diabetes) involving T cell-mediated inflammation and / or tissue damage will be suitable for this approach. For this use, it is necessary to understand the specific peptide epitopes presented by the relevant pMHC.

[0111] Also contemplated is the use of the soluble TCR and / or multivalent TCR complex of the present disclosure in the preparation of a composition for treating cancer or autoimmune disease.

[0112] Also provided is a method of treating cancer or autoimmune disease comprising administering an effective amount of the soluble TCR and / or multivalent TCR complex of the invention to a patient in need thereof.

[0113] As is common in anti-cancer and autoimmune therapies, the sTCRs of the present disclosure can be used in combination with other agents for the treatment of cancer and autoimmune diseases and other related conditions found in similar patient groups.

[0114] III. Adoptive Cell Transfer Therapy

[0115] Provided herein are methods for treating cancer or delaying cancer progression in an individual, comprising applying an effective amount of antigen-specific cell (e.g., autologous or allogeneic T cell (e.g., regulatory T cell, CD4+T cell, CD8+T cell or γ-δT cell), NK cell, constant NK cell, NKT cell, mesenchymal stem cell (MSC) or induced pluripotent stem (iPS) cell) therapy to the individual, such as SLC45A2-specific cell therapy. Also provided herein are adoptive T cell therapies using genetically engineered TCR-transduced T cells (e.g., expressing a TCR comprising one of SEQ ID NOs: 51-70). In other embodiments, provided are methods for treating cancer (e.g., melanoma) comprising immunizing a subject with a purified tumor antigen or an immunodominant tumor antigen-specific peptide. In certain embodiments, adoptive cell transfer therapy is provided to a subject (e.g., a human patient) in combination with a second therapy (such as chemotherapy, radiotherapy, surgery, or a second immunotherapy).

[0116] The embodiments of the present disclosure relate to obtaining TCR engineered cells and administering them to subjects as immunotherapy to target cancer cells. Specifically, TCR engineered cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+T cells, CD8+T cells or γ-δT cells), NK cells, constant NK cells, NKT cells, mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells) are antigen-specific cells (e.g., SLC45A2-specific cells). Several basic schemes for the derivation, activation and amplification of functional anti-tumor effector cells have been described in the past two decades. These include: autologous cells, such as tumor infiltrating lymphocytes (TIL); T cells activated in vitro using autologous DC, lymphocytes, artificial antigen presenting cells (APC) or beads coated with T cell ligands and activating antibodies, or cells separated by capturing target cell membranes; allogeneic cells that naturally express anti-host tumor T cell receptors (TCR); and non-tumor-specific autologous or allogeneic cells that are genetically reprogrammed or "redirected" to express tumor-reactive TCR or chimeric TCR molecules (which exhibit antibody-like tumor recognition capabilities, referred to as "T-bodies"). These protocols have produced many protocols for T cell preparation and immunization, which can be used in the methods described herein.

[0117] AT cell preparation and administration

[0118] In certain embodiments, the T cells are autologous. However, the cells can be allogeneic. In certain embodiments, the T cells are isolated from a patient, so the cells are autologous. If the T cells are allogeneic, T cells can be pooled from several donors. The cells are administered to the target subject in an amount sufficient to control, reduce or eliminate the symptoms and signs of the disease being treated.

[0119] In certain embodiments, the T cells are derived from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In certain aspects, the cells are human cells. The cells are typically primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. In certain embodiments, the cells include one or more subsets of T cells or other cell types, such as a total T cell population, a CD4 + Cells, CD8 + Cells, and subpopulations thereof, such as those defined by: function, activation state, maturity, differentiation potential, amplification, recirculation, location and / or persistence, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion characteristics, and / or degree of differentiation. Related to the subject to be treated, the cell can be allogeneic and / or autologous. In some aspects, for example, for existing technologies, the cell is pluripotent and / or omnipotent, such as stem cells, such as induced pluripotent stem cells (iPSC). In certain embodiments, as described herein, the method includes separating cells from the subject, preparing, processing, culturing and / or engineering them, and reintroducing them into the same patient before or after cryopreservation.

[0120] In T cell subtypes and subsets (e.g., CD4 + and / or CD8 + T cells) are the initial T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (TSC M ) cells, central memory T (TC M ) cells, effector memory T (T EM ) cells or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal layer associated constant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells and δ / γ T cells.

[0121] In certain embodiments, one or more T cell populations are enriched or depleted of cells that are positive for a particular marker (e.g., surface marker), or cells that are negative for a particular marker. In some cases, such markers are absent or expressed at relatively low levels on certain T cell (e.g., non-memory cell) populations, but present or expressed at relatively high levels on certain other T cell (e.g., memory cell) populations.

[0122] In certain embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. + or CD8 + Selection step for separation of CD4 + Helper T cells and CD8 + Cytotoxic T cells. Such CD4 + and CD8 + Populations can be further classified into subpopulations.

[0123] In certain embodiments, CD8 + T cells are further enriched or depleted for naive cells, central memory cells, effector memory cells, and / or central memory stem cells, for example, by positive or negative selection based on surface antigens associated with the corresponding subpopulations. In certain embodiments, central memory T (T CM ) cells to improve efficacy, for example to improve long-term survival, expansion and / or engraftment after administration, which in some aspects is particularly robust in such a subpopulation. See Terakura et al. (2012) Blood. 1: 72-82; Wang et al. (2012) J Immunother. 35(9): 689-701.

[0124] In certain embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension is obtained by any suitable means, such as mechanically (using, for example, gentleMACS TM Single cell suspensions can be obtained by dissociating tumors using a dissociator (Miltenyi Biotec, Auburn, Calif.) or enzymatically (e.g., collagenase or DNase). Single cell suspensions of tumor enzymatic digests are cultured in interleukin-2 (IL-2). Cells are cultured until confluent (e.g., about 2×10 6Lymphocytes), for example, from about 5 days to about 21 days, preferably from about 10 days to about 14 days. For example, the cells can be cultured from 5 days, 5.5 days or 5.8 days to 21 days, 21.5 days or 21.8 days, such as from 10 days, 10.5 days or 10.8 days to 14 days, 14.5 days or 14.8 days.

[0125] The T cells of culture can be merged and rapidly expanded. Rapid expansion can provide the number of antigen-specific T cells in the time of about 10 days to about 14 days at least about 50 times (e.g., 50-, 60-, 70-, 80-, 90- or 100-fold or larger) increase. More preferably, rapid expansion can provide in the time of about 10 days to about 14 days at least about 200 times (e.g., 200-, 300-, 400-, 500-, 600-, 700-, 800-, 900-fold or larger) increase.

[0126] Expansion can be accomplished by any of a number of methods known in the art. For example, T cells can be rapidly expanded using nonspecific T cell receptor stimulation in the presence of feeder lymphocytes and interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. Nonspecific T cell receptor stimulation can include about 30 ng / ml of OKT3 (a mouse monoclonal anti-CD3 antibody available from Ortho- Raritan, NJ). Alternatively, T cells can be rapidly expanded by stimulating peripheral blood mononuclear cells (PBMCs) in vitro with one or more cancer antigens (including antigenic portions thereof, such as epitopes or cells) (which can optionally be expressed from a vector, such as human leukocyte antigen A2 (HLA-A2) binding peptides) in the presence of T cell growth factors (e.g., such as 300 IU / ml IL-2 or IL-15, with IL-2 being preferred). T cells induced in vitro are rapidly expanded by restimulation with the same cancer antigen on antigen presenting cells pulsed to express HLA-A2. Alternatively, T cells can be restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0127] Autologous T cells can be modified to express T cell growth factors that promote the growth and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL) -2, IL-7, IL-15 and IL-12. Suitable modification methods are known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In a particular aspect, modified autologous T cells express T cell growth factors at high levels. T cell growth factor coding sequences, such as the coding sequence of IL-12, are readily available in the art, as are promoters, and their operable connection to the T cell growth factor coding sequence promotes high-level expression.

[0128] In certain embodiments, the T cell growth factor that promotes the growth and activation of autologous T cells is administered to the subject simultaneously with or after the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the growth and activation of autologous T cells. The example of suitable T cell growth factor includes interleukin (IL) -2, IL-7, IL-15 and IL-12, which can be used alone or in different combinations, such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL2. IL-12 is a preferred T cell growth factor.

