Compositions and methods for targeting loxhd1-associated disease

Abstract

The present invention relates to compositions and methods of treating L0XHD1- associated diseases and disorders (e.g., bone cancer, such as Ewing Sarcoma). In some embodiments, the present invention also relates to T cell receptors that bind to specific L0XHD1 peptide fragments in the context of specific HLA types. In some embodiments, the present invention relates to nucleic acid molecules encoding said T cell receptors. In other embodiments, the present invention relates to cells modified to express said T cell receptors or nucleic acid molecules encoding thereof.

Description

[0001] TITLE OF THE INVENTION

[0002] COMPOSITIONS AND METHODS FOR TARGETING LOXHD1 -ASSOCIATED

[0003] DISEASE

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 516,178, filed July 28, 2023, the contents of which are incoiporated hereby by reference herein in their entireties.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

[0007] DEVELOPMENT

[0008] This invention was made with government support under CA204261 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0009] INCORPORATION BY REFERENCE OF A SEQUENCE LISTING SUBMITTED AS AN XML FILE

[0010] The Sequence Listing file in XML format titled “046483_6258_00WO_SequenceListing.xml”, having a creation date of July 24, 2024 and file size of 154,139 bytes in size, is hereby incorporated by reference in its entirety.

[0011] BACKGROUND OF THE INVENTION

[0012] Ewing Sarcoma is the second most common bone cancer affecting children and young adults. Standard of care high-dose chemotherapy has yielded poor results for patients with metastatic disease. Adoptive cellular therapy strategies utilizing T cells redirected against tumor-associated antigens, such as NY-ESO-1, have yielded promising results for the treatment of synovial sarcoma. Ewing Sarcoma is considered an immunologically inert or “cold" tumor and to date no tumor associated antigen has been identified.

[0013] Thus, there is a need in the art for compositions and methods for treating Ewing Sarcoma and other immunologically inert tumors. The present invention satisfies this unmet need. SUMMARY OF THE INVENTION

[0014] In various aspects, the present invention provides a T cell receptor (TCR) or a composition thereof, wherein the TCR specifically binds to a human leukocyte antigen (HLA): lipoxygenase homology PLAT (polycystin / lipoxygenase / alpha-toxin) domains 1 (LOXHDl)-peptide complex.

[0015] In various aspects, the present invention also provides a modified T cell modified to express a TCR, wherein the TCR specifically binds to a HLA :LOXHD1 -peptide complex.

[0016] In some embodiments, the TCR is engineered. In some embodiments, the TCR is naturally occurring.

[0017] In some embodiments, the TCR comprises a TCR a chain and a TCR p chain. In some embodiments, the TCR a chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof. In some embodiments, the TCR P chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

[0018] In various aspects, the present invention also provides an antibody or a composition thereof, wherein the antibody specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the antibody comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof, and at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

[0019] In various aspects, the present invention also provides a modified T cell modified to express a chimeric antigen receptor (CAR), wherein the CAR specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the CAR comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof, and at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

[0020] In some embodiments, the HLA comprises HLA-A *02:01, HLA-B *07:02, or a combination thereof.

[0021] In some embodiments, the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof.

[0022] In various aspects, the present invention also provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a composition that specifically binds to a HLA :LOXH DI -peptide complex to the subject.

[0023] In various aspects, the present invention also provides a method of administering a composition that specifically binds to a HLA:LOXHD1 -peptide complex to a subject in need thereof. In some embodiments, the subject has a LOXHD1 -associated disease or disorder.

[0024] In some embodiments, the composition comprises at least one TCR of the present invention.

[0025] In some embodiments, the composition comprises at least one modified T cell (e.g., a modified T cell modified to express a TCR, modified T cell modified to express a CAR, etc.) of the present invention.

[0026] In some embodiments, the composition comprises at least one antibody of the present invention.

[0027] In some embodiments, the LOXHD1 -associated disease or disorder is a LOXHD1 -associated cancer. In some embodiments, the LOXHD1 -associated cancer is an Ewing Sarcoma.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following detailed description of exemplary embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0030] Figure 1, comprising Figure 1A through Figure 1C, depicts representative images of tissue sections from different sarcoma subtypes, new treatment strategies, and increasing mutational burden. Figure 1 A depicts representative images of tissue sections from Ewing Sarcoma (left) and synovial sarcoma (right), both translocation-associated sarcomas. New treatment strategies comprise epigenetic therapy, immunotherapy (adoptive transfer of autologous dendritic cells / T cells), immunotherapy (tumor vaccines) and targeted therapy (inhibitors of common targetable cancer pathways, multikinase inhibitors). Figure IB depicts representative images of tissue sections embryonal rhabdomyosarcoma (left) and malignant peripheral nerve sheath tumor (right). New treatment strategies comprise epigenetic therapy, immunotherapy (adoptive transfer of autologous dendritic cells / T cells), immunotherapy (tumor vaccines), immunotherapy (immune checkpoint inhibitors), immune stimulation from radiation therapy, and targeted therapy (inhibitors of common targetable cancer pathways, multikinase inhibitors). Figure 1C depicts representative images of tissue sections from osteosarcoma (left) and undifferentiated pleomorphic sarcoma (right). New treatment strategies comprise immunotherapy (immune checkpoint inhibitors), immune stimulation from radiation therapy, and targeted therapy (inhibitors of common targetable cancer pathways, multikinase inhibitors).

[0031] Figure 2 depicts a schematic representation of genomic alterations across childhood (left panel) and adult (right panel) cancers. Representative data from a total number of 879 pediatric cancers and 3281 adult cancers are presented. Representative data from Ewing Sarcoma are presented in the rectangular box.

[0032] Figure 3, comprising Figure 3A through Figure 3C, depicts representative data demonstrating that LOXHD1 was upregulated by EWSR1 -FL1 translocation. Figure 3A depicts representative data demonstrating that LOXHD1 was predominantly expressed only in Ewing Sarcoma tumors and tumor-derived cell lines. LOX HD 1 expression in MET500 + Ewing Sarcoma n = 507, CCLE n = 980, and GTEX n = 11401 transcriptomic datasets (Deng et al., 2022, Cell Reports). Figure 3B depicts representative data demonstrating LOXHD1 expression in 57 Ewing Sarcoma tumors classified as non- metastatic (left, n = 38) or metastatic (right, n = 18). Figure 3C depicts representative Western Blot analysis of LOXHD1 expression in various Ewing Sarcoma tumor cell lines (SK-NM-C, RD-ES, TC-71, and CHLA-10) treated with and without IFN-y (+).

[0033] Figure 4 depicts representative data depicting LOWED 1 expression in primary and metastatic disease via immunofluorescence of cell lines. Representative LOXHD1 (top row), F actin (middle row), and DAP1 (bottom row) immunofluorescence staining in pancreatic cells (left column), SK-NM-C (middle left column) and TC-71 cells (middle right column), and TC-205 cells (right column) are depicted. SK-NM-C and TC-71 cells expressed the EWS:FL1 translocation; TC-205 cells expressed the EWS:FEV translocation. LOXHD1 was highly expressed in SK-NM-C, TC-71 cells and TC-205 cells.

[0034] Figure 5, comprising Figure 5A and Figure 5B, depicts representative data demonstrating that LOXHD1 was expressed in Ewing Sarcoma cell lines that present EWS-FL1, EWS-FEV and EWS-ERG translocations. Figure 5A depicts representative data demonstrating the relative LOXHD1 mRNA expression in Ewing Sarcoma tumor cell lines versus the expression in a non-Ewing Sarcoma LOXHD1 negative pancreatic tumor cell line, and representative results of RT-qPCR analysis of LOXHD1 mRNA expression in a panel of human derived Ewing Sarcoma cell lines (n = 10) and patient derived xenograft (PDX) tumor (n = 1 ) categorized as EWSR1 -FLI 1 , EWSR1 -FEV, or EWSR1-ERG. Non- Ewing Sarcoma (blue) pancreatic cell line, PANC-1, used as a negative control. PANC-1 was calculated according to the ACt. Figure 5B depicts representative immunoblot data demonstrating the detection of stereociliary LOXHD1 protein in Ewing Sarcoma tumor cell lines (n = 10), a prostate cancer cell line (PANC-1) and a CRISPR-Cas9 / LOXHD1 knockout gene edited SK-NM-C cell line. fl-actin was used as loading control. Western immunoblot analysis demonstrates LOXHD1 expression in Ewing Sarcoma cell lines grouped by Ewing Sarcoma chromosomal translocations.

[0035] Figure 6 depicts a schematic representation of identifying LOXHD1 as a nonmutated oncoprotein and novel biomarker for Ewing Sarcoma. The transcriptional basis of malignant Ewing Sarcoma phenotype was identified via EWSR1-ETS knockdown and transcriptional core-signature determination (top left box). Highly expressed genes were determined from a 32-gene signature of the Ewing Sarcoma transcriptome identified from primary, metastatic and normal tissue (top right box). LOXHD1, a non-mutated oncogenic driver in primary and metastatic disease with restricted expression to Ewing Sarcoma, was a 2211 amino acid protein which contains a highly conserved Polycystin- 1, Lipoxygenase, Alpha-Toxin (PLAT) domain which targeted protein to the plasma membrane (lower box).

[0036] Figure 7 depicts a schematic representation of MHC Class 1 molecule mediated cell surface antigen presentation to CD8+ cells following IFN-y stimulated proteasomal degradation. Inflammatory cytokines, such as IFN-y, promote de novo expression of immunoproteasome subunits PSMB9 / LMP2, PSMB8 / LMP7, PSMB10 / MECL1 along with upregulation of MHC Class I expression resulting in changes in the Ewing Sarcoma epitope repertoire.

[0037] Figure 8, comprising Figure 8A and Figure 8B, depicts representative data demonstrating that IFN-y treatment of Ewing Sarcoma cells promoted immunoproteasome subunit expression leading to changes in HLA class I restricted epitope repertoires. Figure 8A depicts representative Western immunoblot analysis of Ewing Sarcoma cell lines treated with lOOIU / mL of IFN-y (+) for 24 h demonstrating upregulation of immunoproteasome subunits PSMB9 ~23kDa, PSMB8 ~30kDa, PSMB10 ~29kDa in comparison to untreated (-) (P-actin loading control). Figure 8B depicts representative FACS histogram plots of Ewing Sarcoma cell lines demonstrating CD99 expression and upregulation of class I expression as detected by BB7.2 (HLA- A*02:01) and W6 / 32 (pan- HLA class I) Abs after IFN-y (24 h, lOOu / mL).

[0038] Figure 9 depicts representative data demonstrating that IFN- y treatment of Ewing Sarcoma cells promoted immunoproteasome subunit expression leading to changes in HLA class I restricted epitope repertoires. Representative data as presented in Figure 8A expanded to additional Ewing Sarcoma cell lines. Western protein expression of immunoproteasome subunits pli / LMP2 (PSMB9) ~23kDa, p5i / LMP7 (PSMB8) ~30kDa, p2i / PSMB10 (PSMB10) ~29kDa in Ewing Sarcoma cell lines treated with IFN-y (+) (1 OOU / mL) for 24 h in comparison to basal untreated (-). 0-actin was used as protein loading control.

[0039] Figure 10 depicts a list of representative sequences of LOXHD1 fragments (SEQ ID NOs: 1-27), their calculated binding affinity, and peptide stability. Figure 11 depicts a representative human LOXHD1 protein sequence (Isoform 1) (SEQ ID NO: 28) highlighting the HLA-A *02:01 VLLSPLSRV (SEQ ID NO: 5), FLGSVQIRV (SEQ ID NO: 1), QLSRELLPV (SEQ ID NO: 10), and KMADVDISTV (SEQ ID NO: 30), and HLA-B*07:02 LPSDSSATL (SEQ ID NO: 29).

[0040] Figure 12, comprising Figure 12A and Figure 12B, depicts representative data demonstrating that LOXHD1 represented an immunological target in Ewing Sarcoma. Figure 12A depicts a representative MS / MS fragmentation pattern of LOXHD1 peptides identified by immunopeptidomics. Five HLA-A*02:01 restricted LOXHD1 -derived peptides were identified: VLLSPLSRV (AA353-361) (SEQ ID NO: 5), FLGSVQIRV (AA766-774) (SEQ ID NO: 1), QLSRELLPV (AA 1134-1142) (SEQ ID NO: 10), SIIRDLFHA (SEQ ID NO: 26), and KMADVDISTV (AA1604-1613) (SEQ ID NO: 30). While FLGSVQIRV (SEQ ID NO: 1) and QLSRELLPV (SEQ ID NO: 10) were detected exclusively under inflammatory and non-inflammatory conditions, VLLSPLSRV (SEQ ID NO: 5) and KMADVDISTV (SEQ ID NO: 30) were detected under all conditions. Figure 12B depicts representative binding affinity data of LOXHD1 specific HLA- A*02:01 peptides as determined using fluorescence polarization competitive peptide binding assays. Top left panel: SEQ ID NO: L top right panel: SEQ ID NO: 10; lower left panel: SEQ ID NO: 5; lower right panel: SEQ ID NO: 30. The IC50 values of test peptides, determined by fitting data to dose-response model using Prism software, was reported as the logl0[IC50(nM)] values. R2 indicated the goodness of fit. Peptides were classified as high affinity (logio[ICso] < 3.7nM), medium affinity (logio[ICso] "T3.7- 4.7nM), low affinity (logl0[lC50] = 4.7-5.5nM), and very low affinity (logiofICso] = 5.5- 6.0nM).

[0041] Figure 13, comprising Figure 13A and Figure 13B, depicts representative data demonstrating LOXHD1 HLA-A*02:01 epitope identification and characterization. Figure 13A depicts a list of representative HLA-A*02:01 restricted 9mer peptides (SEQ ID NOs 1-27, 31, and 32). Figure 13B depicts a list of representative HLA-A*02:01 restrictelOmer LOXHD1 peptides (SEQ ID NOs: 30, 33, and 34-61). Binding affinity and stability of HLA-A*02:01 restricted LOXHD1 candidate peptides were identified using NetMHCpan 4.0 and NetMHCstabpan 1.0. Values were given as IC50 (nM) and stability (h). Identified HLA-A*02:01 restricted LOXHD1 9mer / 10mer epitopes by immunopeptidomics of the SK-NM-C-engineered monoallelic cell line (KMADVDISTV (SEQ ID NO: 30)).

[0042] Figure 14, comprising Figure 14A through Figure 14C, depicts representative HLA-A*02:01 restricted LOXHD1 epitopes by immunopeptidomics. Figure 14A depicts a representative short isoform LOXHD1 protein sequence used to generate HLA- A*02:01 restricted LOXHD1 candidate epitopes (1891 aa) (Deng et al 2022, Cell Reports). Identified HLA- A*02:01 restricted LOXHD1 nonamer / decamer epitopes by immunopeptidomics of the SK-N-MCA*02"01engineered monoallelic cell line (underlined and bolded). Figure 14B depicts a list of representative HLA-A*02:01 restricted LOXHD1 9mer / 10mer epitopes, SEQ ID NOs: 1, 5, 10, 26, and 30-32, by immunopeptidomics of the SK-NM-C-engineered monoallelic cell line. Binding affinity and stability of HLA-A*02:01 restricted LOXHD1 candidate peptides were identified using NetMHCpan 4.0 and NetMHCstabpan 1.0. Values are given as IC50 (nM) and stability (h). Figure 14C depicts representative MS / MS fragmentation pattern of LOXHD1 peptides identified by immunopeptidomics. Seven HLA-A*02:01 restricted LOXHD1 -derived peptides were identified: VLLSPLSRV (AA353-361) (SEQ ID NO: 5), SVGHGNVGV (A A362-370) (SEQ ID NO: 31), FLGSVQ1RV (AA766-774) (SEQ ID NO: 1), QLSRELLPV (AA 1134-1142) (SEQ ID NO: 10), SIIRDLFHA (AA1287- 1295) (SEQ ID NO: 26), KMADVDISTV (AA 1604-1613) (SEQ ID NO: 30), ATDSRAFIFL (AA1642-1651) (SEQ ID NO: 32). While QLSRELLPV (SEQ ID NO: 10), FLGSVQIRV (SEQ ID NO: 1) and SIIRDLFHA (SEQ ID NO: 26) were detected exclusively under non-inflammatoiy and inflammatory conditions, VLLSPLSRV (SEQ ID NO: 5), SVGHGNVGV (SEQ ID NO: 31), KMADVDISTV (SEQ ID NO: 30), and ATDSRAFIFL (SEQ ID NO: 32) were detected under all conditions.

[0043] Figure 15, comprising Figure 15A and Figure 15B, depicts representative data demonstrating LOXHD1 HLA-B*07:01 epitope identification and characterization. Figure 15A depicts a list of representative HLA-B*07:02 restricted 9mer peptides (SEQ ID NOs: 29 and 62-65). Figure 15B depicts a list of representative HLA-B*07:02 restricted 1 Omer peptides (SEQ ID NOs: 66-73). LOXHD1 Binding affinity and stability of HLA-B*07:02 restricted LOXHD1 candidate peptides (< 200nM binding affinity) were identified using NetMHCpan 4.0 and NetMHCstabpan 1.0. Values are given as IC50 (nM) and stability (h).