[0129] T cells can be administered intravenously, intramuscularly, subcutaneously, transdermally, intraperitoneally, intrathecally, parenterally, intrathecally, intracavitary, intraventricularly, intraarterially, or via the cerebrospinal fluid, or by any implantable or semi-implantable permanent or degradable device. The appropriate dose of T cell therapy can be determined based on the type of disease to be treated, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the judgment of the attending physician.

[0130] For discrete, solid, accessible tumors, intratumoral injection or injection into the tumor vasculature is particularly contemplated. Local, regional or systemic administration may also be appropriate. For tumors > 4 cm, the volume to be administered will be about 4-10 ml (particularly 10 ml), while for tumors < 4 cm, a volume of about 1-3 ml (particularly 3 ml) will be used. Multiple injections delivered as a single dose contain a volume of about 0.1 to about 0.5 ml.

[0131] In certain embodiments, naked DNA or a suitable vector encoding CAR can be introduced into the T cells of the subject (e.g., T cells obtained from human patients with cancer or other diseases). Methods for stably transfecting T cells using naked DNA by electroporation are known in the art. See, for example, U.S. Patent No. 6,410,319. Naked DNA generally refers to the DNA encoding the chimeric receptor of the present invention contained in a plasmid expression vector in an appropriate expression direction. In certain embodiments, the use of naked DNA can reduce the time required to produce T cells (which express the CAR produced by the method of the present invention).

[0132] Alternatively, a viral vector (e.g., a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector) can be used to introduce the chimeric construct into T cells. Typically, the vector encoding the CAR for transfecting T cells from a subject should generally not replicate in the T cells of the subject. A large number of viral-based vectors are known in which the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell. Exemplary vectors include pFB-neo vectors. and vectors based on HIV, SV40, EBV, HSV, or BPV.

[0133] Once it is determined that the transfected or transduced T cells can express CAR as a surface membrane protein with the required regulation and the required level, it is possible to determine whether the chimeric receptor has a function in the host cell to provide the required signal induction. Subsequently, the transduced T cells can be reintroduced or administered to the subject to activate the anti-tumor response in the subject. In order to promote administration, the transduced T cells can be made into a pharmaceutical composition or an implant suitable for in vivo administration together with a suitable carrier or diluent, and the carrier or diluent is preferably pharmaceutically acceptable. Methods for preparing such compositions or implants have been described in the art (see, for example, Remington's Pharmaceutical Sciences, 16th edition, Mack, ed. (1980)). Where appropriate, the transduced T cells expressing CAR can be formulated into a semisolid or liquid form of preparation, such as a capsule, solution, injection, inhalant or aerosol in a conventional manner for their corresponding route of administration. Means known in the art can be used to prevent or minimize the release and absorption of the composition before reaching the target tissue or organ, or to ensure the timed release of the composition. Generally, it is preferred to use a pharmaceutically acceptable form that does not affect the cells expressing the chimeric receptor. Thus, ideally, the transduced T cells can be formulated into a pharmaceutical composition containing a balanced salt solution such as Hanks balanced salt solution or normal saline.

[0134] B. Antigen presenting cells

[0135] Antigen presenting cells, which include macrophages, B lymphocytes and dendritic cells, are distinguished by their expression of specific MHC molecules. APCs internalize antigens and re-express a portion of the antigen together with MHC molecules on their outer cell membrane. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. The MHC loci encode two major classes of MHC membrane molecules, called class I and class II MHC. T helper lymphocytes usually recognize antigens bound to MHC class II molecules, while T cytotoxic lymphocytes recognize antigens bound to MHC class I molecules. In humans, MHC is called the HLA complex and in mice it is called the H-2 complex.

[0136] In some cases, artificial antigen presenting cells (aAPC) can be used to prepare CAR-based therapeutic compositions and cell therapy products. For general guidance on the preparation and use of antigen presentation systems, see, for example, U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO2007 / 103009.

[0137] aAPC can be used to expand T cells expressing CAR. When encountering tumor antigens, the signal delivered to T cells by antigen presenting cells can affect T cell programming and its subsequent therapeutic effect. This has stimulated the efforts to develop artificial antigen presenting cells, which allow optimal control of the signal provided to T cells (Turtle et al., 2010). In addition to the antibody or antigen of interest, the aAPC system can also include at least one exogenous auxiliary molecule. Any suitable number and combination of auxiliary molecules can be used. Auxiliary molecules can be selected from auxiliary molecules such as costimulatory molecules and adhesion molecules. Exemplary costimulatory molecules include CD70 and B7.1 (also referred to as B7 or CD80), which can bind to CD28 and / or CTLA-4 molecules on the surface of T cells, thereby affecting, for example, T cell expansion, Th1 differentiation, short-term T cell survival and cytokine secretion, such as interleukin (IL) -2 secretion (see Kim et al., 2004). Adhesion molecules can include: carbohydrate binding glycoproteins, such as selectins; transmembrane binding glycoproteins, such as integrins; calcium-dependent proteins, such as cadherins; and single-pass transmembrane immunoglobulin (Ig) superfamily proteins, such as intercellular adhesion molecules (ICAMs), which promote, for example, cell-to-cell or cell-to-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1. Techniques, methods and reagents that can be used to select, clone, prepare and express exemplary auxiliary molecules (including co-stimulatory molecules and adhesion molecules) are exemplified in, for example, U.S. Patent Nos. 6,225,042, 6,355,479 and 6,362,001.

[0138] C. Nucleic Acids

[0139] In one aspect, the disclosure provides a nucleic acid encoding an isolated TCR, CAR, or soluble peptide that selectively binds to SLC45A2 (e.g., 382-390 or SLC45A2 393-402 The peptides may be immunogenic epitopes) and have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the TCR variable regions disclosed herein (e.g., SEQ ID NOs: 1-50), or the peptides may have 1, 2, 3 or 4 point mutations (e.g., substitution mutations) compared to SEQ ID NOs: 1-50. As described above, the peptides may have a length of, for example, 8-35 amino acids, or any range derived therefrom. In certain embodiments, the tumor antigen-specific peptides correspond to portions of tumor antigen proteins such as SLC45A2. The term "nucleic acid" is intended to include DNA and RNA, and may be double-stranded or single-stranded.

[0140] Some embodiments of the present disclosure provide recombinantly produced tumor antigen-specific peptides (e.g., SLC45A2 peptides) that can specifically bind to HLA-A*0201. Thus, nucleic acids encoding tumor antigen-specific peptides can be operably linked to expression vectors, and the peptides can be produced in suitable expression systems using methods well known in the field of molecular biology. Nucleic acids encoding tumor antigen-specific peptides disclosed herein can be incorporated into any expression vector that ensures good expression of the peptides. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), as long as the vector is suitable for transformation of host cells.

[0141] A recombinant expression vector "suitable for transformation of a host cell" means that the expression vector contains a nucleic acid molecule of the present invention and a regulatory sequence selected based on the host cell to be used for expression, operably linked to the nucleic acid molecule. The terms "operably linked" or "operably linked" are used interchangeably and mean that the nucleic acid is linked to a regulatory sequence in a manner that allows expression of the nucleic acid.

[0142] Therefore, the present invention provides a recombinant expression vector comprising a nucleic acid encoding a tumor antigen-specific peptide and necessary regulatory sequences for transcription and translation of the inserted protein sequence. Suitable regulatory sequences can be derived from a variety of sources, including bacteria, fungi or viral genes (e.g., see the regulatory sequences described in Goeddel (1990)).

[0143] The selection of suitable regulatory sequences usually depends on the selected host cell and can be easily achieved by those of ordinary skill in the art. Examples of such regulatory sequences include: transcription promoters and enhancers or RNA polymerase binding sequences, ribosome binding sequences, which include translation initiation signals. In addition, other sequences (such as replication origins, other DNA restriction sites, enhancers, and sequences that confer transcription induction ability) may also be incorporated into the expression vector, depending on the selected host cell and the vector used. It should also be understood that the necessary regulatory sequences may be provided by native proteins and / or their flanking regions.