[0044] Figure 16, comprising Figure 16A through Figure 16D, depicts representative data demonstrating the identification of HLA-B*07:02 restricted LOXHD1 epitopes by immunopeptidomics. Figure 16A depicts a representative human LOXHD1 Protein sequence (Isoform 1) (SEQ ID NO: 28) used to identify HLA-B*07:02 restricted LOXHD1 candidate epitopes (1891 aa) (Deng et al 2022, Cell Reports). Figure 16B depicts a representative HLA-B*07:02 restricted LOXHD1 9mer epitope (SEQ ID NO: 29) by immunopeptidomics of the SK-NM-C-engineered monoallelic cell line. Binding affinity and stability of HLA-B*07:02 restricted LOXHD1 candidate peptide (< 200nM binding affinity) were identified using NetMHCpan 4.0 and NetMHCstabpan 1.0. Values are given as IC50 (nM) and stability (h). Figure 16C depicts representative MS / MS fragmentation pattern of LOXHD1 peptide identified by immunopeptidomics. One HLA- B*07:02 restricted LOXHD1 -derived peptide was identified: LPSDSSATL (AA 673- 681) (SEQ ID NO: 29) under non-inflammatoiy and inflammatory conditions. Figure 16D depicts representative binding affinity data of LOXHD1 specific HLA-A*07:02 peptide (SEQ ID NO: 29) as determined using fluorescence polarization competitive peptide binding assays. The JC50 values of test peptides is determined by fitting data to dose-response model using Prism software and the logl0[IC50(nM)J values were reported. R indicates the goodness of fit. Peptides were classified as high affinity (logl 0[IC50] < 3.7nM), medium affinity (logl0[IC50] = 3.7-4.7nM), low affinity (logl0[IC50] = 4.7-5.5nM) and very low affinity (logl0[IC50] = 5.5-6.0nM).

[0045] Figure 17, comprising Figure 17A and Figure 17B, depicts representative data demonstrating identification of four HLA-A*02:01 restricted LOXHD1 epitopes by immunopeptidomics. Figure 17A depicts a representative Venn diagram depicting four identified LOXHD1 peptides (SEQ ID NOs: 1, 5, 10, and 30) and grouping them as identified under non-inflammatoiy (left) or inflammatory (right) conditions. Global immunopeptidomic analysis of SK-NM-C A2+ Ewing Sarcoma cell line identified a set of unique and shared epitopes 16,504 and 22,137 epitopes presented in the context of HLA-A*02:01 under non-inflammatoiy and inflammatory (4-IFN-y) conditions respectively. Figure 17B depicts representative data demonstrating the MS / MS fragmentation pattern of LOXHD1 peptides (SEQ ID NOs: 1, 5, 10, and 30) identified by immunopeptidomics. Four HLA-A*02:01 restricted LOXHD1 -derived peptides were identified: VLLSPLSRV (AA353-361) SEQ ID NO: 5), FLGSVQIRV (AA766-774) (SEQ ID NO: 1), QLSRELLPV (AA 1134-1142) (SEQ ID NO: 10), and KMADVDISTV (AA1604-1613) (SEQ ID NO: 30). While FLGSVQIRV (SEQ ID NO: 1) and QLSRELLPV are detected exclusively under inflammatory and non-inflammatory conditions, VLLSPLSRV (SEQ ID NO: 5) and KMADVDISTV (SEQ ID NO: 30) were detected under all conditions.

[0046] Figure 18, comprising Figure 18A through Figure 18C, depicts representative data demonstrating the identification of HLA-B*07:02 restricted LOXHD1 epitopes by immunopeptidomics. Figure 18A depicts representative proteomic analysis of Ewing Sarcoma cell line SK-NM-C under non-inflammatory (left) and inflammatory (+IFN- y / right) conditions (n = 3 replicates). Proteomic analysis identified unique (noninflammatory, n = 3908 and inflammatory, n = 7239) as well as shared (n = 35738) peptide repertoires presented in the context of HLA-B*07:02. Processed and presented LOXHD1 peptides with corresponding AA sequences were represented in Venn diagrams (SEQ ID NO 29 was processed and presented in the context of HLA-B*07:02). Figure 18B depicts representative MS / MS fragmentation pattern of processed and presented HLA-B*07:02 (SEQ ID NO: 29) peptide. Figure 18C depicts representative peptide affinity measurements of LOXHD1 specific HLA-A*02:01 epitopes (SEQ ID NOs: 1, 5, 10, and 30) and HLA-B*07:02 (SEQ ID NO: 29). p-HLA affinities were assessed by fluorescence polarization competitive peptide-binding assays. High peptide affinity was classified as a logl0[IC50] < 3.7 nM and is indicated by the dashed line.

[0047] Figure 19, comprising Figure 19A and Figure 19B, depicts representative affinity measurements of LOXHD1 specific HLA-A*02:01 epitopes. Figure 19A depicts representative data demonstrating the affinity and stability of LOXHD1 epitopes; the four identified LOXHD1 peptides were presented with their respective labels - VLLSPLSRV (AA353-361) (SEQ ID NO: 5), FLGSVQIRV (AA766-774) (SEQ ID NO: 1), QLSRELLPV (AA 1134-1142) (SEQ ID NO: 10), and KMADVDISTV (AA1604-1613) (SEQ ID NO: 30). Figure 19B presents representative data demonstrating the affinity of four LOXHD1 specific HLA-A*02:01 epitopes via fluorescence polarization competitive binding assays; from left to right VLLSPLSRV (AA353-361) (SEQ ID NO: 5), FLGSVQ1RV (AA766-774) (SEQ ID NO: 1), QLSRELLPV (AA 1 134-1142) (SEQ ID NO: 10), and KMADVDISTV (AA1604-1613) (SEQ ID NO: 30). The last two columns on the right depict a high affinity control peptide, with high affinity binding determined as logl0[IC50] < 3.7 nM, horizontal dashed line.

[0048] Figure 20 depicts a schematic representation of a method to obtain antigenspecific T cells from peripheral blood mononuclear cells (PBMCs) of healthy donors.

[0049] Figure 21, comprising Figure 21 A through Figure 21C, depicts representative data demonstrating the LOXHD1 epitopes were immunogenic. Figure 21 A depicts representative data demonstrating that LOXHD1 peptides VLLSPLSRV (SEQ ID NO: 5) and QLSRELLPV (SEQ ID NO: 10) were immunogenic. HLA-A*02:01+ CD8+ T cells obtained from healthy donors were stimulated twice with indicated peptides and responses evaluated in IFN-y ELISPOT assay. IFN-y ELISPOT Spot Forming Cell (SFC) values were subtracted from media controls for each donor and values above dotted line were considered positive. Only responses to two out of four peptides were observed. IFN- y responses to these immunogenic peptides (QLSRELLPV (SEQ ID NO: 10) and VLLSPLSRV (SEQ ID NO: 5) peptides were observed in two donors each. Figure 21B depicts representative data from p-HLA multimer analysis demonstrating positive HLA- A*02:01 -restricted CD8+ T cell responses following in vitro stimulation with VLLSPLSRV (SEQ ID NO: 5) and QLSRELLPV (SEQ ID NO: 10). Cells were stained with p-HLA multimer and anti-CD8 Ab. (non-specific pMHC / multimer used as control). Figure 21C depicts representative LOXHD-1 peptide AA residues recognized, HLA restriction, Va and V0 TCR family, and CDR3 AA sequences of TCRs that recognized immunogenic LOXHD1 epitopes VLLSPLSRV (AA353-361) (SEQ ID NO: 5) and QLSRELLPV (AA 1134-1142) (SEQ ID NO: 10).

[0050] Figure 22, comprising Figure 22A and Figure 22B, depicts representative data demonstrating that T Cell receptor (TCR) 353- and 1134-engineered LOXHD1 -specific CD8+ T cells demonstrate high antigen avidity. Figure 22A depicts representative data demonstrating the expression of TCR 353 and 1134 on primary CD8+ T cells as detected by peptide / HLA multimers. Flow cytometry profiles of CD8+ T cells CRISPR / Cas9- edited for deletion of endogenous TCR (TCRnull) and transduced with lentivirus expressing TCR353 or TCR1134. TCR353 or TCS1134 induce proliferation of CD8+ T cells. Figure 22B depicts representative data demonstrating TCR avidities for LOXHD1 peptide HL A- A* 02:01 complexes. TCR353 or TCR1134 CD8+ (TCRnull) T cells were cocultured with HLA-A*02:01 K562 target cells pulsed with titrated peptide concentrations and lysis of target cells tested as effectors in a 4 h51Cr-release cytotoxicity release assay. TCR avidity (EC50) was determined as the mean peptide concentration required to achieve 50% specific lysis. TCR353 EC50 = 24 nM and TCR1134 EC50 = 2.7 nM.

[0051] Figure 23, comprising Figure 23A and Figure 23B, depicts representative data demonstrating that LOXHD1 peptides p353-361 (SEQ ID NO: 5) and pl 134-1142 (SEQ ID NO: 10) were immunogenic. Figure 23A depicts representative IFN-y ELISPOT assay results following in vitro expansion of healthy donor purified CD8+ T cells stimulated with LOXHD1 peptides. Values above dotted line represent positivity. Inserts: IFN-y ELISPOT Spot Forming Cell (SFC) values were subtracted from media controls for each donor. Figure 23B depicts representative p-HLA multimer analysis data demonstrating positive HLA-A*02:01 -restricted CD8+ T cell responses following in vitro stimulation with SEQ ID NO: 5 (top right panel) and SEQ ID NO: 10 (lower right panel). Cells were stained with p-HLA multimer and anti-CD8 Ab. (non-specific pMHC / multimer used as control).

[0052] Figure 24, comprising Figure 24A and Figure 24B, depicts representative data demonstrating that TCRs specific for p353-361 (SEQ ID NO: 5), TCR353, and pl 134- 1142 (SEQ ID NO: 10), TCR1134, were isolated from T cells shown in Figure 23B using TCR next-generation sequencing followed by molecular cloning; expression of these TCRs on CD8+ T cells (gene-edited to KO endogenous TCR) conferred reactivity against corresponding HLA / pep multimers and conferred cytotoxic activity against targets pulsed with peptide. Figure 24A depicts representative flow cytometry dot plots demonstrating p-HLA multimer binding to TCR353 or TCR1143 lentiviral transduced CD8+ T cells (endogenous TCR KO using CISPR / Cas9 editing). Figure 24B depicts representative data demonstrating in vitro evaluation of antigen recognition. CDS T cells expressing TCR353 and TCR1134 were cocultured with HLA-matched monoallelic K562 cell lines pulsed with titrated peptide concentrations. TCR avidity (EC50) was determined as the mean peptide concentration required to achieve 50% specific lysis (5,Cr assay) activation of TCR-engineered CD8+ T cells. These LOXHD1 -specific TCR engineered CD8+ T cells display avidities of <10nM for antigen.

[0053] Figure 25, comprising Figure 25A through Figure 25G, depicts representative data demonstrating the immunogenicity of LOXHD1 peptides and identification of LOXHD1- specific T cell receptors. Figure 25A depicts a schematic representation of the assessment of LOXHD1 peptides immunogenicity and TCR isolation. Immunogenicity was examined using purified CD8+ T cells from HLA-A*02:01+ healthy donors stimulated with peptide-pulsed autologous mature dendritic cells, followed by a secondary stimulation with peptide-pulsed K562 / A*02:01+ cells (aAPCs), followed by assessment of antigen specificity. LOXHD1 epitopes were validated by the isolation of LOXHD1- specific TCRs. CD8+ T cells were lentivirally transduced with LOXHD1 specific TCRs to study antigen processing and presentation. Figure 25 B depicts representative data demonstrating LOXHD1 TCR isolation. Using expanded, peptide / HLA multimer sorted (99% purity) CD8+T cells isolated from Figure 21 A, TCRct / p sequences were determined by next-generation RNA sequencing. Circus plots represent TCR V[3 and TCR Va composition of VLLSPLSRV (SEQ ID NO: 5)-(designated TCR353) and QLSRELLPV (SEQ ID NO: 10)-(designated TCR1134) specific T cells. Figure 25C depicts representative data demonstrating expression of TCR353 and 1134 on primary CD8+ T cells as detected by peptide / HLA multimers. Flow cytometry plots of p-HLA multimer analyses demonstrated positive VLLSPLSRV (SEQ ID NO: 5)-specific HLA-A*02:01- restricted CD8+ T cell response isolated from ND523. Flow cytometry profiles of CD8+ T cells CRISPR / Cas9-edited for deletion of endogenous TCR (TCR™11) and transduced with lentivirus expressing TCR353 or TCR1134. Figure 25D depicts representative HLA- A*02:01+ CD8+ T cells obtained from healthy donors were stimulated twice with indicated peptides and responses were evaluated in an IFN-y ELISPOT assay. IFN-y ELISPOT Spot Forming Cell (SFC) values were subtracted from media controls for each donor and values > 100 SFCs per 5e5 CD8+ T cells (indicated by the dotted line) were considered positive. Representative data were the pooled results (triplicate) from single experiments of individual donors indicated by respective symbols. Red symbols represent positive donors. Figure 25E depicts representative flow cytometry plot demonstrating p- HLA multimer staining following lentiviral engineering of J*81*90(Jurkat E6.1 cells) with TCR353 constructs (red) compared to T (black). Figure 25F depicts representative J*81*90reporter assay to evaluate antigen recognition. JASP9O reporter cells expressing TCR353 were cocultured with SK-N-MCA*0201cells (red) or with SK-N-MCB2M-KOcells (blue) treated with either lOOIU / mL IFN-y or without (basal) or peptide pulsed with lOug / mL LOXHD1 TCR353 specific peptide VLLSPLSRV (SEQ ID NO: 5). TCR signaling was indicated by eGFP expression upon NF AT activation. Figure 25G depicts representative TCR avidity for LOXHD1 peptide / HLA-A*02:01 complex. TCR353 CD8+ (TCRnull) T cells were cocultured with K562 / A*02:01+ target cells pulsed with titrated peptide concentrations and lysis of target cells was determined in a51Cr release assay. TCR avidity (EC50) was determined as the mean peptide concentration required to achieve 50% specific lysis (TCR353 EC50 = 24 nM).

[0054] Figure 26, comprising Figure 26A through Figure 26C, depicts representative phenotypic characterization of Ewing Sarcoma tumor cell lines and generation of HLA- A*02:01 monoallelic Ewing Sarcoma cell lines. Characterization of HLA-A2 expression in Ewing Sarcoma tumor cell lines. Figure 26A depicts representative Ewing Sarcoma cell lines categorized by translocation EWSR1 -FLU, EWSR1-FEV, EWSR1-ERG, LOXHD1 expression as demonstrated in Figure 5 A and Figure 5B, and expression of HLA-A2 (+) denoted endogenous expression of HLA-A2 (n = 6 / 10) (-) HLA-A2 negative cell line. Figure 26B depicts representative design of HLA-A*02:01 / [32M single chain dimer (SCD) lentiviral vector construct. Figure 26C depicts representative flow cytometric analysis demonstrating expression of HLA-A2 as detected by pan HLA class I-specific antibody W6 / 32 in Ewing Sarcoma cell lines transduced with SCD (n = 4 denoted with A*02:01) or endogenously expressing HLA-A2.

[0055] Figure 27, comprising Figure 27A through Figure 27C, depicts representative results demonstrating disruption of [32M abrogates TCR353 cytotoxic activity. Figure 27 A depicts representative results of flow cytometric analysis demonstrating expression of 02M by MHC class 1 molecules as detected by pan antibody W6 / 32 of parental Ewing Sarcoma cell lines (red) or CRISPR / Cas9 edited gene deletion of 02M (02MKO) (blue) (n = 4). Figure 27B depicts representative data demonstrating that TCR353 enhanced lytic activity against IFN-y treated tumor cells. TCR353+ CD8+ (TCRnull) T cells were tested as effectors in a 4 h5lCr-release cytotoxicity assay using as targets a panel of Ewing Sarcoma cell lines expressing EWS-FL1 (SK-NM-C, TC-71, RD-ES) or EWS- FEV (TC-205) translocations. To evaluate effect of inflammatory conditions on antigen recognition, cell lines w'ere incubated in the absence or presence of IFN-y (24 h, 100 u / mL) prior to assay and stimulated with 10 pg ofVLLSPLSRV (AA353-361) (SEQ ID NO: 5) and / or QLSRELLPV (AA 1134-1142) peptides (SEQ ID NO: 10). Percent- specific lysis is shown in duplicate for each data point. Data are presented as mean ± SD. At an E:T ratio of 10:1 or higher p value is less than p < 0.0001 comparing endogenous levels of presentation (steady state to + IFN-y treated). 02M knockout cells (02MKO), lower panel, are used as a negative control. ****p < 0.0001 : 2wayAnova / Sidak Multiple Compare. Figure 27C depicts representative results of 4 h5,Cr- release cytotoxicity assay of TCR353 demonstrating lytic activity against HLA-matched non-Ewing Sarcoma cell line.

[0056] Figure 28 depicts representative data demonstrating that TCR353 enhanced lytic activity against IFN-y treated tumor cells. TCR353+ CD8+ (TCRnull) T cells were tested as effectors in a 4 h5,Cr-release cytotoxicity assay using as targets a panel of Ewing Sarcoma cell lines expressing EWS-FL1 (A-673, CHLA-9, TC-32) or EWS-ERG (TC- 205) translocations, and control line CHLA-10 lacking HLA-A*02:01. To evaluate effect of inflammatory conditions on antigen recognition, cell lines were incubated in the absence or presence of IFN-y (24 h, 100 u / mL) prior to assay and stimulated with 10 pg ofVLLSPLSRV (AA353-361) (SEQ ID NO: 5) and / or QLSRELLPV (AA 1134-1142) (SEQ ID NO: 10) peptides. Percent-specific lysis is shown in duplicate for each data point. Data are presented as mean ± SD. At an E:T ratio of 10: 1 or higher p value is less than p < 0.0001 comparing endogenous levels of presentation (steady state to + IFN-y treated). 02M knockout cells (02MKO), lower panel, are used as a negative control. ****p < 0.0001: 2wayAnova / Sidak Multiple Compare.

[0057] Figure 29 depicts representative data demonstrating that TCR353 conferred lytic activity against Ewing Sarcoma tumor cells at steady state. TCR353+ CD8+ (TCRnull) T cells (TCR353) or control CD8+ (TCRnull) T cells (TCRKO) were tested as effectors in a cellular impedance assay using as targets a panel of Ewing Sarcoma cell lines expressing EWS-FL1 (SK-NM-C and TC-71) or EWS-FEV (TC-205) translocations in the presence or absence and stimulated with 10 pg of VLLSPLSRV (AA353-361) (SEQ ID NO: 5) and / or QLSRELLPV (AA 1134-1142) (SEQ ID NO: 10) peptides.

[0058] Figure 30 depicts representative data demonstrating the kinetics of tumor cell lysis (cytotoxicity) upon (top) TCR353- and (bottom) TCR1134-expressing T cell recognition of EWS-FLI1 translocation lines SK-NM-C, TC-71, and FEV translation line TC-205 ES. Normalized cell index values over time for E:T ratio of 3: 1 are shown compared to tumor cells cultured in media alone. Data are presented as mean values ± SD. Conditions depicted top panel: tumor cells + TCR, tumor cells +IFN-y + TCR, tumor cells + peptide (SEQ ID NO: 5) + TCR, Tumor cells, tumor cells + IFN-y, tumor cells + peptide (SEQ ID NO: 5). Conditions depicted lower panel: tumor cells + TCR, tumor cells +IFN-y + TCR, tumor cells + peptide (SEQ ID NO: 10) + TCR, Tumor cells, tumor cells + IFN-y, tumor cells + peptide (SEQ ID NO: 10).

[0059] Figure 31 , comprising Figure 31 A through Figure 3 ID, depicts representative data demonstrating that expression of TCR353 on CD8+ T cells conferred lytic activity against Ewing Sarcoma human tumor cells. To evaluate effect of inflammatory conditions on antigen recognition, cell lines were incubated in the absence or presence of IFN-y (24 h, 100 u / mL) prior to assay. Percent-specific lysis is shown in triplicate for each data point. Data are presented as mean ± SD. At an E:T ratio of 10: 1 or higher p value is less than p < 0.0001 comparing endogenous levels of presentation (steady state / black)) to + IFN-y treated (red). ****p < 0.0001: 2wayAnova / Sidak Multiple Compare. Figure 31A depicts representative data demonstrating TCR353+ CD8+ (TCRnull) T cells as effectors in a 4 h$1Cr-release cytotoxicity assay using as targets a panel of Ewing Sarcoma cell lines expressing EWS-FL1 translocations. Figure 31 B depicts representative data demonstrating TCR353+ CD8+ (TCRnull) T cells as effectors in a 4 h5lCr-release cytotoxicity assay using as targets a panel of Ewing Sarcoma cell lines expressing EWS-FEV translocations. Figure 31C depicts representative data demonstrating TCR353+ CD8+ (TCRnull) T cells as effectors in a 4 h51Cr-release cytotoxicity assay using as targets a panel of Ewing Sarcoma cell lines expressing EWS- ERG translocations. Figure 3 ID depicts representative proteomic quantitative data demonstrating VLLSPLSRV (SEQ ID NO: 5) epitope expression by SK-NM-C under non-inflammatory (basal) and inflammatory (treated with IFNg) conditions. Figure 32, comprising Figure 32A and Figure 32B, depicts representative data demonstrating that TNFa promoted recognition of Ewing Sarcoma cancer cell lines by TCR353-engineered CD8+ T cells. Figure 32A depicts representative data demonstrating TCR353+ CD8+ (TCRnull) T cells as effectors in a 4 h5lCr-release cytotoxicity assay using as targets two Ewing Sarcoma cell lines. To evaluate effect of additional inflammatory cytokines on LOXHD1 recognition, Ewing Sarcoma cell lines were incubated in the absence or presence of IFN-y (24 h, lOOu / mL) or TNF-a (24 h, 100 ng / mL) prior to assay. Percent-specific lysis is shown in triplicate for each data point. Data are presented as mean ± SD. At an E:T ratio of 10: 1 or higher p value is less than p < 0.0001 comparing endogenous levels of presentation (circle) to +TNF-a (triangle) and + IFN-y treated (square) peptide pulsed VLLSPLSRV (SEQ ID NO: 5) (upside down triangle). ****p < 0.0001: 2wayAnova / Sidak Multiple Compare. Figure 32B depicts representative data demonstrating upregulation of HLA-A*02:01 (BB7.2 Ab) and pan- HLA class I (W6 / 32 Ab) expression after IFN-y (24 h, lOOu / mL) or +TNF-O (24 h 100 ng / mL).

[0060] Figure 33, comprising Figure 33A through Figure 33G, depicts representative data demonstrating the kinetics of Ewing Sarcoma recognition by TCR353-T cells were enhanced in the presence of inflammatory conditions. Expression of TCR353 on CD8+ T cells conferred lytic activity against Ewing Sarcoma human tumor cells. Figure 33A depicts representative images of live cells under non-inflammatory (- IFN-y), inflammatory (+ IFN-y), and peptide (VLLSPLSRV) (SEQ ID NO: 5) conditions to assess TCR353 activity against SK-NM-C, TCRnull (TCRKO) cells, and media used as controls. Ewing Sarcoma cell lines expressed GFP allowing for the monitoring of cell lysis using imaging (48 h). Time to reach 50% cell death KT50(hrs) of SK-NM-C, TC- 71, and TC-205 cells upon TCR353 recognition at an E:T ratio of 3:1 is reported. Representative images at 48 h comparing SK-N-MCA*02:01cultured with TCR353 (green), TCR KO (TCRnuU) cells (blue), and media (black). Figure 33B depicts representative eGFP quantification of live cell imaging of TCR353 T cells against Ewing Sarcoma cell lines SK-N-MCA*02:I)1over 48 h period is shown. Colored lines correspond to wells with tumor cells cultured with TCR353 (green) compared to TCR KO T cells (blue) and media alone (black). Figure 33C depicts representative eGFP quantification of live cell imaging of TCR353 T cells against Ewing Sarcoma cell lines TC-71 over 48 h period is shown. Colored lines correspond to wells with tumor cells cultured with TCR353 (green) compared to TCR KO T cells (blue) and media alone (black). Figure 33D depicts representative eGFP quantification of live cell imaging of TCR353 T cells against Ewing Sarcoma cell lines TC-205 over 48 h period is shown. Colored lines correspond to wells with tumor cells cultured with TCR353 (green) compared to TCR KO T cells (blue) and media alone (black). Figure 33E depicts representative Comparative kinetics of the time to reach 50% cell death KT50 (hrs) of SK-N-MCA*0201cells upon TCR353 recognition (green). Data represented at an E:T ratio of 3:1. Data are presented as mean values (n = 2). Data are representative of 2 independent experiments. *p<0.05, **p<0.001 t-test analysis comparing target cells in the basal state to target cells pretreated with +IFN-y. Figure 33F depicts representative Comparative kinetics of the time to reach 50% cell death KT50 (hrs) of TC-71 cells upon TCR353 recognition (green). Data represented at an E:T ratio of 3: 1. Data are presented as mean values (n = 2). Data are representative of 2 independent experiments. *p<0.05, **p<0.001 t-test analysis comparing target cells in the basal state to target cells pretreated with +IFN-y. Figure 33G depicts representative Comparative kinetics of the time to reach 50% cell death KT50 (hrs) of TC-205 cells upon TCR353 recognition (green). Data represented at an E:T ratio of 3:1. Data are presented as mean values (n = 2). Data are representative of 2 independent experiments. *p<0.05, **p<0.001 t-test analysis comparing target cells in the basal state to target cells pretreated with +IFN-y.

[0061] Figure 34 depicts a representative data demonstrating the TCR353 cytolytic activity on a panel of EWS-FL1 (A673, CHLA-9, TC-32, TC-71, RD-ES, and SK-NM- C), EWS-FEV (TC-205), and EWS-ERG (COG-E-352) Ewing Sarcoma tumor cell lines.

[0062] Figure 35, comprising Figure 35A and Figure 35B, depicts a representative data demonstrating the kinetics of Ewing Sarcoma tumor cell line growth in NSG mice. Figure 35A depicts a schematic representation of Ewing Sarcoma tumor cell line growth in NSG mice. Figure 35B depicts representative data demonstrating the bioluminescence (BLI) quantitation of engrafted cells at 7 days, 14 days, 21 days, 28 days, and 35 days post engraftment.

[0063] Figure 36, comprising Figure 36A and Figure 36B, depicts representative data demonstrating that overexpression of target antigen increases in vitro cytolytic activity of TCR353. Figure 36A depicts a schematic representation of a target antigen construct comprising a human 02M sequence, VLLSPLSRV peptide (SEQ ID NO: 5), HLA- A*02:01, and two linkers (A2 / LOXHD-1 SCT). Figure 36B depicts representative in vitro data demonstrating the cytolytic activity of TCR353 cells in a 4 h51Cr-release cytotoxicity assay using Ewing Sarcoma cell line TC-71. Compared are TC-71 cells at steady state, TC-71 cells in the presence of IFN-y, TC-71 cells with peptide, and TC-71 cells with SCT.

[0064] Figure 37, comprising Figure 37A through Figure 37C, depicts representative data demonstrating the in vivo antitumor efficacy of TCR353 T cells against target antigen depicted in Figure 36A expressed in TC71. Figure 37A depicts a schematic representation demonstrating that 20 NSG were intravenously engrafted with 5x105bioluminescent TC-7 cells or 5xl05bioluminescent TC-71 cells expressing A2 / LOXHD1 SCT in a lung metastasis model. Figure 37B depicts representative data demonstrating that the intravenous injection of TCR353+ reduced overall tumor volume as indicated by the reduced BLI signal. Figure 37C depicts representative data demonstrating that TCR353+ T cell-treated mice displayed an increased in overall survival relative to non- TCR T cell treated mice.

[0065] Figure 38, comprising Figure 38A through Figure 38G, depicts representative data demonstrating that adoptive transfer of TCR353-T cells led to in vivo eradication of Ewing Sarcoma under non-inflammatoiy and inflammatory conditions. Figure 38A depicts a schematic representation of in vivo Ewing Sarcoma mouse xenograft study and quantitation analysis of processed and presented VLLSPLSRV (SEQ ID NO: 5) / pMHC complexes expressed by SK-N-MCA*02:tncell line under basal and inflammatory conditions. SK-N-MCA*02:01tumors expressing CBR luciferase were engrafted into NSG mice via subcutaneous flank injection. On day -1, mice were either given one injection of PBS (basal) or + IFN-y (500 IU) (inflammatory) intratumorally. On day 0, 2, 4, and 6, mice were left untreated (MockZPBS) ) (n = 8), treated with TCR a0 null (TCR KO) (n = 8) or LOXHD1-TCR engineered CD8+ T cells (TCR353) (n = 8). Tumor burden was assessed weekly by BLI imaging before and after treatment, tumor volume (caliper measurement), and overall survival monitored over time. Figure 38B depicts representative data demonstrating the total flux quantification over time of individual mice under non-inflammatory (-IFN-y) conditions before and after treatment. Dots represent individual mice over time untreated / mock, TCRnull (TCRKO), and TCR353. Figure 38C depicts representative data demonstrating the total Flux quantification over time of individual mice under inflammatory (+ IFN-y) conditions before and after treatment. Dots represent individual mice over time untreated / mock, TCRnull (TCRKO), and TCR353. ****p<.0001, one-way ANOVA followed by Tukey’s HSD post-test comparing TCR353 and Mock under basal conditions and TCR353 and Mock under inflammatory conditions (+IFN-y) at 34 days. No statistical difference between Mock and TCR KO control groups under basal or inflammatory (+IFN-y) conditions. Figure 38D depicts representative bioluminescence imaging (n = 3 representative mice per group) prior to and following treatment. SK-N-MCA,02;01tumors treated with TCR353 T cells under basal conditions compared to control groups. Figure 38E depicts representative bioluminescence imaging (n = 3 representative mice per group) prior to and following treatment. tumors treated with TCR353 T cells under inflammatory conditions (+IFN-y) compared to control groups. Figure 38F depicts representative results of Kaplan-Meier analysis of overall survival of S basal. Colored lines correspond to treatment groups indicated in figure legend. ****p<0.0001. Figure 38G depicts representative results of Kaplan-Meier analysis of overall survival of SK-N- MC**02:01inflammatory (+IFN-y). Colored lines correspond to treatment groups indicated in figure legend. ****p<0.0001; log-rank testing comparing TCR353 and Mock under basal conditions and TCR353 and Mock under inflammatory conditions (+IFN-y). No statistical difference in survival between Mock and TCR KO control groups under basal or inflammatory (+IFN-y) conditions were noted.

[0066] Figure 39, comprising Figure 39A through Figure 39C, depicts representative data demonstrating that intratumor injection of TCR353 T cells conferred antitumor activity. Figure 39A depicts a schematic representation of the murine system that was used to compare the antitumor efficacy of TCR353+ T cells, TCR knock out (KO) cells, PBS, and IFN-y. 8 NSG mice that were subcutaneously injected with 2.5xl05bioluminescent TC-71 SCT353 cells on both left and right flanks. BLI was measured at 4 days post injections and 2 x 106TCR353+ T cells, TCR KO cells, PBS, or IFN-y were injected intratumorally into the right flank tumor engraflment site at 4 days, 6 days, 8 days, and 10 days post TC-71 SCT353 injection. Tumor progression was then measured for 1 week via BL1 quantitation / caliper measurement, overall survival and eye bleed to determine T cell persistence. Immunopeptidomics was performed on SK-NM-C, TC-71, TC-205, and surface pMHC complexes was quantitated. Figure 39B depicts representative data demonstrating that tumor volume, as indicated by BLI signal, was smaller on the left flank engraftment site of mice at 4 days, 10 days, and 17 days post subcutaneous tumor cell injection. Figure 39C depicts representative data demonstrating that the volume of tumor treated with TCR353 T cell were smaller than tumors treated with PBS as indicated by caliper measurement.

[0067] Figure 40 depicts a representative GLE951-Pep5 TCR ORF sequence and cloning system to generate variants thereof (SEQ ID NOs: 106-125).

[0068] Figure 41 depicts a representative GLE1034-pepl0 TCR ORF sequence and cloning system to generate variants thereof (SEQ ID NOs: 126-145).

[0069] Figure 42 depicts a representative translation of GLE951-Pep5 TCR ORF sequence (SEQ ID NOs: 146-148).

[0070] Figure 43, comprising Figure 43A through Figure 43D, depicts representative results demonstrating that intratumoral administration of TCR353 ablated tumor growth in vivo. Orthogonal assessment of tumor burden in the adoptive transfer of TCR353 in vivo. Weekly assessment of tumor volume by caliper measurement. Mice were monitored for clinical signs and weight. There is no significant difference in the weight of mice overtime under basal and inflammatory conditions (+IFN-y) compared to control groups (Mock and TCRKO). 2wayAnova / Sidak Multiple Compare between Mock and TCR353 treated mice **p<0.001, ****p<0.0001. Figure 43A depicts representative tumor volume of SK-N-MCA*02:01tumors treated with TCR353 T cells under basal conditions compared to control groups (Mock and TCRKO). Figure 43B depicts representative tumor volume of SK-N-MCA*02:01tumors treated with TCR353 T cells under inflammatory conditions (+IFN-y) compared to control groups (Mock and TCRKO). Figure 43C depicts representative weights of mice having SK-N-MCA*°2:01tumors treated with TCR353 T cells under basal conditions compared to control groups (Mock and TCRKO). Figure 43D depicts representative weights of mice having SK-N-MCA*02:01tumors treated with TCR353 T cells under inflammatory conditions (+IFN-y) compared to control groups (Mock and TCRKO).

[0071] DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention is based, in part, on the unexpected results that specifically targeting LOXHD1 in the context of one or more LOXHD1 peptides associated with HLA-A *02:01 and / or HLA-B *07:02 decreased the level of Ewing Sarcoma tumor cells.

[0073] As such, in one aspect, the present invention provides at least one T cell receptor (TCR) that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the at least one TCR specifically binds to a HLA-A*02:01:LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some aspects, the present invention provides a composition comprising at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprises at least one TCR that specifically binds to a HLA- A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex.

[0074] In some embodiments, the TCR is engineered. In some embodiments, the TCR is naturally occurring. In some embodiments, the at least one TCR comprises one or more TCR chains (e.g., TCR alpha chain, TCR beta chain, TCR delta chain, TCR gamma chain, etc.) that, either alone or together, specifically bind to HLA:LOXHD1 -peptide complex. For example, in some embodiments, the at least one TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR specifically binds to HLA:LOXHD1 -peptide complex. In some embodiments, the at least one TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR specifically binds to a HLA- A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex.