[0144] The recombinant expression vector may also contain a selective marker gene that facilitates the selection of host cells transformed or transfected with the recombinant tumor antigen-specific peptides disclosed herein (e.g., SLC45A2 peptides). Examples of selective marker genes are genes encoding, for example, proteins that confer resistance to certain drugs, such as G418 and hygromycin; β-galactosidase; chloramphenicol acetyltransferase; or firefly luciferase. The transcription of the selective marker gene is monitored by changes in the concentration of a selective marker protein such as β-galactosidase, chloramphenicol acetyltransferase or firefly luciferase. If the selective marker gene encodes a protein that confers antibiotic resistance (e.g., neomycin resistance), transformant cells can be selected with G418. Cells that have incorporated the selective marker gene will survive, while other cells will die. This makes it possible to visualize and measure the expression of the recombinant expression vector, and specifically determine the effects of mutations on expression and phenotype. It should be understood that the selective marker can be introduced onto a vector separate from the target nucleic acid.

[0145] The recombinant expression vector can be introduced into a host cell to produce a transformant host cell. The term "transformant host cell" is intended to include prokaryotic and eukaryotic cells that have been transformed or transfected with the recombinant expression vector of the present invention. The terms "transformed with ... ", "transfected with ... ", "conversion" and "transfection" are intended to include the introduction of nucleic acid (e.g., vector) into a cell by one of many possible techniques known in the art. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. For example, the protein of the present invention can be expressed in bacterial cells (e.g., E. coli), insect cells (using baculovirus), yeast cells, or mammalian cells.

[0146] Nucleic acid molecules of the invention can also be chemically synthesized using standard techniques. A variety of methods for chemically synthesizing polydeoxynucleotides are known, including solid phase synthesis, which, similar to peptide synthesis, has been fully automated in commercially available DNA synthesizers (see, e.g., U.S. Pat. Nos. 4,598,049; 4,458,066; 4,401,796; and 4,373,071).

[0147] II. Pharmaceutical Preparations

[0148] In selected embodiments, it is contemplated that cells expressing a TCR as disclosed herein, proteins containing the variable regions of a TCR, or DNA encoding the variable regions of a TCR of the invention may be included in a vaccine composition and administered to a subject to induce a therapeutic immune response in the subject against a cancer expressing SLC45A2 (e.g., melanoma). A therapeutic composition for pharmaceutical use in a subject may comprise a TCR composition disclosed herein, such as a soluble TCR (optionally linked to an imaging agent) and a pharmaceutically acceptable carrier.

[0149] The phrases "drug", "pharmaceutically acceptable", or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when properly administered to animals, such as humans. "Pharmaceutically acceptable carriers" as used herein include any and all solvents, dispersion media, coating agents, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials and combinations thereof, as known to those of ordinary skill in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, Pharmaceutical Press, 2011, incorporated herein by reference). Unless any conventional carrier is incompatible with the active ingredient, its use in the vaccine composition of the present invention is considered.

[0150] As used herein, "protective immune response" refers to the response of the immune system of a mammalian host to cancer. Protective immune response can provide therapeutic effects for the treatment of cancer, such as reducing tumor size, increasing survival, etc.

[0151] Those of ordinary skill in the medical arts will appreciate that the actual dosage of the therapeutic composition administered to an animal or human patient can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, previous or concurrent therapeutic interventions, idiopathic conditions of the patient, and route of administration. In any case, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for an individual subject.

[0152] The therapeutic compositions disclosed herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, and by inhalation, injection, infusion, continuous infusion, lavage, and local perfusion. The therapeutic compositions can also be administered to a subject by catheter, in an emulsion, in a lipid composition, by ballistic microparticle delivery, or by other methods or any combination of the foregoing methods, as known to those of ordinary skill in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams and Wilkins, 2005, incorporated herein by reference).

[0153] Although any suitable carrier known to those of ordinary skill in the art can be used in the pharmaceutical composition of the present invention, the type of carrier will vary with the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, fat, wax or buffer. For oral administration, any of the above-mentioned carriers or solid carriers can be used, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum powder, cellulose, glucose, sucrose and magnesium carbonate. Biodegradable microspheres (such as polylactic acid galactose) can also be used as the carrier of the pharmaceutical composition of the present invention. Suitable biodegradable microspheres are disclosed in, for example, U.S. Patents 4,897,268 and 5,075,109.

[0154] In certain embodiments, the vaccine composition may be administered via microstructured transdermal or ballistic microparticle delivery. Microstructures as carriers for vaccine formulations are ideal configurations for vaccine applications and are well known in the art (Gerstel and Place 1976 (U.S. Pat. No. 3,964,482); Ganderton and McAinsh 1974 (U.S. Pat. No. 3,814,097); U.S. Pat. Nos. 5,797,898, 5,770,219 and 5,783,208 and U.S. Patent Application 2005 / 0065463). Such vaccine compositions formulated for ballistic microparticle delivery may comprise an isolated SLC45A2 peptide disclosed herein immobilized on the surface of a support substrate. In these embodiments, the support substrate may include, but is not limited to, microcapsules, microparticles, microspheres, nanocapsules, nanoparticles, nanospheres, or combinations thereof.

[0155] The microstructures or ballistic microparticles disclosed herein that serve as support substrates for TCRs (such as soluble TCRs) can be composed of biodegradable and non-biodegradable materials, and such support substrates can be composed of synthetic polymers, silica, lipids, carbohydrates, proteins, lectins, ionic agents, cross-linking agents, and other microstructure components available in the art. Protocols and reagents for immobilizing the peptides of the present invention onto support substrates composed of such materials are widely available commercially and in the art.

[0156] In other embodiments, the vaccine composition comprises an immobilized or encapsulated TCR or soluble TCR disclosed herein and a supporting substrate. In these embodiments, the supporting substrate may include, but is not limited to, lipid microspheres, lipid nanoparticles, ethosomes, liposomes, niosomes, phospholipids, sphingosomes, surfactants, transferosomes, emulsions, or combinations thereof. The formation and use of liposomes and other lipid nanocarriers and microcarrier preparations are generally known to those of ordinary skill in the art, and the application of liposomes, microparticles, nanocapsules, etc. has been widely used in the delivery of therapeutic agents (e.g., U.S. Patent No. 5,741,516, particularly incorporated herein by reference in its entirety). Numerous methods of liposomes and liposome-like preparations as potential drug carriers have been reviewed, including the encapsulation of peptides (U.S. Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587, each of which is particularly incorporated herein by reference in its entirety).

[0157] In addition to the delivery methods described herein, a number of alternative techniques for administering the disclosed vaccine compositions are contemplated. As non-limiting examples, the vaccine compositions may be administered as follows: by sonophoresis (i.e., ultrasound), which has been used and described in U.S. Patent No. 5,656,016 to increase the rate and efficacy of drug penetration into and through the circulatory system; intraosseous injection (U.S. Patent No. 5,779,708); or feedback-controlled delivery (U.S. Patent No. 5,697,899), and each of the patents in this paragraph is specifically incorporated herein by reference in its entirety.

[0158] Any of a variety of adjuvants can be used in the vaccine of the present invention to non-specifically enhance the immune response. Most adjuvants contain substances intended to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, and non-specific stimulants of immune response, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis. Suitable adjuvants are commercially available, for example, as Freund's incomplete adjuvant and Freund's complete adjuvant (Difco Laboratories, Detroit, Mich.) and Merck adjuvant 65 (Merck and Company, Inc., Rahway, NJ). Other suitable adjuvants include alum, biodegradable microspheres, monophosphoryl lipid A and quil A.

[0159] The soluble TCR can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins), and they are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed with free carboxyl groups can also be derived from inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or ferric hydroxide) and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0160] In any case, the composition may contain various antioxidants to prevent oxidation of one or more components. In addition, the action of microorganisms may be prevented by preservatives such as various antibacterial and antifungal agents including, but not limited to, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

[0161] Aseptic injectable solution is prepared as follows: the active peptide of the required amount is mixed in a suitable solvent together with the required various other ingredients mentioned above, and then sterilized by filtration. Usually, dispersion is prepared as follows: various sterilized active ingredients are mixed in the sterile vehicle containing basic dispersion medium and / or other ingredients. With regard to the sterile powder for preparing aseptic injectable solution, suspension or emulsion, the preferred preparation method is vacuum drying or freeze drying technology, which produces active ingredient plus any additional desired component powder of its liquid medium from previous aseptic filtration. If necessary, the liquid medium should be appropriately buffered, and before injection with enough saline or glucose, the liquid diluent is first made isotonic. The preparation of high-concentration compositions for direct injection is also considered, wherein it is envisioned that DMSO is used as a solvent to cause extremely fast penetration, thereby delivering high concentrations of active agents to small areas.

[0162] The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. It will be appreciated that endotoxin contamination should be minimized to safe levels, for example less than 0.5 ng / mg protein.

[0163] In specific embodiments, prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents that delay absorption, for example, aluminum monostearate, gelatin, or a combination thereof.

[0164] A. Combination therapy

[0165] In certain embodiments, the compositions and methods of the embodiments of the present invention relate to a population of antigen-specific cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+T cells, CD8+T cells, or γ-δT cells), NK cells, constant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells) in combination with at least one additional therapy. The additional therapy can be radiation therapy, surgery (e.g., lesion resection and mastectomy), chemotherapy, gene therapy, DNA therapy, virotherapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy can be in the form of adjuvant therapy or neoadjuvant therapy.

[0166] In certain embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In certain embodiments, the additional therapy is the administration of a side effect limiter (e.g., an agent intended to reduce the occurrence and / or severity of the side effects of treatment, such as an anti-nausea agent, etc.). In certain embodiments, the additional therapy is radiation therapy. In certain embodiments, the additional therapy is surgery. In certain embodiments, the additional therapy is a combination of radiation therapy and surgery. In certain embodiments, the additional therapy is gamma irradiation. In certain embodiments, the additional therapy is chemotherapy, for example, dacarbazine or temozolomide. The additional therapy can be one or more of chemotherapeutic agents known in the art.

[0167] T cell therapy can be administered before, during, after or in different combinations relative to other cancer therapies (such as immune checkpoint therapy). The administration can be performed at intervals from simultaneously to several minutes to several days to several weeks. In the embodiment in which T cell therapy is provided to the patient separately from other therapeutic agents, it is generally ensured that a considerable period of time will not be spent between the time of each delivery, so that the two compounds can still play a favorable combination effect on the patient. In such a case, it is considered that within about 12 hours to 24 hours or 72 hours, and more particularly, within about 6 to 12 hours, antibody therapy and anticancer therapy can be provided to the patient. In some cases, it may be desirable to significantly extend the time period of treatment, wherein between each administration, a few days (2, 3, 4, 5, 6 or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8 weeks) are spaced.

[0168] A variety of combinations can be used. For the following examples, antigen-specific T cell therapy, peptide or TCR is "A" and anti-cancer therapy is "B":

[0169] A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B

[0170] B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A

[0171] B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0172] Administration of any compound or therapy of the present embodiments to a patient will follow the general regimen for administration of such compounds, taking into account the toxicity, if any, of the agent. Thus, in certain embodiments, there is a step of monitoring toxicity attributable to the combination therapy.

[0173] 1. Chemotherapy

[0174] A variety of chemotherapeutic agents can be used according to embodiments of the present invention. The term "chemotherapy" means the use of drugs to treat cancer. "Chemotherapeutic agent" is used to refer to a compound or composition used in the treatment of cancer. These agents or drugs are classified by their intracellular activity patterns (e.g., whether they affect the cell cycle and at what stage they affect the cell cycle). Alternatively, agents are characterized based on their ability to directly crosslink DNA, embed into DNA, or induce chromosome and mitotic aberrations by affecting nucleic acid synthesis.

[0175] Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; tatin); CC-1065 (including its synthetic analogs adolesin, kazelesin and bitrazelasin); Nostoc cyclic peptides (especially Nostoc cyclic peptide 1 and Nostoc cyclic peptide 8); dolastatin; dukamycin (including synthetic analogs KW-2189 and CB1-TM1); soft coral alcohol; pancratistatin; sarcodictyin; spongestatin; nitrogen mustards such as chlorambucil, naphthyl nitrogen mustard, cholophosphamide, estramustine, ifosfamide, nitrogen mustard, nitrogen oxide mustard hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosfamide and uramustine; nitroureas such as carmustine, chlorambucil ... urea, fotemustine, lomustine, nimustine and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, especially calicheamicin gamma lI and calicheamicin omega lI); danemycins, including danemycin A; bisphosphonates such as clodronate; esperamicins; and the neocarcinogen chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authrarnycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin, carmomycin, chromomycin, dactinomycin, daunorubicin, detopicin, 6-diazo-5-oxo-L-leuproin aminidine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivetomycin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozocin, streptozocin, tuberculocidin, ubenimex, zoloft, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as folinic acid, pteropterin and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiabendazole and thioguanine;Pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as captestosterone, drostanolone propionate, cyclothiocarb, melastane, and testolactone; antiadrenal agents such as mitotane and trilostane; folic acid supplements such as folinic acid; aceglucuronolide; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; colcemid; diazocone; elformithine; elformithine; epothilone; epothilone; etoglucose; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansines such as maytansine and ansamitocin ; mitoxantrone; mopidarol; nitrilosine; pentostatin; methambucil; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizoran; spirogermanium; tenuzolic acid; triazinon; 2,2′,2″-trichlorotriethylamine; trisporin; trisporin; urethran; urethranol ... an); vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; taxanes, such as paclitaxel and docetaxel; gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Chunruibin; Noxolin; Teniposide; Edatrexate; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (e.g., CPT-11); Topoisomerase inhibitor RFS2000; Difluoromethylornithine (DMFO); Retinoids, such as retinoic acid; Capecitabine; Carboplatin, procarbazine, plicamycin, gemcitabine, Navelbine, farnesyl-protein transferase inhibitors, transplatinum, and any of the above pharmaceutically acceptable salts, acids or derivatives. ;

[0176] 2. Radiation therapy

[0177] Other factors that cause DNA damage and have been widely used include those commonly referred to as gamma rays, X-rays and / or the directional delivery of radioisotopes to tumor cells. Other forms of DNA damage factors have also been considered, such as microwaves, proton beam irradiation (USPs 5,760,395 and 4,870,287) and UV irradiation. Most likely, all these factors cause wide-range damage to DNA, DNA precursors, replication and repair of DNA and chromosomal assembly and maintenance. The dosage range of X-rays is from a daily dose of 50-200 roentgens for a long period of time (3 to 4 weeks) to a single dose of 2000-6000 roentgens. The dosage range of radioisotopes varies widely, and depends on the half-life of the isotope, the intensity and type of the radiation emitted and the uptake of tumor cells.

[0178] 3. Immunotherapy

[0179] The skilled artisan will appreciate that additional immunotherapies may be combined or used in conjunction with the methods of the embodiments. In the context of cancer treatment, immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab It is such an example.Immune effectors can be antibodies specific to some markers on the tumor cell surface, for example. The antibody can serve as the effector of treatment individually, or it can recruit other cells to actually affect cell killing. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and serve as targeting agents. Alternatively, effectors can be lymphocytes carrying surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0180] A breakthrough approach has emerged in the development of antibody-drug conjugates as cancer therapeutics. Cancer is one of the leading causes of death in the world. Antibody-drug conjugates (ADCs) comprise a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of the MAb for its antigen target with a highly potent cytotoxic drug, resulting in an "armed" MAb that delivers the cargo (drug) to tumor cells that have abundant levels of the antigen. Targeted delivery of the drug also minimizes its exposure to normal tissues, resulting in reduced toxicity and an improved therapeutic index. The FDA approval of two ADC drugs (in 2011) and the approval of the ADC drug by the FDA (in 2013) have resulted in a significant increase in the number of ADCs. (Brentuximab vedotin) and 2013 (trastuzumab emtansine or T-DM1)) validated this approach. There are currently more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-cargo optimization become more and more mature, the discovery and development of new ADCs increasingly rely on the identification and validation of new targets suitable for this approach and the generation of targeted MAbs. Two criteria for ADC targets are upregulated / high levels of expression and robust internalization in tumor cells.

[0181] In one aspect of immunotherapy, tumor cells must carry some markers suitable for targeting, that is, the markers are not present on most other cells. There are many tumor markers, and any of these tumor markers may be suitable for targeting in the context of embodiments of the present invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is to combine anticancer effects with immunostimulatory effects. There are also immunostimulatory molecules, including: cytokines, such as IL-2, IL-4, IL-12, GM-CSF, γ-IFN, chemokines, such as MIP-1, MCP-1, IL-8, and growth factors, such as FLT3 ligands.