[0075] In one aspect, the present invention provides at least one modified cell (e.g., a modified T cell), which is modified to express at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the at least one modified cell (e.g., a modified T cell) is modified to express at least one TCR that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some embodiments, the present invention provides a composition comprising at least one modified cell (e.g., a modified T cell), which is modified to express the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprises at least one modified cell (e.g., a modified T cell), which is modified to express the at least one TCR that specifically binds to a HLA- A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex.

[0076] In one aspect, the present invention provides at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some embodiments, the present invention provides a composition comprising at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHD1- peptide complex. In some embodiments, the composition comprises at least one antigenbinding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex.

[0077] In one embodiment, the present invention provides at least one CAR that specifically binds to a HLA:LOXHD1 -peptide complex. In one embodiment, the at least one CAR specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some embodiments, the present invention provides a composition comprising at least one CAR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprising at least one CAR specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex.

[0078] In one aspect, the present invention provides at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLAzLOXHDl -peptide complex. In some embodiments, the at least one modified cell (e.g., a modified T cell) is modified to express a CAR that CAR specifically binds to a HLA-A*()2:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXH DI -peptide complex. In some embodiments, the present invention provides a composition comprising at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprises at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some embodiments, the at least one modified cell (e.g., a modified T cell), which is modified to express a CAR, is a CAR-T cell.

[0079] In one aspect, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a composition that specifically binds to a HLA:LOXHD1 peptide complex. In some embodiments, the method comprises administering to the subject a composition comprising at least one TCR that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex, at least one modified cell (e.g., a modified T cell), which is modified to express the at least one TCR that specifically binds to a HLA- A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, at least one CAR that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA-A*02:01:LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, or any combination thereof.

[0080] In a further embodiment, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof comprising adoptive therapy. In some embodiments, the adoptive therapy comprises isolating at least one cell from a subject; culturing the at least one cell ex vivo; genetically modifying the isolated cell to express at least one TCR of the present invention, wherein the TCR specifically binds to a HLA:LOXHD1 peptide complex (e.g., a HLA-A*02:01 :LOXHD1- pcptidc complex and / or a HLA-B *07:02:LOXHD1 -peptide complex); and administering the genetically modified cell to the subject. In some embodiments, the at least one cell isolated from a subject is a T cell.

[0081] In another embodiment, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof comprising adoptive therapy. In some embodiments, the adoptive therapy comprises isolating at least one cell from a subject; culturing the at least one cell ex vivo; genetically modifying the isolated cell to express at least one CAR of the present invention, wherein the CAR specifically binds to a HLA:LOXHD1 peptide complex (e.g., a HLA-A*02:01:LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex); and administering the genetically modified cell to the subject. In some embodiments, the at least one cell isolated from a subject is a T cell.

[0082] In one embodiment, the present invention provides a method for treating or preventing a LOXHD1 -associated disease or disorder in a subject having, suspected of having, or at risk for having a LOXHD1 -associated disease or disorder. Exemplary LOXHD1 -associated disease or disorder that is treatable or preventable by way of the compositions and methods of the present invention include, but is not limited to, a cancer, such as a bone cancer (e.g., Ewing Sarcoma), or any cancer characterized by cells that present a HLA:LOXHD1 -peptide complex.

[0083] In various embodiments, the compositions and methods described herein can be used to kill at least one cell of interest (e.g., a cancer cell, such as Ewing Sarcoma tumor cell), decrease tumor size, inhibit tumor growth, inhibit tumor metastasis, slow tumor progression or severity, and the like.

[0084] Definitions

[0085] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section.

[0086] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element

[0087] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0088] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, in some embodiments, the animal is a mammal, and in some embodiments, the mammal is a human, having a complement system, including a human in need of therapy for, or susceptible to, a condition or its sequelae. The individual may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, and mice and humans.

[0089] The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected / homeostatic) respective characteristic. Characteristics which are normal or expected for one cell, tissue type, or subject, might be abnormal for a different cell or tissue type.

[0090] The terms “effective amount” and “pharmaceutically effective amount” refer to a sufficient amount of an agent to provide the desired biological result. That result can be reduction and / or alleviation of at least one sign, symptom, or cause of a disease or disorder, or any other desired alteration of a biological system. An appropriate effective amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0091] A “therapeutic treatment” is a treatment administered to a subject who exhibits at least one sign or symptom of a disease or disorder, for the purpose of diminishing or eliminating the at least one sign or symptom.

[0092] As used herein, “treating a disease or disorder” means reducing the frequency and / or severity of at least one sign and / or symptom of the disease or disorder is experienced by a patient.

[0093] A “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject’s health continues to deteriorate.

[0094] In contrast, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health.

[0095] The term “cancer” as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

[0096] A disease or disorder is “alleviated” if the severity of at least one sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

[0097] The terms “inhibit” and “inhibition,” as used herein, means to reduce, suppress, diminish or block an activity or function by at least about 10% relative to a control value. In some embodiments, the activity is suppressed or blocked by at least about 50% compared to a control value. In some embodiments, the activity is suppressed or blocked by at least about 75%. In some embodiments, the activity is suppressed or blocked by at least about 95%.

[0098] The term “anti-tumor effect” as used herein, refers to a biological effect, which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of die peptides, polynucleotides, cells, and antibodies of the invention in prevention of the occurrence or recurrence of a tumor in the first place.

[0099] The phrase “biological sample", “sample” or “specimen" as used herein, is intended to include any sample comprising a cell, a tissue, or a bodily fluid in which expression of a nucleic acid or polypeptide can be delected. The biological sample may contain any biological material suitable for detecting a desired biomarker and may comprise cellular and / or non-cellular material obtained from the individual. Examples of such biological samples include but are not limited to blood, lymph, bone marrow, biopsies, and smears. Sanpies that are liquid in nature are referred to herein as “bodily fluids." Biological samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to obtain bodily fluids. Methods for collecting various body samples are well known in the art.

[0100] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0101] “Allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0102] “Xenogeneic" refers to a graft derived from an animal of a different species.

[0103] “Operably linked” or “operatively linked” as used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter may be positioned 5* (upstream) or 3’ (downstream) of a gene under its control. The distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.

[0104] “Activation”, as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division. As used herein, an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.

[0105] As used herein, the term “immune cell” includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils, and granulocytes.

[0106] As used herein, the term “immune response” includes T cell mediated and / or B cell mediated immune responses that are influenced by modulation of T cell costimulation. The term immune response further includes immune responses that are indirectly effected by T cell activation such as antibody production (humoral responses) and the activation of cytokine responsive cells such as macrophages.

[0107] As used herein, the term “T cell immune response” refers to activation of antigen specific T cells as measured by proliferation or expression of molecules on the cell surface or secretion of proteins such as cytokines.

[0108] As used herein, the term “T cell” refers to a lymphocyte (e.g., white blood cell) that functions in cell-mediated immunity. In some embodiments, the presence of a T cell receptor (TCR) on the cell surface distinguishes T cells from other lymphocytes. As is known in the art, T cells typically do not present antigens, and rely on other lymphocytes (e.g., natural killer cells and B cells) to aid in antigen presentation. Types of T cells include: T helper cells (TH cells), Memory T cells (Tcm, Tem, or Temra), Regulatory T cells (Treg), Cytotoxic T cells (CTLs), Natural killer T cells (NK cells), gamma delta T cells, and Mucosal associated invariant T cells (MAIT).

[0109] As used herein, the term “TCR” refers to “T cell receptor.” A T cell receptor is a molecule on the surface of T lymphocytes (“T cells”). In one embodiment, the receptor is an a0-TCR receptor, meaning that the T cell receptor comprises an alpha (a) and beta (0) chain, which is typically expressed as part of a complex with CD3 chain molecules.

[0110] “CDRs” are defined as the complementarity determining region amino acid sequences of a TCR or TCR chain.

[0111] By the term “specifically binds,” as used herein with respect to a polypeptide (e.g., a TCR or TCR chain), is meant a polypeptide which recognizes and binds to a specific target molecule, but does not substantially recognize or bind other molecules in a sample. In some instances, the terms “specific binding” or “specifically binding,” is used to mean that the recognition and binding is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the target molecule.

[0112] The term “antibody” includes any antibody protein construct comprising at least one antibody variable domain comprising at least one antigen-binding site (ABS). Antibodies include, but are not limited to, immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). The overall structure of Immunoglobulin G (IgG) antibodies assembled from two identical heavy (H)-chain and two identical light (L)- chain polypeptides is well established and highly conserved in mammals (Padlan (1994) Mol. Immunol. 31:169-217).

[0113] A conventional antibody or immunoglobulin (Ig) is a protein comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy (H) chain variable domains are abbreviated herein as VH, and the light (L) chain variable domains are abbreviated herein as VL. These domains, domains related thereto, and domains derived therefrom, may be referred to herein as immunoglobulin chain variable domains. The VH and VL domains (also referred to as VH and VL regions) can be further subdivided into regions, termed “complementarity determining regions” (“CDRs”), interspersed with regions that are more conserved, termed “framework regions” (“FRs”). The framework and complementarity determining regions have been precisely defined (Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition U.S. Department of Health and Human Services, (1991) NIH Publication Number 91 -3242). There are also alternative numbering conventions for CDR sequences, for example those set out in Chothia et al. (1989) Nature 342: 877-883 or as summarized by IMGT.org. In a conventional antibody, each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-temiinus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The conventional antibody tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains is formed with the heavy and the light immunoglobulin chains inter-connected by e.g., disulphide bonds, and the heavy chains similarly connected. The heavy chain constant region includes three domains, CH1 , CH2, and CH3. The light chain constant region is comprised of one domain, CL. The variable domain of the heavy chains and the variable domain of the light chains are binding domains that interact with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl q) of the classical complement system.

[0114] “Specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antibody or antigen-binding fragment thereof can bind. The specificity of an antibody is the ability of the antibody to recognize a particular antigen as a unique molecular entity and distinguish it from another. An antibody that “specifically binds” to an antigen or an epitope is a term well understood in the art. A molecule is said to exhibit “specific binding” if it reacts more frequently, more rapidly, with greater duration and / or with greater affinity with a particular target antigen or epitope, than it does with alternative targets. An antibody “specifically binds” to a target antigen or epitope if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances.

[0115] In some embodiments of the invention, the polypeptides or antibodies are bispecific polypeptides or antibodies. A “bispecific polypeptide” or “bispecific antibody” is a polypeptide or antibody that is capable of binding two different epitopes simultaneously and / or sequentially. Generally, the epitopes will not be on the same antigen. Hence bispecific polypeptides or antibodies have the capability to selectively bind to two different epitopes present on two different antigens via two different binding domains. This contrasts with conventional monospecific polypeptides or antibodies which do not have this capability. Hence, the bispecific polypeptides or antibodies of the invention bind two different antigens simultaneously and / or sequentially.

[0116] “Affinity,” represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding polypeptide (KD), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antibody (or antigenbinding fragment thereof): the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding polypeptide. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD. Affinity can be determined by known methods, depending on the specific antigen of interest. For example, in some embodiments, KD may be determined by surface plasmon resonance. Any KD value less than 10-6 is considered to indicate binding. Specific binding of an antibody, or antigen-binding fragment thereof, to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (BIA), and sandwich competition assays, equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay) and the different variants thereof known in the art.

[0117] “Avidity” is the measure of the strength of binding between an antibody, or antigen-binding fragment thereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antibody and the number of pertinent binding sites present on the antibody.

[0118] A “coding region” of a gene consists of the nucleotide residues of the coding strand of the gene and the nucleotides of the non-coding strand of the gene, which are homologous with or complementary to, respectively, the coding region of an mRNA molecule which is produced by transcription of the gene. A “coding region” of a mRNA molecule also consists of the nucleotide residues of the mRNA molecule, which are matched with an anti-codon region of a transfer RNA molecule during translation of the mRNA molecule or which encode a stop codon. The coding region may thus include nucleotide residues comprising codons for amino acid residues, which are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0119] “Differentially decreased expression” or “down regulation” refers to biomarker product levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% lower or less, and / or 2.0 fold, 1.8 fold, 1.6 fold, 1.4 fold, 1.2 fold, 1.1 fold or less lower, and any and all whole or partial increments therebetween than a control.

[0120] “Differentially increased expression” or “up regulation” refers to biomarker product levels which are at least 10% or more, for example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% higher or more, and / or 1.1 fold, 1 .2 fold, 1.4 fold, 1 .6 fold, 1.8 fold, 2.0 fold higher or more, and any and all whole or partial increments therebetween than a control.

[0121] “Complementary” as used herein to refer to a nucleic acid, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and or at least about 75%, or at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In some embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0122] The term “DNA” as used herein is defined as deoxyribonucleic acid.

[0123] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0124] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA methods.

[0125] “Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0126] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0127] The term “hybridoma,” as used herein refers to a cell resulting from the fusion of a B-lymphocyte and a fusion partner such as a myeloma cell. A hybridoma can be cloned and maintained indefinitely in cell culture and is able to produce monoclonal antibodies. A hybridoma can also be considered to be a hybrid cell.

[0128] “Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in its normal context in a living subject is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is “isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0129] An “isolated nucleic acid" refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

[0130] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0131] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0132] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0133] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, hctcrodimcrs, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0134] The term “RNA” as used herein is defined as ribonucleic acid.

[0135] As used herein, “conjugated” refers to covalent attachment of one molecule to a second molecule.

[0136] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0137] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91 %, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 80%, at least 75%, or at least 70% identical to the reference sequence.

[0138] The term “regulating” as used herein can mean any method of altering the level or activity of a substrate. Non-limiting examples of regulating with regard to a protein include affecting expression (including transcription and / or translation), affecting folding, affecting degradation or protein turnover, and affecting localization of a protein. Nonlimiting examples of regulating with regard to an enzyme further include affecting the enzymatic activity. “Regulator" refers to a molecule whose activity includes affecting the level or activity of a substrate. A regulator can be direct or indirect. A regulator can function to activate or inhibit or otherwise modulate its substrate.

[0139] “Vector” as used herein may mean a nucleic acid sequence containing an origin of replication. A vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector may be a DNA or RNA vector. A vector may be either a self-replicating extrachromosomal vector or a vector which integrates into a host genome.

[0140] As used herein, a “substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0141] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0142] Description

[0143] The present invention is based, in part, on the unexpected results that specifically targeting LOXHD1 in the context of one or more LOXHD1 peptides associated with HLA-A *02:01 and / or HLA-B *07:02 decreased the level of Ewing Sarcoma tumor cells.

[0144] As such, in one aspect, the present invention provides at least one TCR that specifically binds to a HLA:LOXH DI -peptide complex. In some embodiments, the at least one TCR specifically binds to a HLA-A*02:01 :LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some aspects, the present invention provides a composition comprising at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprises at least one TCR that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex.

[0145] In some embodiments, the TCR is engineered. In some embodiments, the TCR is naturally occurring. In some embodiments, the at least one TCR comprises one or more TCR chains (e.g., TCR alpha chain, TCR beta chain, TCR delta chain, TCR gamma chain, etc.) that, either alone or together, specifically bind to HLA:LOXHD1 -peptide complex. For example, in some embodiments, the at least one TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR specifically binds to HLA:LOXHD1 -peptide complex. In some embodiments, the at least one TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR specifically binds to a HLA- A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex.

[0146] In one aspect, the present invention provides at least one modified cell (e.g., a modified T cell), which is modified to express at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the at least one modified cell (e.g., a modified T cell) is modified to express at least one TCR that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-pcptidc complex. In some embodiments, the present invention provides a composition comprising at least one modified cell (e.g., a modified T cell), which is modified to express the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprises at least one modified cell (e.g., a modified T cell), which is modified to express the at least one TCR that specifically binds to a HLA- A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex.

[0147] In one aspect, the present invention provides at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHDl-peptide complex. In some embodiments, the at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) specifically binds to a HLA-A*02:01 :LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some embodiments, the present invention provides a composition comprising at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHD1- peptide complex. In some embodiments, the composition comprises at least one antigenbinding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex.

[0148] In one embodiment, the present invention provides at least one CAR that specifically binds to a HLA:LOXHD1 -peptide complex. In one embodiment, the at least one CAR specifically binds to a HLA-A*02:01 :LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some embodiments, the present invention provides a composition comprising at least one CAR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprising at least one CAR specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02 :LOXHD1 -peptide complex.

[0149] In one aspect, the present invention provides at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA:LOXHDl-peptide complex. In some embodiments, the at least one modified cell (e.g., a modified T cell) is modified to express a CAR that CAR specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some embodiments, the present invention provides a composition comprising at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLArLOXHDl -peptide complex. In some embodiments, the composition comprises at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some embodiments, the at least one modified cell (e.g., a modified T cell), which is modified to express a CAR, is a CAR-T cell.

[0150] In one aspect, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a composition that specifically binds to a HLA:LOXHD1 peptide complex. In some embodiments, the method comprises administering to the subject a composition comprising at least one TCR that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex, at least one modified cell (e.g., a modified T cell), which is modified to express the at least one TCR that specifically binds to a HLA- A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc. ) that specifically binds to a HLA-A*02:01 :LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, at least one CAR that specifically binds to a HLA-A*02:01 :LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex, at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA-A*02:()1:LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex, or any combination thereof.

[0151] In a further embodiment, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof comprising adoptive therapy. In some embodiments, the adoptive therapy comprises isolating at least one cell from a subject; culturing the at least one cell ex vivo; genetically modifying the isolated cell to express at least one TCR of the present invention, wherein the TCR specifically binds to a HLA:LOXHD1 peptide complex (e.g., a HLA-A*02:01:LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex); and administering the genetically modified cell to the subject. In some embodiments, the at least one cell isolated from a subject is a T cell.