[0182] Examples of immunotherapies currently under investigation or in use are immune adjuvants, such as Mycobacterium bovis, Plasmodium falciparum), dinitrochlorobenzene, and aromatic compounds (U.S. Pat. Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapy, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945); and monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Pat. No. 5,824,311). It is contemplated that one or more anti-cancer therapies may be used with the antibody therapies described herein.

[0183] In certain embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints raise signals (e.g., co-stimulatory molecules) or lower signals. Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include: adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activation gene-3 (LAG3), programmed death 1 (PD-1), T-cell immunoglobulin domain and mucin domain 3 (TIM-3) and V domain Ig inhibitor of T cell activation (VISTA). Specifically, immune checkpoint inhibitors target PD-1 axis and / or CTLA-4.

[0184] Immune checkpoint inhibitors can be drugs, such as small molecules, recombinant forms of ligands or receptors, or in particular antibodies, such as human antibodies (e.g., International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4):252-64, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or analogs thereof can be used, in particular chimeric, humanized or human antibodies can be used. As will be known to the skilled person, alternative and / or equivalent names can be used for certain antibodies mentioned in the present disclosure. In the context of the present disclosure, such alternative and / or equivalent names are interchangeable. For example, it is known that lambrolizumab is also known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0185] In certain embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, for example, as described in US Patent Application Nos. US20140294898, US2014022021, and US20110008369, all of which are incorporated herein by reference.

[0186] In certain embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In certain embodiments, the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, and CT-011. In certain embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin containing an extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In certain embodiments, the PD-1 binding antagonist is AMP-224. Nivolumab (also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and ) is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab (also known as MK-3475, Merck 3475, pembrolizumab, and SCH-900475) is an anti-PD-1 antibody described in WO2009 / 114335. CT-011 (also known as hBAT or hBAT-1) is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 (also known as B7-DCIg) is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0187] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is present on the surface of T cells and acts as a "close" switch when combined with CD80 or CD86 on the surface of antigen presenting cells. CTLA4 is a member of the immunoglobulin superfamily, which is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to T cell co-stimulatory protein CD28, and these two molecules bind CD80 and CD86 (also referred to as B7-1 and B7-2) on antigen presenting cells. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulating signals. Intracellular CTLA4 is also present in regulatory T cells and may be important to its function. T cell activation by T cell receptor and CD28 can lead to increased CTLA-4 (inhibitory receptor of B7 molecule) expression.

[0188] In certain embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (eg, a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0189] Anti-human-CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in the methods of the invention can be generated using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in the following can be used in the methods disclosed herein: US 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly tesimumab), U.S. Pat. No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17):10067-10071; Camacho et al. (2004) J Clin Oncology 22(145):Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304. The teachings of each of the above publications are hereby incorporated by reference. Antibodies that compete with any of these antibodies known in the art for binding to CTLA-4 may also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001014424, WO2000037504, and U.S. Patent No. 8,017,114 (all incorporated herein by reference).

[0190] An exemplary anti-CTLA-4 antibody is ipilimumab (also known as 10D1, MDX-010, MDX-101, and ) or antigen-binding fragments and variants thereof (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes with the above antibodies for binding to the same epitope on CTLA-4 and / or binds to the same epitope on CTLA-4. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the above antibodies (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0191] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors described, for example, in U.S. Pat. Nos. 5,844,905, 5,885,796 and International Patent Application Nos. WO1995001994 and WO1998042752 (all incorporated herein by reference), and immunoadhesins described, for example, in U.S. Pat. No. 8,329,867 (incorporated herein by reference).

[0192] 4. Surgery

[0193] Approximately 60% of people with cancer will undergo some type of surgery, including preventive, diagnostic or staging, curative, and palliative surgery. Curative surgery includes resection in which all or a portion of cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies (e.g., treatment according to embodiments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapy). Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and surgery controlled with a microscope (Mohs surgery).

[0194] After removing a portion or all of the cancerous cells, tissues or tumors, a cavity can be formed in vivo. Treatment can be achieved by perfusion, direct injection or other anticancer therapy applied locally to the region. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6 or 7 days, or every 1, 2, 3, 4 and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. These treatments can also have different doses.

[0195] 5. Other medicines

[0196] It is contemplated that other agents may be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic effect of treatment. These other agents include agents that affect the upregulation of cell surface receptors and GAP connections, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of overproliferation cells to apoptosis inducers, or other biological agents. The increase in intercellular signaling achieved by increasing the number of GAP connections will increase the anti-overproliferation effect on adjacent overproliferation cell colonies. In other embodiments, cell growth inhibitors or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-overproliferation efficacy of treatment. Cell adhesion inhibitors are considered to improve the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents (e.g., antibody c225) that increase the sensitivity of overproliferation cells to apoptosis may be used in combination with certain aspects of the embodiments of the present invention to improve therapeutic efficacy.

[0197] III. Embodiment

[0198] The following examples are included to demonstrate the preferred embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present invention and thus can be considered to constitute the preferred modes of its practice. However, it will be appreciated by those skilled in the art that many changes can be made in the disclosed specific embodiments and still similar or similar results can be obtained without departing from the spirit and scope of the present invention in light of the present disclosure.

[0199] Example 1

[0200] Human SLC45A2 T cell receptor (TCR) clone

[0201] method

[0202] Generation of T cell clones

[0203] TCR clones were generated by exposing cells to specific SLC45A2 peptides. SLC45A2 was selectively expressed in melanoma compared to normal tissues. SLC45A2 peptides SLC45A2 382-390 and SLC45A2 393-402The peptides are immunogenic epitopes that can selectively bind to HLA-A*0201 (HLA-A2) and HLA-A*2402 (HLA-A24), respectively, and cytotoxic T lymphocytes (CTLs) amplified using these peptides can effectively kill a variety of melanoma cells, including a variety of cutaneous melanomas, uveal melanomas, mucosal melanomas, and metastatic melanomas. The SLC45A2 peptide can exhibit antigen-specific and HLA-A*0201 or HLA A*2402-restricted SLC45A2-specific CD8 T cell responses.

[0204] Full-length VCX3A RNA was transfected into mature dendritic cells (DCs). In the presence of IL-21, RNA-transfected DCs were co-cultured with autologous naive T cells at a ratio of DC:T=1:10. After one week, RNA-transfected DCs were used to stimulate T cells again. After two rounds of stimulation, CD8+ and tetramer+ double-positive T cell populations were sorted and amplified using a rapid expansion protocol (REP). T cell clones were generated using a limiting dilution method. Highly active CTL clones were screened by tumor cell killing assays.

[0205] T cell receptor (TCR) cloning and retroviral expression vector construction

[0206] According to the manual of the kit, TCR (including α chain and β chain) was cloned using the 5'-RACE method. TCR V-α and TCR V-β were identified using the IMGT / V-QUEST annotation tool. In addition, TCR V-β was also identified using flow detection using the TCR VβRepertoire kit. TCR V-α was identified using PCR using a set of specific primers annealing to the 5' end of different TCR V-α. For TCR expression retroviral vector construction, forward primers were designed according to TCR V-α or β usage. Reverse primers were designed according to the sequence of the TCR α or β constant region. Expression cassettes containing α- and β-TCR chains separated by the P2A linker peptide were generated, and the full-length PCR products were cloned into the retroviral vector pMSGV1. The cloned DNA sequence was verified by sequencing.

[0207] Retroviral Generation and Infection of Human Peripheral Blood Lymphocytes (PBL)

[0208] The pMSGV1 vector containing the TCR and the envelope vector RD114 were co-transfected into the packaging cell line GP2-293. After 6-8 hours of transfection, the culture medium was renewed. The supernatant was harvested 24 hours later and added to a 6-well plate coated with 20 mg / mL RetroNectin, followed by centrifugation (2000×g) for 2 hours at 32°C. The supernatant was then removed, and PBL activated for 2 days with 50 ng / ml OKT3 and 300 U / ml IL-2 were added to a plate loaded with retrovirus, followed by centrifugation (1000×g) for 10 minutes at 32°C. The cells were then incubated overnight at 32°C, and the procedure was repeated the next day (a total of two transductions). Thereafter, the cells were expanded at 37°C in a 5% CO2 incubator and split as needed.

[0209] Generation of T cell clones with TCR engineering

[0210] After infection, CD8+ and tetramer+ T cell populations were sorted and T cell clones were generated by limiting dilution. Highly active CTL clones were screened by tumor cell killing assay. High tumor killing activity T cell clones were further expanded with REP.

[0211] IFN-γ release assay

[0212] IFN-γ release from T cells was detected by ELISA. T cells were incubated with target cells at a ratio of 10:1 in a 96-well plate containing 0.2 ml of culture medium at 37° C. After co-culture overnight, the supernatant was harvested and the IFN-γ concentration was detected using ELISA according to the kit manual.

[0213] Intracellular cytokine staining (ICS) assay

[0214] T cells were incubated with target cells at a ratio of 10:1 at 37°C overnight in the presence of Brefeldin A (BFA). After co-cultivation, T cells were harvested and washed. First, cells were stained with flow antibodies against surface markers. Thereafter, cells were washed and fixed with a fixation buffer and then permeabilized using a permeabilization solution. The permeabilized cells were then stained with intracellular cytokine flow antibodies. Finally, FACS was used to analyze the cytokine production levels in the cells.

[0215] Peptide-MHC tetramer staining

[0216] By using SLC45A2 382-390 Peptide / MHC complex (for HLA A*0201) or SLC45A2 393-402Tetramer staining of peptide / MHC complexes (for HLA A*2402) confirmed SLC45A2-specific CD8 T cells. CD8 T cells were incubated with PE-conjugated tetramers for 20 minutes, washed, and then stained with APC-conjugated CD8 antibody for 15 minutes at room temperature. After washing, cells were analyzed by flow cytometry (LSRFortessa X-20 analyzer). 382-390 SLC45A2 393-402 Tetramers of HLA-A*A0201 and HLA-A*A2402 were purchased from Fred Hutchinson Cancer Research Center.

[0217] 51 Chromium release assay

[0218] Use standard 51 The killing ability of TCR-engineered T cells or CTL clones to lyse HLA-A2 tumor targets was measured by Cr release assay. Tumor cells or normal cells were incubated at 37°C with 200 μCi of 51 Cr-labeled for 2 hours, and after washing 3 times, the labeled targets were plated in triplicate at 2000 targets / well. The labeled target cells were washed and then incubated with effector cells at different ratios in 0.2 ml complete medium at 37°C for 4 hours. The harvested supernatants were counted using an automated gamma counter. The maximum and spontaneous cytokines were determined by incubating the labeled target cells in trypan blue lysis buffer or medium at 37°C for 4 hours. 51 Cr release. Each data point was determined as the average of quadruplicate wells. Specific lysis % was calculated as follows: % killing = ((specific release - spontaneous release) / (total release - spontaneous release)) x 100.

[0219] Results: The TCR sequences of several SLC45A2 CD8 T cell clones were determined. The CDR3 sequences of these TCR clones (including β3, β22, #24, #39, and #76) are shown in Table 2. T cells were transfected with each of these TCR clones and expressed using Mel526 (HLA A2 + ) and Mel888 (HLA A2 - ) cells were evaluated for cytotoxic activity by chromium release assay and compared with the activity of the parental T cell clones. For all clones, TCR-transfected T cells were observed to lyse the HLA-A24 matched target Mel888, but not the HLA-A24 mismatched target Mel526 ( Figure 2A -B, Figure 4A -B and Fig. 6A -B). Parental T cells exhibited similar cytotoxicity.

[0220] SLC45A2 tetramer and CD8 staining were also performed on all TCR clones and parental cells. Activated autologous PBMCs were transduced with retrovirus containing the TCR gene. After 8 days, T cells were stained with SLC45A2-PE conjugated tetramers. SLC45A2 tetramer-positive T cells were sorted and REP ( Figure 3A -B, 5A-B and 7A-B).

[0221] Table 2: SLC45A2 TCR gene information.

[0222]

[0223]

[0224] Example 2

[0225] Functionality of Human SLC45A2 T Cell Receptor (TCR) Clone #39

[0226] Tetramer staining of TCR engineered T cells. TCR from SLC45A2 CTL (clone #39) was cloned into the retroviral expression vector pMSGV1, and recombinant retrovirus was generated for infection of PBMCs. After infection, tetramer+ populations of CD8+ and CD4+ T cells appeared. CD8+tetramer+ and CD4+tetramer+ populations were sorted and amplified using the rapid expansion protocol (REP). After amplification, the purity of the CD8+tetramer+ population reached 96% ( Figure 8 However, the tetramer+ population of CD4+ T cells was lost after REP ( Figure 8 ).

[0227] Peptide binding titration assay of TCR engineered T cells. T2 cells were pulsed with different concentrations of SLC45A2 peptide (from 10 pg / mL to 10 μg / mL) and treated with 51 Cr labeling. CD8+ or CD4+ TCR engineered T cells were used as effector cells and co-cultured with T2 cells. The expression of cytokines was detected after 4 hours of co-culture. 51 Cr release. CD8+TCR engineered T cells showed high affinity, but CD4+TCR engineered T cells did not ( Fig. 9 ).

[0228] CD8+TCR engineered T cells recognize endogenously presented epitopes. CD8+TCR engineered T cells were able to kill Mel526 (HLA-A2+, SLC45A2+) and Mel888-A2 (forced expression of HLA-A2, SLC45A2+) tumor cell lines, but not A375 (HLA-A2+, SLC45A2-) or Mel624 (HLA-A2+, SLC45A2+) tumor cell lines ( Fig.10 ). However, T cells were able to kill A375 cells pulsed with SLC45A2 peptide, indicating that Mel526 and Mel888-A2 naturally present endogenous epitopes and TCR-engineered T cells can recognize it. Mel624 may present low levels of epitopes on the cell surface, although it expresses SLC45A2.

[0229] CD4+TCR engineered T cells recognized endogenously presented epitopes. Although CD4+TCR engineered T cells did not significantly generate tetramer+ populations after REP, they still killed tumor cells after long-term co-culture (20 h) ( Fig.11 ). Thus, they can recognize epitopes that are presented endogenously at low levels.

[0230] TCR engineered T cells respond specifically when they encounter target cells. An internal cytokine staining (ICS) assay was performed to detect the specific response of TCR engineered T cells when they encounter target cells. Mel526 (naturally presenting an endogenous epitope of SLC45A2), A375 (SLC45A2 negative), T2 pulsed with SLC45A2 peptide, and T2 pulsed with M26 peptide (negative control) were co-cultured with TCR engineered T cells (CD8+ or CD4+, E:T=10:1). After overnight incubation, TNF-α ( Fig. 12A )、CD107a( Fig. 12B ), IFN-γ( Fig. 12C )、CD137( Fig.12D ) and IL-2( Fig.12E Compared with A375 and T2 pulsed with M26 peptide, both CD8+ and CD4+ TCR engineered T cells expressed significantly higher levels of TNF-α, CD107a, IFN-γ, CD137 and IL-2 when co-cultured with Mel526 and T2 pulsed with SLC45A2 peptide, indicating that TCR engineered T cells are functional and exhibit specific responses when they encounter target cells.

[0231] According to the above, the present application provides the following implementation schemes:

[0232] 1. An engineered T cell receptor (TCR) comprising an α chain CDR3 having an amino acid sequence of SEQ ID NO: 5, 15, 25, 35 or 45 and / or a β chain CDR3 having an amino acid sequence of SEQ ID NO: 10, 20, 30, 40 or 50.

[0233] 2. The TCR of embodiment 1, wherein the engineered TCR binds to HLA-A2.

[0234] 3. A TCR according to embodiment 2, wherein the engineered TCR binds to HLA-A*0201.

[0235] 4. The TCR of embodiment 1, wherein the engineered TCR binds to HLA-A24.

[0236] 5. The TCR of embodiment 4, wherein the engineered TCR binds to HLA-A*2402.

[0237] 6. A TCR according to embodiment 6, wherein the TCR comprises an α chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32 or 42 and / or a β chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37 or 47.

[0238] 7. A TCR according to embodiment 6, wherein the TCR comprises an α chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32 or 42 and / or a β chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37 or 47.