[0152] In another embodiment, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof comprising adoptive therapy. In some embodiments, the adoptive therapy comprises isolating at least one cell from a subject; culturing the at least one cell ex vivo; genetically modifying the isolated cell to express at least one CAR of the present invention, wherein the CAR specifically binds to a HLA:LOXHD1 peptide complex (e.g., a HLA-A*02:01 :LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex); and administering the genetically modified cell to the subject. In some embodiments, the at least one cell isolated from a subject is a T cell.

[0153] In one embodiment, the present invention provides a method for treating or preventing a LOXH DI -associated disease or disorder in a subject having, suspected of having, or at risk for having a LOXHD1 -associated disease or disorder. Exemplary LOXHD1 -associated disease or disorder that is treatable or preventable by way of the compositions and methods of the present invention include, but is not limited to, a cancer, such as a bone cancer (e.g., Ewing Sarcoma), or any cancer characterized by cells that present a HLA:LOXHD1 -peptide complex.

[0154] In various embodiments, the compositions and methods described herein can be used to kill at least one cell of interest (e.g., a cancer cell, such as Ewing Sarcoma tumor cell), decrease tumor size, inhibit tumor growth, inhibit tumor metastasis, slow tumor progression or severity, and the like.

[0155] T cell receptors (TCRs)

[0156] In one embodiment, the present invention provides at least one T cell receptor (TCR) that specifically binds to a HL A: LOXH DI -peptide complex. In some embodiments, the at least one TCR specifically binds to a HLA-A*02:01 :LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some embodiments, the present invention provides a composition comprising at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex. In embodiments, the present invention provides a composition comprising at least one TCR that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex.

[0157] In some embodiments, the TCR is engineered. In some embodiments, the TCR is naturally occurring.

[0158] In some embodiments, the TCR comprises one or more TCR chains (e.g., TCR alpha chain, TCR beta chain, TCR delta chain, TCR gamma chain, etc.) that, either alone or together, specifically bind to HLA:LOXHD1 -peptide complex. For example, in one embodiment, the TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR specifically binds to HLA:LOXHD1 -peptide complex. In one embodiment, the TCR comprises a TCR alpha chain and a TCR beta chain, wherein the TCR specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1- peptide complex.

[0159] T cell receptor (TCR) 353

[0160] TCR 353 proteins

[0161] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex, comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR alpha chain.

[0162] In one embodiment, the CDR1 of the TCR alpha chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 78, or a variant or a fragment thereof. For example, in one embodiment, the CDR1 of the TCR alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 78.

[0163] In one embodiment, the CDR2 of the TCR alpha chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 79, or a variant or a fragment thereof. For example, in one embodiment, the CDR2 of the TCR alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 79.

[0164] In one embodiment, the CDR3 of the TCR alpha chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 80, or a variant or a fragment thereof. For example, in one embodiment, the CDR3 of the TCR alpha chain comprises the amino acid sequence as set forth in SEQ ID NO: 80.

[0165] Thus, in some embodiments, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 78, or a variant or a fragment thereof, a CDR2 of a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 79, or a variant or a fragment thereof, and a CDR3 of a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 80, or a variant or a fragment thereof. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 78, a CDR2 of a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 79, and a CDR3 of a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 80.

[0166] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR alpha chain. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 74, or a variant or a fragment thereof. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 74.

[0167] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0168] In one embodiment, the CDR1 of the TCR beta chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 84, or a variant or a fragment thereof. For example, in one embodiment, the CDR1 of the TCR beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 84.

[0169] In one embodiment, the CDR2 of the TCR beta chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 85, or a variant or a fragment thereof. For example, in one embodiment, the CDR2 of the TCR beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 85.

[0170] In one embodiment, the CDR3 of the TCR beta chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 86, or a variant or a fragment thereof. For example, in one embodiment, the CDR3 of the TCR beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 86.

[0171] Thus, in some embodiments, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 84, or a variant or a fragment thereof, a CDR2 of a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 85, or a variant or a fragment thereof, and a CDR3 of a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 86, or a variant or a fragment thereof. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 84, a CDR2 of a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 85, and a CDR3 of a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 86.

[0172] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR beta chain. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 76, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLAzLOXHDl -peptide complex comprises a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 76. In one embodiment, the at least one TCR that specifically binds to a HLAzLOXHDl -peptide complex comprises (a) one or more of: SEQ ID NOs: 78-80, or a variant or a fragment thereof, and (b) one or more of SEQ ID NOs: 84-86, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising one or more of: SEQ ID NOs: 78-80, or a variant or a fragment thereof, and (b) a TCR beta chain comprising one or more of SEQ ID NOs: 84-86, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1- peptide complex comprises (a) a TCR alpha chain comprising one or more of: SEQ ID NOs: 78-80, or a variant or a fragment thereof, and (b) a TCR beta chain comprising one or more of SEQ ID NOs: 84-86, or a variant or a fragment thereof, wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof, and the HLA molecule comprises HLA- A*02:01 and / or HLA-A*07:02.

[0173] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) SEQ ID NOs: 78-80, or a variant or a fragment thereof, and (b) SEQ ID NOs: 84-86, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising SEQ ID NOs: 78-80, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NOs: 84-86, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising SEQ ID NOs: 78-80, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NOs: 84-86, or a variant or a fragment thereof, and wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof, and wherein the H LA molecule comprises HLA-A*02:01 and / or HLA- A*07:02.

[0174] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) SEQ ID NO: 74, or a variant or a fragment thereof, and (b) SEQ ID NO: 76, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising SEQ ID NO: 74, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NO: 76, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex (a) a TCR alpha chain comprising SEQ ID NO: 74, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NO: 76, or a variant or a fragment thereof, wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof, and the HLA molecule comprises HLA-A*02:01 and / or HLA-A*07:02.

[0175] TCR 353 nucleic acids

[0176] In one embodiment, a nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain. In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain, a nucleotide sequence encoding CDR2 of a TCR alpha chain, and a nucleotide sequence encoding CDR3 of a TCR alpha chain. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding CDR1 of a TCR alpha chain, a nucleotide sequence encoding CDR2 of a TCR alpha chain, and a nucleotide sequence encoding CDR3 of a TCR alpha chain.

[0177] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain that comprises one or more of: SEQ ID NOs: 78- 80 or a variant thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain that comprises one or more of: CDR1 comprising the amino acid sequence of SEQ ID NO: 78, or a variant or a fragment thereof, CDR2 comprising the amino acid sequence of SEQ ID NO: 79, or a variant or a fragment thereof, and CDR3 comprising the amino acid sequence of SEQ ID NO: 80, or a variant or a fragment thereof.

[0178] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 78, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 78.

[0179] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 79, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 79.

[0180] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 80, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 80.

[0181] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 78, or a variant or a fragment thereof, a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 79, or a variant or a fragment thereof, and a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 80, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 78, a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 79, and a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 80.

[0182] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain, a nucleotide sequence encoding CDR2 of a TCR alpha chain, and a nucleotide sequence encoding CDR3 of a TCR alpha chain.

[0183] In one embodiment, the nucleotide sequence encoding CDR1 of a TCR alpha chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 81, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR1 of a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 81.

[0184] In one embodiment, the nucleotide sequence encoding CDR2 of a TCR alpha chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 82, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR2 of a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 82.

[0185] In one embodiment, the nucleotide sequence encoding CDR3 of a TCR alpha chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 83, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR3 of a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 83.

[0186] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 81, or a variant or a fragment thereof, a nucleotide sequence encoding CDR2 of a TCR alpha chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 82, or a variant or a fragment thereof, and a nucleotide sequence encoding CDR3 of a TCR alpha chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 83, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 81, a nucleotide sequence encoding CDR2 of a TCR alpha chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 82, and a nucleotide sequence encoding CDR3 of a TCR alpha chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 83.

[0187] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain that comprises SEQ ID NO: 74, or a variant or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 74, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 74.

[0188] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a TCR alpha chain. In one embodiment, the nucleotide sequence encoding a TCR alpha chain comprises at least about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 75, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 75.

[0189] In one embodiment, a nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR beta chain. In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain, a nucleotide sequence encoding CDR2 of a TCR beta chain, and a nucleotide sequence encoding CDR3 of a TCR beta chain. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding CDR1 of a TCR beta chain, a nucleotide sequence encoding CDR2 of a TCR beta chain, and a nucleotide sequence encoding CDR3 of a TCR beta chain.

[0190] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR beta chain that comprises one or more of: SEQ ID NOs: 84- 86, or a variant or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a TCR comprising a TCR beta chain that comprises one or more of: CDR1 comprising the amino acid sequence of SEQ ID NO: 84, or a variant or a fragment thereof, CDR2 comprising the amino acid sequence of SEQ ID NO: 85, or a variant or a fragment thereof, and CDR3 comprising the amino acid sequence of SEQ ID NO: 86, or a variant or a fragment thereof.

[0191] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 84, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 84. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 85, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 85.

[0192] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 86, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 86.

[0193] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 84, or a variant or a fragment thereof, a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 85, or a variant or a fragment thereof, and a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 86, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 84, a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 85, and a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 86. In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain, a nucleotide sequence encoding CDR2 of a TCR beta chain, and a nucleotide sequence encoding CDR3 of a TCR beta chain.

[0194] In one embodiment, the nucleotide sequence encoding CDR1 of a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO:

[0195] 87, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR1 of a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 87.

[0196] In one embodiment, the nucleotide sequence encoding CDR2 of a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO:

[0197] 88, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR2 of a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 88.

[0198] In one embodiment, the nucleotide sequence encoding CDR3 of a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO:

[0199] 89, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR3 of a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 89.

[0200] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 87, or a variant or a fragment thereof, a nucleotide sequence encoding CDR2 of a TCR beta chain that comprises at least about 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 88, or a variant or a fragment thereof, and a nucleotide sequence encoding CDR3 of a TCR beta chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 89, or a variant or a fragment thereof. For example, in one embodiment, nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 87, a nucleotide sequence encoding CDR2 of a TCR beta chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 88, and a nucleotide sequence encoding CDR3 of a TCR beta chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 89.

[0201] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a TCR comprising a TCR beta chain that comprises SEQ ID NO: 76, or a variant or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 76, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 76.

[0202] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a TCR beta chain. In one embodiment, the nucleotide sequence encoding a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 77, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 77. T cell receptor (TCR) 1134

[0203] TCR1134 proteins

[0204] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex, comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR alpha chain.

[0205] In one embodiment, the CDR1 of the TCR alpha chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 94, or a variant or a fragment thereof. For example, in one embodiment, the CDR1 of the TCR alpha chain comprises the amino acid sequence as set forth in SEQ ID NO:

[0206] 94.

[0207] In one embodiment, the CDR2 of the TCR alpha chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 95, or a variant or a fragment thereof. For example, in one embodiment, the CDR2 of the TCR alpha chain comprises the amino acid sequence as set forth in SEQ ID NO:

[0208] 95.

[0209] In one embodiment, the CDR3 of the TCR alpha chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 96, or a variant or a fragment thereof. For example, in one embodiment, the CDR3 of the TCR alpha chain comprises the amino acid sequence as set forth in SEQ ID NO:

[0210] 96.

[0211] Thus, in some embodiments, the at least one TCR that specifically binds to a H LA: LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 94, or a variant or a fragment thereof, a CDR2 of a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 95, or a variant or a fragment thereof, and a CDR3 of a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 96, or a variant or a fragment thereof. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 94, a CDR2 of a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 95, and a CDR3 of a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 96.

[0212] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR alpha chain. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR alpha chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 90, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR alpha chain comprising the amino acid sequence as set forth in SEQ ID NO: 90.

[0213] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1, a CDR2, and a CDR3 of a TCR beta chain.

[0214] In one embodiment, the CDR1 of the TCR beta chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 100, or a variant or a fragment thereof. For example, in one embodiment, the CDR1 of the TCR beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 100.

[0215] In one embodiment, the CDR2 of the TCR beta chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 101, or a variant or a fragment thereof. For example, in one embodiment, the CDR2 of the TCR beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 101.

[0216] In one embodiment, the CDR3 of the TCR beta chain comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 102, or a variant or a fragment thereof. For example, in one embodiment, the CDR3 of the TCR beta chain comprises the amino acid sequence as set forth in SEQ ID NO: 102.

[0217] Thus, in some embodiments, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 100, or a variant or a fragment thereof, a CDR2 of a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ I D NO: 101 , or a variant or a fragment thereof, and a CDR3 of a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 102, or a variant or a fragment thereof. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises one or more of: a CDR1 of a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 100, a CDR2 of a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 101 , and a CDR3 of a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 102.

[0218] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR beta chain. For example, in one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR beta chain comprising an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 92, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises a TCR beta chain comprising the amino acid sequence as set forth in SEQ ID NO: 92.

[0219] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) one or more of: SEQ ID NOs: 94-96, or a variant or a fragment thereof, and (b) one or more of SEQ ID NOs: 100-102, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising one or more of: SEQ ID NOs: 94-96, or a variant or a fragment thereof, and (b) a TCR beta chain comprising one or more of SEQ ID NOs: 100-102, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXH DI -peptide complex comprises (a) a TCR alpha chain comprising one or more of: SEQ ID NOs: 94-96, or a variant or a fragment thereof, and (b) a TCR beta chain comprising one or more of SEQ ID NOs: 100-102, or a variant or a fragment thereof, wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-A*02:01 and / or HLA-A*07:02. In one embodiment, the at least one TCR that specifically binds to a HLAzLOXHDl -peptide complex comprises (a) SEQ ID NOs: 94-96, or a variant or a fragment thereof, and (b) SEQ ID NOs: 100-102, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising SEQ ID NOs: 94-96, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NOs: 100-102, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising SEQ ID NOs: 94-96, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NOs: 100-102, or a variant or a fragment thereof, wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-A*02:01 and / or HLA- A*07:02.

[0220] In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) SEQ ID NO: 90, or a variant or a fragment thereof, and (b) SEQ ID NO: 92, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising SEQ ID NO: 90, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NO: 92, or a variant or a fragment thereof. In one embodiment, the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprises (a) a TCR alpha chain comprising SEQ ID NO: 90, or a variant or a fragment thereof, and (b) a TCR beta chain comprising SEQ ID NO: 92, or a variant or a fragment thereof, wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-A*02:01 and / or HLA-A*07:02.

[0221] TCR.1134 nucleic acids

[0222] In one embodiment, a nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain. In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain, a nucleotide sequence encoding CDR2 of a TCR alpha chain, and a nucleotide sequence encoding CDR3 of a TCR alpha chain. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding CDR1 of a TCR alpha chain, a nucleotide sequence encoding CDR2 of a TCR alpha chain, and a nucleotide sequence encoding CDR3 of a TCR alpha chain.

[0223] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain that comprises one or more of: SEQ ID NOs: 94- 96, or a variant or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain that comprises one or more of: CDR1 comprising the amino acid sequence of SEQ ID NO: 94, or a variant or a fragment thereof, CDR2 comprising the amino acid sequence of SEQ ID NO: 95, or a variant or a fragment thereof, and CDR3 comprising the amino acid sequence of SEQ ID NO: 96, or a variant or a fragment thereof.

[0224] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 94, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 94 or a fragment thereof.

[0225] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 95, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 95.

[0226] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 96, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 96.

[0227] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 94, or a variant or a fragment thereof, a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 95, or a variant or a fragment thereof, and a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 96, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 94, a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 95, and a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 96.

[0228] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain, a nucleotide sequence encoding CDR2 of a TCR alpha chain, and a nucleotide sequence encoding CDR3 of a TCR alpha chain.

[0229] In one embodiment, the nucleotide sequence encoding CDR1 of a TCR alpha chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 97, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR1 of a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 97.

[0230] In one embodiment, the nucleotide sequence encoding CDR2 of a TCR alpha chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 98, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR2 of a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 98 or a fragment thereof.

[0231] In one embodiment, the nucleotide sequence encoding CDR3 of a TCR alpha chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 99, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR3 of a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 99.

[0232] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 97, or a variant or a fragment thereof, a nucleotide sequence encoding CDR2 of a TCR alpha chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 98, or a variant or a fragment thereof, and a nucleotide sequence encoding CDR3 of a TCR alpha chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 99, or a variant or a fragment thereof. For example, in one embodiment, nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR alpha chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 97, a nucleotide sequence encoding CDR2 of a TCR alpha chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 98, and a nucleotide sequence encoding CDR3 of a TCR alpha chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 99.

[0233] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR alpha chain that comprises SEQ ID NO: 90, or a variant or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 90, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 90.

[0234] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a TCR alpha chain. In one embodiment, the nucleotide sequence encoding a TCR alpha chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 91, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding a TCR alpha chain comprises the nucleotide sequence as set forth in SEQ ID NO: 91.

[0235] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR beta chain. In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain, a nucleotide sequence encoding CDR2 of a TCR beta chain, and a nucleotide sequence encoding CDR3 of a TCR beta chain. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding CDR1 of a TCR beta chain, a nucleotide sequence encoding CDR2 of a TCR beta chain, and a nucleotide sequence encoding CDR3 of a TCR beta chain.