[0239] 8. A TCR according to embodiment 6, wherein the TCR comprises an α chain that is at least 99% identical to the amino acid sequence of SEQ ID NO:2, 12, 22, 32 or 42 and / or a β chain that is at least 99% identical to the amino acid sequence of SEQ ID NO:7, 17, 27, 37 or 47.

[0240] 9. A TCR according to embodiment 1, wherein the TCR comprises an α chain of SEQ ID NO: 2, 12, 22, 32 or 42 and / or a β chain of SEQ ID NO: 7, 17, 27, 37 or 47.

[0241] 10. A TCR according to embodiment 6, wherein the TCR comprises an α chain having at least 95% identity with the nucleotide sequence of SEQ ID NO:1, 11, 21, 31 or 41 and / or a β chain having at least 95% identity with the nucleotide sequence of SEQ ID NO:6, 16, 26, 36 or 46.

[0242] 11. A TCR according to embodiment 6, wherein the TCR comprises an α chain containing a nucleotide sequence of SEQ ID NO: 1, 11, 21, 31 or 41 and / or a β chain containing a nucleotide sequence of SEQ ID NO: 6, 16, 26, 36 or 46.

[0243] 12. A TCR according to embodiment 1, wherein the TCR is further defined as a soluble TCR, wherein the soluble TCR does not contain a transmembrane domain.

[0244] 13. The TCR of any one of embodiments 1-12, further comprising a detectable label.

[0245] 14. A TCR according to any one of embodiments 1-12, wherein the TCR is covalently bound to a therapeutic agent.

[0246] 15. A TCR according to embodiment 14, wherein the therapeutic agent is an immunotoxin or a chemotherapeutic agent.

[0247] 16. A multivalent TCR complex comprising a plurality of TCRs according to any one of embodiments 1-12.

[0248] 17. A complex according to embodiment 16, wherein the multivalent TCR comprises 2, 3, 4 or more TCRs that bind to each other.

[0249] 18. A complex according to embodiment 17, wherein the multivalent TCR is present in a lipid bilayer, in a liposome, or attached to a nanoparticle.

[0250] 19. A complex according to embodiment 17, wherein the TCRs are bound to each other via a linker molecule.

[0251] 20. A polypeptide encoding the TCR of any one of embodiments 1-19.

[0252] 21. A polynucleotide encoding the polypeptide of embodiment 20.

[0253] 22. An expression vector encoding the TCR of any one of embodiments 1-19.

[0254] 23. An expression vector according to embodiment 22, wherein the sequence encoding the TCR is under the control of a promoter.

[0255] 24. An expression vector according to embodiment 22, wherein the expression vector is a viral vector.

[0256] 25. An expression vector according to embodiment 24, wherein the viral vector is a retroviral vector.

[0257] 26. An expression vector according to embodiment 22, wherein the vector further encodes a linker domain.

[0258] 27. An expression vector according to embodiment 26, wherein the linker domain is located between the α chain and the β chain.

[0259] 28. An expression vector according to embodiment 26, wherein the linker domain comprises one or more cleavage sites.

[0260] 29. An expression vector according to embodiment 28, wherein the one or more cleavage sites are furin cleavage sites and / or P2A cleavage sites.

[0261] 30. The expression vector of embodiment 29, wherein the furin cleavage site is RAKR.

[0262] 31. An expression vector according to embodiment 29, wherein the furin cleavage site is ATNFSLLKQAGDVEENPG (SEQ ID NO: 51).

[0263] 32. An expression vector according to embodiment 26, wherein the one or more cleavage sites are separated by spacers.

[0264] 33. An expression vector according to embodiment 32, wherein the spacer is SGSG or GSG.

[0265] 34. A host cell engineered to express the TCR of any one of embodiments 1-12.

[0266] 35. A host cell according to embodiment 34, wherein the cell is a T cell, a NK cell, a constant NK cell, a NKT cell, a mesenchymal stem cell (MSC) or an induced pluripotent stem (iPS) cell.

[0267] 36. A host cell according to embodiment 34, wherein the host cell is an immune cell.

[0268] 37. The host cell of embodiment 34, wherein the host cell is isolated from an umbilical cord.

[0269] 38. The host cell according to embodiment 35, wherein the T cell is a CD8 + T cells, CD4+ T cells or γδ T cells.

[0270] 39. A host cell according to embodiment 35, wherein the T cell is a regulatory T cell (Treg).

[0271] 40. A host cell according to embodiment 34, wherein the cell is autologous.

[0272] 41. A host cell according to embodiment 34, wherein the cell is allogeneic.

[0273] 42. A method for engineering the host cell of embodiment 34, comprising contacting the immune cell with the TCR of any one of embodiments 1-12 or the expression vector of any one of embodiments 22-33.

[0274] 43. A method according to embodiment 42, wherein the immune cells are T cells or peripheral blood lymphocytes.

[0275] 44. The method of embodiment 42, wherein the contacting is further defined as transfection or transduction.

[0276] 45. The method of any one of embodiments 42-44, wherein transfection comprises electroporating RNA encoding the TCR of any one of embodiments 1-12 into immune cells.

[0277] 46. ​​The method of any one of embodiments 44, further comprising producing a viral supernatant from the expression vector of embodiment 22 before transducing the immune cells.

[0278] 47. A method according to any one of embodiments 42-46, wherein the immune cells are stimulated lymphocytes.

[0279] 48. A method according to embodiment 47, wherein the stimulated lymphocytes are human lymphocytes.

[0280] 49. The method of embodiment 47, wherein stimulation comprises contacting the immune cells with OKT3 and / or IL-2 or incubating the immune cells in OKT3 and / or IL-2.

[0281] 50. According to any one of embodiments 42-49, the method further comprises sorting the immune cells to isolate TCR-engineered T cells.

[0282] 51. The method of embodiment 50, further comprising performing T cell cloning by serial dilution.

[0283] 52. The method of embodiment 51, further comprising expanding T cell clones by a rapid expansion protocol.

[0284] 53. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the TCR engineered cells of any one of embodiments 34-40.

[0285] 54. A method according to embodiment 53, wherein the subject is identified as having the HLA-A*0201 allele.

[0286] 55. A method according to embodiment 53, wherein the subject is identified as having the HLA-A*2402 allele.

[0287] 56. A method according to embodiment 53, wherein the TCR engineered cells are T cells or peripheral blood lymphocytes.

[0288] 57. The method according to embodiment 53, wherein the T cells are CD8 + T cells, CD4 + T cells or Tregs.

[0289] 58. A method according to embodiment 53, wherein the cancer is melanoma.

[0290] 59. The method of embodiment 58, wherein the melanoma is cutaneous melanoma, uveal melanoma, mucosal melanoma, or metastatic melanoma.

[0291] 60. A method according to embodiment 53, wherein the subject is human.

[0292] 61. A method according to embodiment 53, wherein the TCR engineered cells are autologous or allogeneic.

[0293] 62. The method of embodiment 53, further comprising depleting the subject's lymphocytes prior to administering the SLC45A2-specific T cells.

[0294] 63. The method of embodiment 62, wherein lymphocyte depletion comprises administration of cyclophosphamide and / or fludarabine.

[0295] 64. The method of any one of embodiments 53-63, further comprising administering a second anti-cancer therapy.

[0296] 65. The method of embodiment 64, wherein the therapy is chemotherapy, immunotherapy, surgery, radiotherapy or biological therapy.

[0297] 66. The method of any one of embodiments 53-64, wherein the TCR engineered cells and / or at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, percutaneously, subcutaneously, topically, or by direct injection or infusion.

[0298] 67. The method of any one of embodiments 53-66, wherein the subject is determined to have cancer cells that overexpress SLC45A2.

[0299] According to the present disclosure, all methods disclosed and claimed in this article can be realized and executed without too much experimentation. Although the compositions and methods of the present invention have been described in the form of preferred embodiments, it is obvious to those skilled in the art that the steps or step sequences of the methods described herein and the methods described herein can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it is obvious that certain medicaments that are chemically and physiologically related can be used to replace the medicaments described herein, and achieve the same or similar results. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to fall within the spirit, scope and concept of the present invention as defined by the appended claims.

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Claims

1. An engineered T cell receptor (TCR), comprising: (a) an α chain and a β chain, the α chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 3, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 4, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 5; the β chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 8, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 9, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; (b) an α chain and a β chain, the α chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 13, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 14, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 15; the β chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 18, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 19, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 20; (c) an α chain and a β chain, the α chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 23, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 24, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 25; the β chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO: 28, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 29, and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 30; or (d) an α chain and a β chain, the α chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO:43, a CDR2 consisting of the amino acid sequence of SEQ ID NO:44, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:45; the β chain comprising: a CDR1 consisting of the amino acid sequence of SEQ ID NO:48, a CDR2 consisting of the amino acid sequence of SEQ ID NO:49, and a CDR3 consisting of the amino acid sequence of SEQ ID NO:

50.

2. The TCR of claim 1, wherein the engineered TCR binds to HLA-A24.

3. The TCR of claim 2, wherein the engineered TCR binds to HLA-A*2402.

4. The TCR of claim 1, wherein the TCR comprises: (a) an alpha chain having at least 90%, 95% or 99% identity to the amino acid sequence of SEQ ID NO: 2 and a beta chain having at least 90%, 95% or 99% identity to the amino acid sequence of SEQ ID NO: 7; (b) an alpha chain that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 12 and a beta chain that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 17; (c) an alpha chain that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 22 and a beta chain that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO: 27; or (d) an alpha chain that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO:42 and a beta chain that is at least 90%, 95% or 99% identical to the amino acid sequence of SEQ ID NO:

47.

5. The TCR of claim 1, wherein the TCR comprises: (a) the α chain of SEQ ID NO: 2 and the β chain of SEQ ID NO: 7; (b) the α chain of SEQ ID NO: 12 and the β chain of SEQ ID NO: 17; (c) the α chain of SEQ ID NO:22 and the β chain of SEQ ID NO:27; or (d) the α chain of SEQ ID NO:42 and the β chain of SEQ ID NO:

47.

6. The TCR of claim 4, wherein the TCR comprises: (a) an alpha chain encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 1 and a beta chain encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 6; (b) an alpha chain encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 11 and a beta chain encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO: 16; (c) an alpha chain encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 21 and a beta chain encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence of SEQ ID NO: 26; or (d) an α chain encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO:41 and a β chain encoded by a nucleotide sequence having at least 95% identity to the nucleotide sequence of SEQ ID NO:

46.

7. The TCR of claim 4, wherein the TCR comprises: (a) an α chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 1 and a β chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 6; (b) an α chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 11 and a β chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 16; (c) an alpha chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 21 and a beta chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 26; or (d) an α chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:41, and a β chain encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:

46.

8. The TCR of claim 1, wherein the TCR is further defined as a soluble TCR, wherein the soluble TCR does not comprise a transmembrane domain.

9. The TCR according to any one of claims 1-8, further comprising a detectable label.

10. The TCR of any one of claims 1-8, wherein the TCR covalently binds a therapeutic agent.

11. The TCR of claim 10, wherein the therapeutic agent is an immunotoxin or a chemotherapeutic agent.

12. A multivalent TCR complex comprising a plurality of TCRs according to any one of claims 1-8.

13. The complex of claim 12, wherein the multivalent TCR comprises 2, 3, 4 or more TCRs that bind to each other.

14. The complex of claim 13, wherein the multivalent TCR is present in a lipid bilayer, in a liposome, or attached to a nanoparticle. The complex of claim 13 , wherein the TCRs are bound to each other via a linker molecule.

16. A polypeptide comprising the TCR according to any one of claims 1-15. A polynucleotide encoding the polypeptide of claim 16.

18. An expression vector encoding the TCR according to any one of claims 1-15.

19. The expression vector of claim 18, wherein the sequence encoding the TCR is under the control of a promoter.

20. The expression vector of claim 18, wherein the expression vector is a viral vector.

21. The expression vector of claim 20, wherein the viral vector is a retroviral vector.

22. The expression vector of claim 18, wherein the vector further encodes a linker domain.

23. The expression vector of claim 22, wherein the linker domain is located between the α chain and the β chain.

24. The expression vector of claim 22, wherein the linker domain comprises one or more cleavage sites.

25. The expression vector of claim 24, wherein the one or more cleavage sites are a furin cleavage site and / or a P2A cleavage site.

26. The expression vector of claim 25, wherein the furin cleavage site is RAKR.

27. The expression vector of claim 25, wherein the furin cleavage site is ATNFSLLKQAGDVEENPG (SEQ ID NO: 51).

28. The expression vector of claim 22, wherein the one or more cleavage sites are separated by a spacer.

29. The expression vector of claim 28, wherein the spacer is SGSG or GSG.

30. A host cell engineered to express the TCR of any one of claims 1-8.

31. The host cell of claim 30, wherein the cell is a T cell, a NK cell, a mesenchymal stem cell (MSC), or an induced pluripotent stem (iPS) cell.

32. The host cell of claim 30, wherein the host cell is a constant type NK cell or a NKT cell.

33. The host cell of claim 30, wherein the host cell is an immune cell.

34. The host cell of claim 30, wherein the host cell is isolated from an umbilical cord.

35. The host cell according to claim 31, wherein the T cell is a CD8 + T cells, CD4+ T cells or γδ T cells.

36. The host cell of claim 31, wherein the T cell is a regulatory T cell (Treg).

37. The host cell of claim 30, wherein the cell is autologous.

38. The host cell of claim 30, wherein the cell is allogeneic.

39. A method for engineering the host cell of claim 33, comprising contacting the immune cell with the TCR of any one of claims 1-8 or the expression vector of any one of claims 18-29.

40. The method of claim 39, wherein the immune cells are T cells or peripheral blood lymphocytes.

41. The method of claim 39, wherein contacting is further defined as transfection or transduction.

42. The method of claim 41, wherein transfection comprises electroporating RNA encoding the TCR of any one of claims 1-8 into an immune cell.

43. The method of claim 41, further comprising producing a viral supernatant from the expression vector of claim 18 prior to transducing the immune cells.

44. The method of claim 39, wherein the immune cells are stimulated lymphocytes.

45. The method of claim 44, wherein the stimulated lymphocytes are human lymphocytes.

46. ​​The method of claim 44, wherein stimulating comprises contacting the immune cells with OKT3 and / or IL-2 or incubating the immune cells in OKT3 and / or IL-2.

47. The method of any one of claims 39-46, further comprising sorting immune cells to isolate TCR-engineered T cells.

48. The method of claim 47, further comprising performing T cell cloning by serial dilution.

49. The method of claim 48, further comprising expanding the T cell clones by a rapid expansion protocol.

50. Use of the TCR-engineered cell of any one of claims 30-38 in the preparation of a medicament for treating a SLC45A2-positive cancer in a subject, wherein the treatment comprises administering to the subject an effective amount of the TCR-engineered cell of any one of claims 30-38, wherein the subject is identified as having an HLA-A*2402 allele, and wherein the cancer is melanoma.

51. The use according to claim 50, wherein the TCR engineered cells are T cells or peripheral blood lymphocytes.

52. The use according to claim 50, wherein the T cells are CD8 + T cells, CD4 + T cells or Tregs.

53. The use according to claim 50, wherein the melanoma is cutaneous melanoma, uveal melanoma, mucosal melanoma or metastatic melanoma.

54. The use according to claim 50, wherein the subject is a human.

55. The use according to claim 50, wherein the TCR engineered cells are autologous or allogeneic.

56. The use according to claim 50, wherein the treatment further comprises lymphocyte depletion of the subject prior to administration of SLC45A2-specific T cells.

57. The use according to claim 56, wherein lymphocyte depletion comprises administration of cyclophosphamide and / or fludarabine.

58. The use of claim 50, wherein the treatment further comprises administering a second anti-cancer therapy.

59. The use according to claim 58, wherein the therapy is chemotherapy, immunotherapy, surgery, radiotherapy or biological therapy.

60. The use of claim 58, wherein the TCR engineered cells and / or at least a second therapeutic agent are administered intravenously, intraperitoneally, intratracheally, intratumorally, intramuscularly, endoscopically, intralesionally, transdermally, subcutaneously, topically, or by direct injection or infusion.

61. The use according to any one of claims 50-60, wherein the subject is determined to have cancer cells that overexpress SLC45A2.

Images