[0236] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR beta chain that comprises one or more of: SEQ ID NOs: 100- 102, or a variant or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a TCR comprising a TCR beta chain that comprises one or more of: CDR1 comprising the amino acid sequence of SEQ ID NO: 100, or a variant or a fragment thereof, CDR2 comprising the amino acid sequence of SEQ ID NO: 101, or a variant or a fragment thereof, and CDR3 comprising the amino acid sequence of SEQ ID NO: 102, or a variant or a fragment thereof.

[0237] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 100, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 100.

[0238] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 101, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 101.

[0239] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 102, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 102.

[0240] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 100, or a variant or a fragment thereof, a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 101, or a variant or a fragment thereof, and a nucleotide sequence encoding an amino acid al least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 102, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 100, a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 101, and a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 102.

[0241] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain, a nucleotide sequence encoding CDR2 of a TCR beta chain, and a nucleotide sequence encoding CDR3 of a TCR beta chain.

[0242] In one embodiment, the nucleotide sequence encoding CDR1 of a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 103, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR1 of a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 103.

[0243] In one embodiment, the nucleotide sequence encoding CDR2 of a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO:

[0244] 104, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR2 of a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 104.

[0245] In one embodiment, the nucleotide sequence encoding CDR3 of a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO:

[0246] 105, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding CDR3 of a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 105.

[0247] In one embodiment, the nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 103, or a variant or a fragment thereof, a nucleotide sequence encoding CDR2 of a TCR beta chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 104, or a variant or a fragment thereof, and a nucleotide sequence encoding CDR3 of a TCR beta chain that comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 105, or a variant or a fragment thereof. For example, in one embodiment, nucleic acid molecule comprises one or more of: a nucleotide sequence encoding CDR1 of a TCR beta chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 103, a nucleotide sequence encoding CDR2 of a TCR beta chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 104, and a nucleotide sequence encoding CDR3 of a TCR beta chain that comprises the nucleotide sequence as set forth in SEQ ID NO: 105.

[0248] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex comprising a TCR beta chain that comprises SEQ ID NO: 92 or a variant or a fragment thereof. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding an amino acid at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence as set forth in SEQ ID NO: 92, or a variant or a fragment thereof. For example, in one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding the amino acid sequence as set forth in SEQ ID NO: 92.

[0249] In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding a TCR beta chain. In one embodiment, the nucleotide sequence encoding a TCR beta chain comprises at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the nucleotide sequence as set forth in SEQ ID NO: 93, or a variant or a fragment thereof. For example, in one embodiment, the nucleotide sequence encoding a TCR beta chain comprises the nucleotide sequence as set forth in SEQ ID NO: 93.

[0250] LOXHD 1 -peptides

[0251] In various embodiments, the present invention provides at least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD 1 peptide comprises at least one amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof. For example, in some embodiments, the LOXHD 1 peptide comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from SEQ ID NOs: 1-27 and 29-73. In some embodiments, the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 29-73 or a variant thereof. In some embodiments, the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from SEQ ID NOs: 1, 5, 10, 29, and 30. In some embodiments, the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1, 5, 10, 29, and 30.

[0252] In various embodiments, the present invention provides at least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises an amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 28, or a variant or a fragment thereof. For example, in some embodiments, the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to SEQ ID NO: 28. In some embodiments, the LOXHD1 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 28.

[0253] In various embodiments, the present invention provides at least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises about 5 to about 24 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 8 to about 24 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 9 to about 11 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 8 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 9 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 10 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide about 11 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 12 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 13 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 14 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 15 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 16 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 17 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 18 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 19 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 20 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 21 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 22 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 23 amino acid residues of the sequence as set forth in SEQ ID NO: 28. In one embodiment, the LOXHD1 peptide comprises about 24 amino acid residues of the sequence as set forth in SEQ ID NO: 28.

[0254] In one embodiment, the present invention provides a nucleic acid molecule encoding one or more of the LOXHD1 peptides described herein. In various embodiments, the nucleic acid molecule is an isolated nucleic acid molecule. For example, in some embodiments, the nucleic acid molecule is a DNA, RNA, or mRNA, encoding one or more of the LOXHD1 peptides described herein.

[0255] In one embodiment, the nucleic acid molecule encodes one more of the antigenic LOXHD1 peptides that comprise at least one amino acid sequence selected from SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof.

[0256] In one embodiment, the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence having substantial homology to at least one amino acid sequence selected from SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof. For example, in certain one embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding at least one amino acid sequence that is at least about 60%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof. For example, in some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding at least one amino acid sequence as set forth in SEQ ID NOs: 1-27 and 29-73.

[0257] HLA-A*02:01

[0258] In one embodiment, the present invention provides for at least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the HLA molecule comprises HLA-A*02:01. For example, in one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-A*02:01. In one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) specifically binds to a HLA: LOXHD1 -peptide complex, wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 1 , 5, 10, 26, and 30-32, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-A*02:01.

[0259] In one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1 -27 and 30-61 , or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA- A*02:01. In one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) specifically binds to a HLA:LOXHD1 -peptide complex comprises a LOXHD1 peptide comprising at least one amino acid sequence as set forth in SEQ ID NOs: 1, 5, 10, 26, and 30-32, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-A*02:01. In one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 1, 5, 10, and 30, or a variant or a fragment thereof, and the HLA molecule comprises HLA- A*02:01.

[0260] HLA-B*07:02

[0261] In one embodiment, the present invention provides for at least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the HLA molecule comprises HLA-B*07:02. For example, in one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 29 and 62-73, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-B*07:02 molecule. In one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) specifically binds to a HLArLOXHDl -peptide complex wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 29, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-B*07:02.

[0262] In one embodiment, the at least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises at least one amino acid sequence as set forth in SEQ ID NOs: 29 and 62-73, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-B*07:02 molecule. In one embodiment, the al least one molecule (e.g., TCR, antibody, CAR, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex wherein the LOXHD1 peptide comprises the amino acid sequence as set forth in SEQ ID NO: 29, or a variant or a fragment thereof, and wherein the HLA molecule comprises HLA-B*07:02.

[0263] Modified T cells

[0264] In one embodiment, the present invention provides at least one modified cell, such as at least one modified T cell, which is modified to express at least one TCR wherein the at least one TCR specifically binds to a HLAzLOXHDl -peptide complex. In one embodiment, the present invention provides at least one modified cell, such as at least one modified T cell, which is modified to express at least one TCR wherein the at least one TCR specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some embodiments, the present invention provides a composition comprising at least one modified cell, such as at least one modified T cell, which is modified to express the at least one TCR, wherein the TCR specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the present invention provides a composition comprising at least one modified cell, such as at least one modified T cell, which is modified to express the at least one TCR, wherein the TCR specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex.

[0265] In one embodiment, the at least one modified T cell is modified to express at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex, wherein the TCR comprises a TCR a chain and a TCR 0 chain, wherein the TCR a chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80. 90, and 94-96, or a variant or a fragment thereof, and wherein the TCR 0 chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

[0266] In one embodiment, the at least one modified T cell is modified to express at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex, wherein the TCR comprises a TCR a chain and a TCR 0 chain, wherein the TCR a chain comprises a nucleotide sequence encoding at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof, and wherein the TCR P chain comprises a nucleotide sequence encoding at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

[0267] In one embodiment, the at least one modified T cell is modified to express at least one TCR that specifically binds to a HLA:LOXHD1 -peptide complex, wherein the TCR comprises a TCR a chain and a TCR P chain, wherein the TCR a chain comprises a nucleotide molecule comprising at least one nucleic acid sequence that is at least about 70% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 75, 81-83, 91, and 97-99, or a variant or a fragment thereof, and wherein the TCR P chain comprises a nucleotide molecule comprising at least one nucleic acid sequence that is at least about 70% identical to the nucleotide sequence selected from the group consisting of SEQ ID NOs: 77, 87-89, 93, and 103-105, or a variant or a fragment thereof.

[0268] In one embodiment, the at least one modified T cell is modified to express one or more TCRs or fragments thereof as provided elsewhere herein.

[0269] In some embodiments, the at least one modified cell may be of any suitable cell type that can express the at least one TCR wherein the at least one TCR specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the at least one modified cell is used in a method where the cell is introduced into a recipient. In some embodiments, the at least one modified cell is autologous, allogeneic, syngeneic or xenogeneic with respect to recipient. In some embodiments, the at least one modified cell is derived from a stem cell or precursor cell. In some embodiments, the stem cell or precursor cell from which the modified cell is derived is autologous, allogeneic, syngeneic or xenogeneic with respect to recipient.

[0270] In some embodiments, the at least one modified cell is modified to not express at least one selected from a beta-2-microglobulin (B2M), class II major histocompatibility complex transactivator (CIITA), or T cell receptor (TCR). In some embodiments, the modified cell is a triple knockout (TKO) cell that does not express at least one selected from a major histocompatibility complex (MHC) I, MHC II, or TCR.

[0271] In one embodiment, the at least one modified cell is an immune cell. In some embodiments, the at least one modified cell comprises an immune cell that comprises or expresses one or more TCRs described elsewhere herein. Exemplary immune cells that may comprise or express one or more TCRs described herein include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, and gamma delta T cells), natural killer (NK) cells, and NK T cells. Exemplary immune cells that may comprise or express one or more peptides or polypeptides described herein include, but are not limited to, an antigen presenting cell, dendritic cell, B cell, macrophage, Langerhans cell, T cell, NK cell, NK T cell. Exemplary immune cells include, but are not limited to, T cells (including killer T cells, helper T cells, regulatory T cells, and gamma delta T cells), B cells, antigen presenting cells (APCs), natural killer (NK) cells, and NK T cells.

[0272] In some embodiments, the at least one modified cell is modified via one or more nucleic acid molecules delivered into the at least one modified cell using a retroviral or lentiviral vector. For example, retroviral and lentiviral vectors expressing a peptide of the invention can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transduced cells as carriers or cell-free local or systemic delivery of encapsulated, bound or naked vectors. The method used can be for any purpose where stable expression is required or sufficient.

[0273] In other embodiments, the nucleic acid sequence is delivered into the at least one modified cell using in vitro transcribed mRNA. In vitro transcribed mRNA can be delivered into different types of eukaryotic cells as well as into tissues and whole organisms using transfected cells as carriers or cell-free local or systemic delivery of encapsulated, bound or naked mRNA. The method used can be for any purpose where transient expression is required or sufficient.

[0274] Prior to expansion and genetic modification of the at least one modified T cell of the invention, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the present invention, any number of T cell linos available in the art, may be used. In some embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Again, surprisingly, initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semiautomated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2*-free, Mg2*-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0275] In another embodiment. T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.<?., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 min. In a further embodiment, the time period ranges from 30 min to 36 h or longer and all integer values there between. In a further embodiment, the time period is at least 1. 2, 3, 4, 5, or 6 h. In yet another embodiment, the time period is 10 to 24 h. In one embodiment, the incubation time period is 24 h. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 h, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In some embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

[0276] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, GDI lb, CD16, HLA-DR, and CDS. In some embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4", CD25~, CD62Lhl, GITR+, and FoxP3*. Alternatively, in some embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

[0277] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In some embodiments, it may be desirable to significantly decrease the volume in which beads and cells arc mixed together (i.c., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / mL is used. In one embodiment, a concentration of 1 billion cells / mL is used. In a further embodiment, greater than 100 million cells / mL is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / mL is used. In further embodiments, concentrations of 125 or 150 million cells / mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+T cells that normally have weaker CD28 expression.

[0278] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+T cells express higher levels of CD28 and are more efficiently captured than CD8+T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5 X 106 / mL. In other embodiments, the concentration used can be from about 1 X 105 / mL to 1 X 106 / mL, and any integer value in between.

[0279] In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature.

[0280] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Scrum Albumin and 7.5% DMSO, or 31.25% Plasmalytc-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of 1 °C per min and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 °C or in liquid nitrogen.

[0281] In some embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one h at room temperature prior to activation using the methods of the present invention.

[0282] Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or an apheresis is taken from a generally healthy subject. In some embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In some embodiments, the T cells may be expanded, frozen, and used at a later time. In some embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu ct al., Cell 66:807-815, 1991; Henderson ct al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the cells are isolated for a patient and frozen for later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

[0283] In a further embodiment of the present invention, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in some embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and / or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0284] Whether prior to or after genetic modification of the T cells to express a peptide of the invention, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. Generally, the T cells of the invention are expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In one embodiment, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+T cells or CD8+T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B- T3, XR-CD28 (Diaclone, Besan$on, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1 -2):53-63, 1999).

[0285] In some embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co- stimulatory signal is bound to a cell surface and the agent providing the primaiy activation signal is in solution or coupled to a surface. In some embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 2004 / 0101519 and 2006 / 0034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

[0286] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 :1 ratio of each antibody bound to the beads for CD4+T cell expansion and T cell growth is used. In some embodiments of the present invention, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1:1. In one embodiment, an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between. In one embodiment of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3.CD28 is less than one. In some embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1. In one embodiment, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used.

[0287] Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In some embodiments, the ratio of cells to particles ranges from 1:100 to 100:1 and any integer values in-between and in further embodiments, the ratio comprises 1:9 to 9:1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain values include 1 : 100, 1 :50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1 with one exemplary ratio being at least 1 :1 particles per T cell. In one embodiment, a ratio of particles to cells of 1:1 or less is used. In one embodiment, the particle: cell ratio is 1:5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1 :1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (based on cell counts on the day of addition). In one embodiment, the ratio of particles to cells is 1 : 1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1 :5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 : 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In one embodiment, ratios will vary depending on particle size and on cell size and type.

[0288] In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent- coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0289] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells. In one embodiment, the cells (for example, 104to 109T cells) and beads (for example, DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a ratio of 1:1) are combined in a buffer, such as PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention. In some embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / mL is used. In further embodiments, concentrations of 125 or 150 million cells / mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in some embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0290] In one embodiment of the present invention, the mixture may be cultured for several h (about 3 h) to about 14 days or any integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention, the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g„ Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF0, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPM1 1640. AIM-V, DMEM, MEM, ct-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).

[0291] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (Tc, CD8*). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of Tc cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of Tc cells has been isolated it may be beneficial to expand this subset to a greater degree.

[0292] Further, in addition to CD4 and CDS markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

[0293] Antibodies

[0294] In one embodiment, the present invention provides at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex. In one embodiment, the at least one antigenbinding molecule specifically binds to a HLA-A*02:01 :LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some embodiments, the present invention provides a composition comprising at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the composition comprising at least one antigen-binding molecule specifically binds to a HLA-A*O2:O1:L€)XHD1- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex.

[0295] In one embodiment, the present invention provides at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that binds a HLA:LOXHD1 -peptide complex, wherein the LOXHD1 peptide thereof comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 1 -27 and 29-73, or a variant or a fragment thereof.

[0296] In one embodiment, the present invention provides at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that binds to a HLAzLOXHDl -peptide complex wherein the HLA molecule comprises HLA- A *02:01, and wherein the LOXHD1 peptide thereof comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 1-27, and 30-61, or a variant or a fragment thereof.

[0297] In one embodiment, the present invention provides at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that binds to a HLA :LOXH DI -peptide complex, wherein the HLA molecule comprises HLA-B *07:02, and wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 29 and 62-73, or a variant or a fragment thereof.

[0298] In another embodiment, the at least one antigen-binding molecule is an antibody fragment selected from Fv, Fab, Fab', F(ab')2, Fc, or a ScFv. In another embodiment, the at least one antigen-binding molecule is an IgA, IgD, IgE, IgG or IgM or isotype thereof. In another embodiment, the at least one antigen-binding molecule is a human antibody or a humanized antibody. In some embodiments, the antigen-binding molecule is a bispecific antibody, for example an artificial bispecific monoclonal antibody or bispecific T-cell engager (BiTe).

[0299] In one embodiment, the at least one antigen-binding molecule is an isolated monoclonal antibody or fragment thereof wherein the isolated monoclonal antibody or fragment thereof is a monoclonal antibody or a polyclonal antibody. In one embodiment, the isolated antibody or fragment thereof is a human or humanized antibody. In one embodiment, the isolated antibody or fragment thereof is a chimeric antibody. In one embodiment, the isolated antibody or fragment thereof comprises a human heavy chain constant region and a human light chain constant region. In one embodiment, the isolated antibody is an IgG isotype. In another embodiment, the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, lgG4, IgAl and IgA2) or derivative thereof (e.g., IgGlf LALA). In one embodiment, the antibodies are of IgGlf LALA isotype. In one embodiment, said isolated antibody or fragment thereof is selected from the group consisting of a Fab, F(ab2)', F(ab)2' and scFV. In one embodiment, the isolated antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, and a synthetic antibody. In one embodiment, the antibody or fragment thereof is a human or humanized antibody.

[0300] In one embodiment, the present invention provides at least one nucleic acid molecule that encodes at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHD1 -peptide complex.

[0301] In one embodiment, the present invention provides at least one nucleic acid molecule that encodes at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-A*02:01 :LOXHD1- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex.

[0302] In one embodiment, the present invention provides at least one nucleic acid molecule that encodes at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-A*02:01 rLOXHDl- peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex wherein the LOXHD1 peptide thereof comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 1 -27 and 29-73, or a variant or a fragment thereof.

[0303] In one embodiment, the present invention provides at least one nucleic acid molecule that encodes at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-A*02:01:LOXHD1- peptide complex, wherein the LOXHD1 peptide thereof comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof.

[0304] In one embodiment, the present invention provides at least one nucleic acid molecule that encodes at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-B *07:02:LOXHD1- peptide complex wherein the LOXHD1 peptide thereof comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in in SEQ ID NOs: 29 and 62-73, or a variant or a fragment thereof.

[0305] In another embodiment, the at least one nucleic acid molecule encodes at least one antigen-binding molecule that comprises an antibody fragment selected from Fv, Fab, Fab', F(ab')2, Fc, or a ScFv. In another embodiment, the at least one antigen-binding molecule is an IgA, IgD, IgE, IgG or IgM or isotype thereof. In another embodiment, the at least one nucleic acid molecule encodes at least one antigen-binding molecule that comprises a human antibody or a humanized antibody. In some embodiments, the at least one nucleic acid molecule encodes an antigen-binding molecule that comprises a bispecific antibody, for example an artificial bispecific monoclonal antibody or bispecific T-cell engager (Bile).

[0306] In one embodiment, the at least one nucleic acid molecule encodes at least one antigen-binding molecule wherein the antigen-binding molecule comprises an isolated monoclonal antibody or fragment thereof wherein the isolated monoclonal antibody or fragment thereof is a monoclonal antibody or a polyclonal antibody. In one embodiment, the isolated antibody or fragment thereof is a human or humanized antibody. In one embodiment, the isolated antibody or fragment thereof is a chimeric antibody. In one embodiment, the isolated antibody or fragment thereof comprises a human heavy chain constant region and a human light chain constant region. In one embodiment, the isolated antibody is an IgG isotype. In another embodiment, the antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or derivative thereof (e.g., IgGlf LALA). In one embodiment, the antibodies are of IgGlf LALA isotype. In one embodiment, said isolated antibody or fragment thereof is selected from the group consisting of a Fab, F(ab2)', F(ab)2' and scFV. In one embodiment, the isolated antibody is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, and a synthetic antibody. In one embodiment, the antibody or fragment thereof is a human or humanized antibody.

[0307] Chimeric Antigen Receptors f CARs) and CAR-T cells

[0308] In one embodiment, the present invention provides a chimeric antigen receptor (CAR) that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the present invention provides at least one CAR that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some embodiments, the present invention provides a composition comprising at least one CAR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the present invention provides a composition comprising at least one CAR that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex.

[0309] In one embodiment, the present invention provides at least one modified cell (e.g., modified T cell), modified to express a CAR that specifically binds to a HLA:LOXHD1- peptide complex. In some embodiments, the present invention provides at least one modified cell (e.g., modified T cell), modified to express a CAR that specifically binds to a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex. In some embodiments, the present invention provides a composition comprising at least one modified cell (e.g., modified T cell), modified to express a CAR that specifically binds to a HLA:LOXHD1 -peptide complex. In some embodiments, the present invention provides a composition comprising at least one modified cell (e.g., modified T cell), modified to express CAR that specifically binds to a HLA- A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex. In some embodiments, the at least one modified T cell, modified to express a CAR, is a CAR-T cell.

[0310] In one embodiment, the CAR binds a HLA:LOXH DI -peptide complex, wherein the LOXHD1 peptide thereof comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 1-73, or a variant or a fragment thereof. In one embodiment, the CAR binds to a HLA:LOXHD1 -peptide complex wherein the HLA molecule comprises HLA-A *02:01, and wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof.

[0311] In one embodiment, the CAR binds to a HLA:LOXHD1 -peptide complex, wherein the HLA molecule comprises HLA-B *07:02, and wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to a sequence as set forth in SEQ ID NOs: 29 and 62-73, or a variant or a fragment thereof.

[0312] In various embodiments, the portion comprising an antibody or antibody fragment of the CAR composition is engineered by modifying one or more amino acids within one or both variable regions (i.e., VH and / or VL). For example, the antibody or antibody fragment of the CAR composition is engineered by modifying one or more amino acids within one or more CDR regions and / or within one or more framework regions.

[0313] In some embodiments, the CAR comprises an extracellular receptor, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the extracellular receptor is connected to the intracellular signaling domain. In one embodiment, the extracellular receptor is connected to the intracellular signaling domain through the transmembrane domain to the intracellular signaling domain.

[0314] In various embodiments, the CAR can be any CAR molecule including, but not limited to, a “first generation,” “second generation,” “third generation," “fourth generation” or “fifth generation” CAR (see, for example, Sadelain et al., Cancer Discov. 3(4):388-398 (2013); Jensen et al., Immunol. Rev. 257:127-133 (2014); Sharpe et al., Dis. Model Meeh. 8(4):337-350 (2015); Brentjens et al., Clin. Cancer Res. 13:5426-5435 (2007); Gade et al., Cancer Res. 65:9080-9088 (2005); Maher et al., Nat. Biotechnol. 20:70-75 (2002); Kershaw et al., J. Immunol. 173:2143-2150 (2004); Sadelain et al., Curr. Opin. Immunol. (2009); Hollyman et al., J. Immunother. 32:169-180 (2009)).

[0315] “First generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to a transmembrane domain, which is fused to a cytoplasmic / intracellular domain of a T cell receptor chain. “First generation” CARs typically have the intracellular domain from the CD3£-chain, which is the primary transmitter of signals from endogenous T cell receptors (TCRs). “First generation” CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3C chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation.

[0316] “Second-generation” CARs for use in the invention comprise an antigen binding domain, for example, a single-chain variable fragment (scFv), fused to an intracellular signaling domain capable of activating T cells and a co-stimulatory domain designed to augment T cell potency and persistence (Sadelain et al., Cancer Discov. 3:388-398 (2013)). CAR design can therefore combine antigen recognition with signal transduction, two functions that are physiologically home by two separate complexes, the TCR heterodimer and the CD3 complex. “Second generation” CARs include an intracellular domain from various co-stimulatory molecules, for example, CD28, 4-1BB, ICOS, 0X40, and the like, in the cytoplasmic tail of the CAR to provide additional signals to the cell.

[0317] “Second generation” CARs provide both co-stimulation, for example, by CD28 or 4-1BB domains, and activation, for example, by a CD3£ signaling domain. Preclinical studies have indicated that “Second Generation” CARs can improve the anti-tumor activity of T cells. For example, robust efficacy of “Second Generation” CAR modified T cells was demonstrated in clinical trials targeting the CD 19 molecule in patients with chronic lymphoblastic leukemia (CLL) and acute lymphoblastic leukemia (ALL) (Davila et al., Oncoimmunol. 1(9):1577-1583 (2012)).

[0318] “Third generation” CARs provide multiple co-stimulation, for example, by comprising both CD28 and 4- IBB domains, and activation, for example, by comprising a CD3£ activation domain.

[0319] “Fourth generation” CARs provide co-stimulation, for example, by CD28 or 4- 1BB domains, and activation, for example, by a CD3C signaling domain in addition to a constitutive or inducible chemokine component.

[0320] “Fifth generation” CARs provide co-stimulation, for example, by CD28 or 4- IBB domains, and activation, for example, by a CD3£ signaling domain, a constitutive or inducible chemokine component, and an intracellular domain of a cytokine receptor, for example, IL-2R0.

[0321] In various embodiments, the CAR can be included in a multivalent CAR system, for example, a DualCAR or “TandemCAR” system. Multivalent CAR systems include systems or cells comprising multiple CARs and systems or cells comprising bivalent / bispecific CARs targeting more than one antigen.

[0322] The present invention encompasses a number of CAR cells engineered for inhibiting or reducing the level of at least one cell of interest (e.g., alloresponsive cell, immune cell, T cell, B cell, natural killer cell, white blood cell, myeloid cell, plasma cell, etc.). In some embodiments, the CAR cell comprises at least one peptide and / or nucleic acid molecule of the present invention.

[0323] For example, in various embodiment, the CAR constructs can comprise a variety of CAR constructs known in the art, including but not limited to CAR disclosed in U.S. Patent No. 8,906,682 B2, U.S. Patent No. 8,911,993 B2, U.S. Patent No. 8,916,381 Bl, U.S. Patent No. 8,975,071 Bl, U.S. Patent No. 9,101,584 B2, U.S. Patent No. 9,102,760 B2, U.S. Patent No. 9,102,761 B2, U.S. Patent No. 9,328,156 B2, U.S. Patent No. 9,464,140 B2, U.S. Patent No. 9,481,728 B2, U.S. Patent No. 9,499,629 B2, U.S. Patent No. 9,518,123 B2, U.S. Patent No. 9,540,445 B2, U.S. Patent No. 9,573,988 B2, U.S. Patent No. 10,040,846 B2, U.S. Patent No. 10,117,896 B2, U.S. Patent No. 10,174,095 B2, U.S. Patent No. 10,221,245 B2, U.S. Patent No. 10,308,717 B2, U.S. Patent Application No. 20170137783 Al, and / or U.S. Patent Application No. 20180258149 Al.

[0324] In one embodiment, the scFv portion of a CAR of the invention is encoded by a transgene whose sequence has been codon optimized for expression in a mammalian cell. In one embodiment, the entire CAR construct of the invention is encoded by a transgene whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least US Patent Numbers 5,786,464 and 6,114,148.

[0325] In some embodiments, the CAR cell comprises an antibody or a fragment thereof engineered for enhanced binding to at least one target of interest. The target-specific binding domain can be any domain that binds to a specific target including, but not limited to, target-specific binding domains derived from any one or more of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, HLA I molecules, HLA 11 molecules, MHC 1 molecules, MHC II molecules, and fragments thereof, including, but not limited to, single-domain antibodies, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH) of camelid derived nanobody, and to alternative scaffolds known in the art that function as antigen binding domains, such as recombinant fibronectin domains, and the like. In some instances, it is beneficial for the target-specific binding domain to be derived from the same species in which the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the target-specific binding domain of the CAR to comprise human or humanized residues for the target-specific binding domain of an antibody or a fragment thereof. Thus, in one embodiment, the targetspecific binding domain portion comprises a humanized antibody or a fragment thereof.

[0326] In some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. In one embodiment, the targetspecific binding domain portion is humanized. In one embodiment, the antigen binding domain portion is humanized.

[0327] A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5.530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al, 1994, PNAS, 91:969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005 / 0042664, U.S. Patent Application Publication No. US2005 / 0048617, U.S. Pat. No. 6.407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska ct al., Protein Eng., 9(10):895-904 (1996), Couto ct al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)

[0328] A humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. As provided herein, humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline. Multiple techniques for humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522- 525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference herein in their entirety). In such humanized chimeric antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106: EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference herein in their entirety.

[0329] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).

[0330] In various embodiments, the portion of the CAR composition that comprises an antibody or a fragment thereof is humanized with retention of high affinity for the target antigen, target cell bearing antigen, target alloresponsive cell bearing antigen, target TCR, target T cell bearing TCR, target alloresponsive T cell bearing TCR, target BCR, target B cell bearing BCR, target alloresponsive B cell bearing BCR, target immune cell, target T cell, target B cell, target natural killer cell, target white blood cell, target myeloid cell, target plasma cell, or any combination thereof, and other favorable biological properties.

[0331] In one embodiment, humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models arc commonly available and arc familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0332] A humanized antibody or antibody fragment retains a similar antigenic specificity as the original antibody. However, the methods disclosed herein may further improve the affinity and / or specificity of binding of the antibody for human cell bearing antigen. In one embodiment, the target-specific binding domain is characterized by particular functional features or properties of an antibody or antibody fragment.

[0333] In one embodiment, the invention relates to an antigen binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.), wherein the antigen binding molecule specifically binds to a target of interest or fragment thereof. In one embodiment, the scFv is contiguous with and in the same reading frame as a leader sequence, (e.g., an antibody, antibody moiety, antibody fragment, etc.)

[0334] In one embodiment, the antibody fragment provided herein is a single chain variable fragment (scFv). In various embodiments, the antibodies of the invention may exist in a variety of other forms including, for example, Fv, Fab, and (Fab’) 2, as well as bi-fimctional (i.e., bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)).

[0335] ScFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise flexible polypeptide linker (e.g., a Ser-Gly linker) with an optimized length and / or amino acid composition. The flexible polypeptide linker length can greatly affect how the variable regions of an scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids, intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT publication Nos. W02006 / 020258 and W02007 / 024715, is incorporated herein by reference.

[0336] The scFv can comprise a polypeptide linker sequence of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The flexible polypeptide linker sequence may comprise any naturally occurring amino acid. In some embodiments, the flexible polypeptide linker sequence comprises amino acids glycine and serine . In another embodiment, the flexible polypeptide linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. Variation in the flexible polypeptide linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.

[0337] Nucleic acid modification techniques

[0338] The nucleic acid sequence encoding a peptide, polypeptide, or protein of the present invention can be obtained using any of the many recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0339] The nucleic acid may comprise any type of nucleic acid, including, but not limited to DNA and RNA. For example, in one embodiment, the composition comprises an isolated DNA molecule, including for example, an isolated cDNA molecule, encoding a polypeptide of the invention, or functional fragment thereof. In one embodiment, the composition comprises an isolated RNA molecule encoding the polypeptide of the invention, or a functional fragment thereof.

[0340] The nucleic acid molecules of the present invention can be modified to improve stability in serum or in growth medium for cell cultures. Modifications can be added to enhance stability, functionality, and / or specificity and to minimize immunostimulatory properties of the nucleic acid molecule of the invention. For example, in order to enhance the stability, the 3 ’-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2 ’-deoxythymidine is tolerated and does not affect function of the molecule.

[0341] In one embodiment of the present invention, the nucleic acid molecule may contain at least one modified nucleotide analogue. For example, the ends may be stabilized by incorporating modified nucleotide analogues.

[0342] Non-limiting examples of nucleotide analogues include sugar- and / or backbone- modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In some backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In some sugar-modified ribonucleotides, the 2’ OH- group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, Cl, Br, or I.

[0343] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6- methyl adenosine are suitable. It should be noted that the above modifications may be combined.

[0344] In some instances, the nucleic acid molecule comprises at least one of the following chemical modifications: 2’-H, 2’-O-methyl, or 2’-OH modification of one or more nucleotides. In some embodiments, a nucleic acid molecule of the invention can have enhanced resistance to nucleases. For increased nuclease resistance, a nucleic acid molecule, can include, for example, 2 ’-modified ribose units and / or phosphorothioate linkages. For example, the 2’ hydroxyl group (OH) can be modified or replaced with a number of different “oxy" or “deoxy" substituents. For increased nuclease resistance the nucleic acid molecules of the invention can include 2’-O-methyl, 2’-fluorine, 2’-O- methoxyethyl, 2’-O-aminopropyl, 2’-amino, and / or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2’-4’-ethylene- bridged nucleic acids, and certain nucleobase modifications such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp modifications, can also increase binding affinity to a target.

[0345] In one embodiment, the nucleic acid molecule includes a 2 ’-modified nucleotide, e.g., a 2’-deoxy, 2’-deoxy-2’-fluoro, 2’-O-methyl, 2’-O-methoxyethyl (2’-O-MOE), 2*- O-aminopropyl (2’-O-AP), 2’-0-dimethylaminoethyl (2’-O-DMAOE), 2’-O- dimethylaminopropyl (2’-O-DMAP), 2’-O-dimethylaminoethyloxyethyl (2’-O- DMAEOE), or 2’-O-N-methylacetamido (2’-0-NMA). In one embodiment, the nucleic acid molecule includes at least one 2’-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides of the nucleic acid molecule include a 2’-O-methyl modification.

[0346] The present invention also includes a vector in which the isolated nucleic acid of the present invention is inserted. The art is replete with suitable vectors that are useful in the present invention.

[0347] In brief summary, the expression of natural or synthetic nucleic acids encoding a peptide of the invention is typically achieved by operably linking a nucleic acid encoding the peptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors to be used are suitable for replication and, optionally, integration in eukaryotic cells. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0348] The vectors of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therapy vector.

[0349] The nucleic acid of the invention can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0350] Further, the vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, heipes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0351] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0352] For example, vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. In one embodiment, the composition includes a vector derived from an adeno-associated virus (AAV). Adeno-associated viral (AAV) vectors have become powerful gene delivery tools for the treatment of various disorders. AAV vectors possess a number of features that render them ideally suited for gene therapy, including a lack of pathogenicity, minimal immunogenicity, and the ability to transduce postmitotic cells in a stable and efficient manner. Expression of a particular gene contained within an AAV vector can be specifically targeted to one or more types of cells by choosing the appropriate combination of AAV serotype, promoter, and delivery method.

[0353] In some embodiments, the vector also includes conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and / or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0354] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor -la (EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian vims 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr vims immediate early promoter, a Rous sarcoma vims promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0355] Enhancer sequences found on a vector also regulates expression of the gene contained therein. Typically, enhancers are bound with protein factors to enhance the transcription of a gene. Enhancers may be located upstream or downstream of the gene it regulates. Enhancers may also be tissue-specific to enhance transcription in a specific cell or tissue type. In one embodiment, the vector of the present invention comprises one or more enhancers to boost transcription of the gene present within the vector.

[0356] In order to assess the expression of a peptide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Usefill selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0357] Reporter genes arc used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEES Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0358] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0359] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New Yoric). One exemplary method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0360] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0361] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0362] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0363] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al, 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0364] Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0365] In one embodiment, the isolated nucleic acid encoding a peptide of the invention comprises in vitro transcribed (TVT) RNA. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.

[0366] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full length gene of interest of a portion of a gene. The gene can include some or all of the 5’ and / or 3’ untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including the 5’ and 3’ UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0367] Genes that can be used as sources of DNA for PCR include genes that encode polypeptides that provide a therapeutic or prophylactic effect to an organism or that can be used to diagnose a disease or disorder in an organism. Some genes are genes which are useful for a short term treatment, or where there are safety concerns regarding dosage or the expressed gene. For example, for treatment of cancer, autoimmune disorders, parasitic, viral, bacterial, fungal or other infections, the transgene(s) to be expressed may encode a polypeptide that functions as a ligand or receptor for cells of the immune system, or can function to stimulate or inhibit the immune system of an organism. In some embodiments, it is not desirable to have prolonged ongoing stimulation of the immune system, nor necessary to produce changes which last after successful treatment, since this may then elicit a new problem. For treatment of an autoimmune disorder, it may be desirable to inhibit or suppress the immune system during a flare-up, but not long term, which could result in the patient becoming overly sensitive to an infection.

[0368] PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. “Substantially complementary”, as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5’ and 3* UTRs. The primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs. Primers useful for PCR are generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.

[0369] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.

[0370] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. In some examples, the RNA has 5* and 3’ UTRs. In one embodiment, the 5’ UTR is between zero and 3000 nucleotides in length. The length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0371] The 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3* UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3’ UTR sequences can decrease the stability of mRNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0372] In one embodiment, the 5* UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5’ UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5’ UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.

[0373] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0374] In a one embodiment, the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is after transcription.

[0375] On a linear DNA template, phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0376] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with poly AT 3’ stretch without cloning highly desirable.

[0377] The polyA / T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly( A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.

[0378] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly( A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0379] 5’ caps on also provide stability to RNA molecules. In one embodiment, RNAs produced by the methods disclosed herein include a 5’ cap. The 5* cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al, Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0380] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.

[0381] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Arnaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Then, 12(8):861-70 (2001).

[0382] In another embodiment, the RNA construct can be delivered into the cells by electroporation. See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285 Al, US 2004 / 0092907A1. The various parameters including electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in the field. See e.g., U.S. Pat. No. 6,678,556, U.S. Pat. No. 7,171,264, and U.S. Pat. No. 7,173,116. Apparatus for therapeutic application of electroporation are available commercially, e.g., the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif.), and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation may also be used for transfection of cells in vitro as described e.g., in US20070128708A1. Electroporation may also be utilized to deliver nucleic acids into cells in vitro. Accordingly, electroporation-mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell. Protein modification methods

[0383] The peptides, polypeptides or proteins of the present invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J ¥ et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. The peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

[0384] The variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (for example, a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g,, residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and / or (v) one in which the polypeptide is fused with another polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translational ly, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0385] As known in the art the “similarity” between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to a sequence of a second polypeptide. Variants are defined to include polypeptide sequences different from the original sequence, for example different from the original sequence in less than 40% of residues per segment of interest, or different from the original sequence in less than 25% of residues per segment of interest, or different by less than 10% of residues per segment of interest, or different from the original protein sequence in just a few residues per segment of interest and at the same time sufficiently homologous to the original sequence to preserve the functionality of the original sequence and / or the ability to bind to ubiquitin or to a ubiquitylatcd protein. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences can be determined, by way of one example, by using the BLAST? algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S„ et al., J. Mol. Biol. 215: 403-410 (1990)].

[0386] The peptides or polypeptides of the present invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.

[0387] The peptides of the invention may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation. By way of example, special tRNAs, such as tRNAs which have suppressor properties, suppressor tRNAs, have been used in the process of site-directed non-native amino acid replacement (SNAAR). In SNAAR, a unique codon is required on the mRNA and the suppressor tRNA, acting to target a non-native amino acid to a unique site dining the protein synthesis (described in WO 90 / 05785). However, the suppressor tRNA must not be recognizable by the aminoacyl tRNA synthetases present in the protein translation system. In certain cases, a non-native amino acid can be formed after the tRNA molecule is aminoacylated using chemical reactions which specifically modify the native amino acid and do not significantly alter the functional activity of the aminoacylated tRNA. These reactions are referred to as post-aminoacylation modifications. For example, the epsilon-amino group of the lysine linked to its cognate tRNA (tRNAivs), could be modified with an amine specific photoaffinity label.

[0388] The peptides of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.

[0389] Stability and Mutations

[0390] The stability of scFv molecules (e.g., soluble scFv) can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or a full length antibody. In one embodiment, the humanized soluble scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a control binding molecule (e.g., a conventional scFv molecule) in the described assays.

[0391] The improved thermal stability of the scFv is subsequently conferred to the entire CAR construct, leading to improved therapeutic properties of the CAR construct. The thermal stability of the scFv can be improved by at least about 2 °C or 3 °C as compared to a conventional antibody. In one embodiment, the scFv has a 1 °C improved thermal stability as compared to a conventional antibody. In another embodiment, the scFv has a 2 °C improved thermal stability as compared to a conventional antibody. In another embodiment, the scFv has a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 °C improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm can be measured. Methods for measuring Tm and other methods of determining protein stability are described in more detail below.

[0392] Mutations in scFv (arising through humanization or direct mutagenesis of the soluble scFv) alter the stability of the scFv and improve the overall stability of the scFv and the CAR construct. Stability of the humanized scFv is compared against the murine scFv using measurements such as Tm, temperature denaturation and temperature aggregation. The residues introduced by humanization thereby have improved the Tm of the scFv by more than 10 °C.

[0393] In one embodiment, the scFv comprises at least one mutation arising from the humanization process such that the mutated scFv confers improved stability to the CAR construct. In various embodiments, the scFv comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv confers improved stability to the CAR construct. The binding capacity of the mutant scFvs can be determined using any of the assays described herein.

[0394] Methods of Evaluating Protein Stability

[0395] To assess the stability of scFv constructs, the stability of the minimal domain of a multidomain protein, i.e., a scFv of a CAR construct, is identified using the methods and those described below'. Such methods allow for the determination of multiple thermal unfolding transitions where the least stable domain either unfolds first or limits the overall stability threshold of a multidomain unit that unfolds cooperatively (i.e., a multidomain protein which exhibits a single unfolding transition). The least stable domain can be identified in a number of additional ways. Mutagenesis can be performed to probe which domain limits the overall stability. Additionally, protease resistance of a multidomain protein can be performed under conditions where the least stable domain is known to be intrinsically unfolded via DSC or other spectroscopic methods (Fontana, et al., 1997, Fold. Des., 2: R17-26; Dimasi et al., 2009, J. Mol. Biol. 393: 672-692). Once the least stable domain is identified, the sequence encoding this domain (or a portion thereof) may be employed as a test sequence in the methods. a) Thermal Stability

[0396] The thermal stability of the compositions may be analyzed using a number of non-limiting biophysical or biochemical techniques known in the art. In some embodiments, thermal stability is evaluated by analytical spectroscopy.

[0397] An exemplary analytical spectroscopy method is Differential Scanning Calorimetry (DSC). DSC employs a calorimeter which is sensitive to the heat absorbances that accompany the unfolding of most proteins or protein domains (e.g., Sanchez-Ruiz et al., 1988, Biochemistry, 27: 1648-1652). To determine the thermal stability of a protein, a sample of the protein is inserted into the calorimeter and the temperature is raised until the Fab or scFv unfolds. The temperature at which the protein unfolds is indicative of overall protein stability.

[0398] Another exemplary analytical spectroscopy method is Circular Dichroism (CD) spectroscopy. CD spectrometry measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. A disordered or unfolded structure results in a CD spectrum very different from that of an ordered or folded structure. The CD spectrum reflects the sensitivity of the proteins to the denaturing effects of increasing temperature and is therefore indicative of a protein’s thermal stability (van Mierlo and Steemsma, J. BiotechnoL, 2000, 79:281-298).

[0399] Another exemplary analytical spectroscopy method for measuring thermal stability is Fluorescence Emission Spectroscopy (van Mierlo and Steemsma, supra). Yet another exemplary analytical spectroscopy method for measuring thermal stability is Nuclear Magnetic Resonance (NMR) spectroscopy (e.g., van Mierlo and Steemsma, supra).

[0400] The thermal stability of a composition can be measured biochemically. An exemplary biochemical method for assessing thermal stability is a thermal challenge assay. In a “thermal challenge assay”, a composition is subjected to a range of elevated temperatures for a set period of time. For example, in one embodiment, test scFv molecules or molecules comprising scFv molecules are subject to a range of increasing temperatures, e.g., for 1 -1.5 h. The activity of the protein is then assayed by a relevant biochemical assay. For example, if the protein is a binding protein (e.g., an scFv or scFv- containing polypeptide ) the binding activity of the binding protein may be determined by a functional or quantitative ELISA.

[0401] Such an assay may be done in a high-throughput format and those disclosed in the Examples using E. coli and high throughput screening. A library of scFv variants may be created using methods known in the art. scFv expression may be induced an scFvs may be subjected to thermal challenge. The challenged test samples may be assayed for binding and those scFvs which are stable may be scaled up and further characterized.

[0402] Thermal stability is evaluated by measuring the melting temperature (Tm) of a composition using any of the above techniques (e.g., analytical spectroscopy techniques). The melting temperature is the temperature at the midpoint of a thermal transition curve wherein 50% of molecules of a composition are in a folded state (e.g., Dimasi et al., 2009, J. Mol Biol. 393: 672-692). In one embodiment, Tm values for a scFv are about 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, 75 °C, 76 °C, 77 °C, 78 °C, 79 °C, 80 °C, 81 °C, 82 °C, 83 °C, 84 °C, 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 91 °C, 92 °C, 93 °C, 94 °C. 95 °C, 96 °C, 97 °C, 98 °C, 99 °C, 100 °C. In one embodiment, Tm values for an IgG is about 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, 75 °C, 76 °C, 77 °C, 78 °C, 79 °C, 80 °C, 81 °C, 82 °C, 83 °C, 84 °C, 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C, 96 °C, 97 °C, 98 °C, 99 °C, 100 °C. In one embodiment, Tm values for an multivalent antibody is about 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, 45 °C, 46 °C, 47 °C, 48 °C, 49 °C, 50 °C, 51 °C, 52 °C, 53 °C, 54 °C, 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C. 67 °C, 68 °C, 69 °C, 70 °C, 71 °C, 72 °C, 73 °C, 74 °C, 75 °C, 76 °C, 77 °C, 78 °C, 79 °C, 80 °C, 81 °C, 82 °C, 83 °C, 84 °C, 85 °C, 86 °C, 87 °C, 88 °C, 89 °C, 90 °C, 91 °C, 92 °C, 93 °C, 94 °C, 95 °C, 96 °C, 97 °C, 98 °C, 99 °C, 100 °C.

[0403] Thermal stability is also evaluated by measuring the specific heat or heat capacity (Cp) of a composition using an analytical calorimetric technique (e.g., DSC). The specific heat of a composition is the energy (e.g., in kcal / mol) is required to rise by 1 °C, the temperature of 1 mol of water. As large Cp is a hallmark of a denatured or inactive protein composition. The change in heat capacity (ACp) of a composition is measured by determining the specific heat of a composition before and after its thermal transition. Thermal stability may also be evaluated by measuring or determining other parameters of thermodynamic stability including Gibbs free energy of unfolding (AG), enthalpy of unfolding (AH), or entropy of unfolding (AS). One or more of the above biochemical assays (e.g., a thermal challenge assay) are used to determine the temperature (i.e., the TC value) at which 50% of the composition retains its activity (e.g., binding activity).

[0404] In addition, mutations to the scFv alter the thermal stability of the scFv compared with the unmutated scFv. When the humanized scFv is incorporated into a CAR construct, the humanized scFv confers thermal stability to the overall anti-CAR construct. In one embodiment, the scFv comprises a single mutation that confers thermal stability to the scFv. In another embodiment, the scFv comprises multiple mutations that confer thermal stability to the scFv. In one embodiment, the multiple mutations in the scFv have an additive effect on thermal stability of the scFv. b) % Aggregation

[0405] The stability of a composition can be determined by measuring its propensity to aggregate. Aggregation can be measured by a number of non-limiting biochemical or biophysical techniques. For example, the aggregation of a composition may be evaluated using chromatography, e.g., Size-Exclusion Chromatography (SEC). SEC separates molecules on the basis of size. A column is filled with semi-solid beads of a polymeric gel that will admit ions and small molecules into their interior but not large ones. When a protein composition is applied to the top of the column, the compact folded proteins (i.e., non-aggregated proteins) are distributed through a larger volume of solvent than is available to the large protein aggregates. Consequently, the large aggregates move more rapidly through the column, and in this way the mixture can be separated or fractionated into its components. Each fraction can be separately quantified (e.g., by light scattering) as it elutes from the gel. Accordingly, the % aggregation of a composition can be determined by comparing the concentration of a fraction with the total concentration of protein applied to the gel. Stable compositions elute from the column as essentially a single fraction and appear as essentially a single peak in the elution profile or chromatogram. c) Binding Affinity

[0406] The stability of a composition can be assessed by determining its target binding affinity. A wide variety of methods for determining binding affinity are known in the art. An exemplary method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125- 131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.

[0407] In various embodiments, the portion of a CAR composition of the invention comprising an antibody or antibody fragment further comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the exemplary antibodies described herein, and wherein the antibodies retain the desired functional properties of the antibodies of the invention. In one embodiment, the CAR composition comprises an antibody fragment. In one embodiment, the antibody fragment comprises an scFv.

[0408] Methods

[0409] In one embodiment, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering to the subject a composition that specifically binds to a HLA:LOXHD1 peptide complex.

[0410] In some embodiments, the method comprises administering to the subject a composition comprising at least one TCRthat specifically binds to a HLA:LOXHD1 peptide complex, at least one modified cell (e.g., a modified T cell), which is modified to express the at least one TCRthat specifically binds to a HLA:LOXHD1 peptide complex, at least one antigen-binding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA:LOXHD1 peptide complex, at least one CAR that specifically binds to a HLA:LOXHD1 peptide complex, at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA:LOXHD1 peptide complex, or any combination thereof.

[0411] In some embodiments, the method comprises administering to the subject a composition comprising at least one TCR that specifically binds to a HLA- A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, at least one modified cell(e.g., a modified T cell), which is modified to express the at least one TCR that specifically binds to a HLA-A*02:01 :LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex, at least one antigenbinding molecule (e.g., an antibody, antibody moiety, antibody fragment, etc.) that specifically binds to a HLA-A*02:01 :LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex, at least one CAR that specifically binds to a HLA- A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHDl-peptide complex, at least one modified cell (e.g., a modified T cell), which is modified to express a CAR that specifically binds to a HLA-A*02:01:LOXHDl-peptide complex and / or a HLA-B *07:02 :LOXHD1 -peptide complex, or any combination thereof.

[0412] In a further embodiment, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof, comprising adoptive therapy.

[0413] In some embodiments, the adoptive therapy comprises isolating at least one cell from a subject; culturing the at least one cell ex vivo; genetically modifying the isolated cell to express at least one TCR of the present invention, wherein the TCR specifically binds to a HLA:LOXHD1 peptide complex (e.g., a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex); and administering the genetically modified cell to the subject.

[0414] In some embodiments, the adoptive therapy comprises isolating at least one cell from a subject; culturing the at least one cell ex vivo; genetically modifying the isolated cell to express at least one CAR of the present invention, wherein the CAR specifically binds to a HLA:LOXHD1 peptide complex (e.g., a HLA-A*02:01:LOXHD1 -peptide complex and / or a HLA-B *07:02:LOXHD1 -peptide complex); and administering the genetically modified cell to the subject.

[0415] In some embodiments, the at least one cell isolated from a subject is a T cell. In one embodiment, the present invention provides a method for treating or preventing a LOXHD1 -associated disease or disorder in a subject having, suspected of having, or at risk for having a LOXHD1 -associated disease or disorder.

[0416] In some aspects, the present invention provides a method of administering a composition that specifically binds to a HLAzLOXHDl -peptide complex to a subject in need thereof. In some embodiments, the composition that specifically binds to a HLA:LOXHD1 -peptide complex is any composition described herein. In some embodiments, the subject has, is suspected of having, or is at risk for having a LOXHD1 - associated disease or disorder.

[0417] Exemplary LOXHD1 -associated disease or disorder that is treatable or preventable by way of the compositions and methods of the present invention include, but is not limited to, a cancer, such as a bone cancer (e.g., Ewing Sarcoma), or any cancer characterized by cells that present a HLA:LOXHD1 -peptide complex.

[0418] In various embodiments, the compositions and methods described herein can be used to kill at least one cell of interest (e.g., a cancer cell, such as Ewing Sarcoma tumor cell), decrease tumor size, inhibit tumor growth.

[0419] Exemplary tumors and types of cancer treatable by way of the present invention includes, but is not limited to, bone cancer, such as Ewing Sarcoma, pancreatic cancer, pancreatic ductal adenocarcinoma (PDA), colon cancer, colorectal adenocarcinoma, gastric adenocarcinoma and cervical adenocarcinoma, renal papillary cancer, prostate cancer, and small intestine neuroendocrine tumor (NET). The following are non-limiting examples of cancers that can be treated by the disclosed methods and compositions: acute lymphoblastic; acute myeloid leukemia; adrenocortical carcinoma; adrenocortical carcinoma, childhood; appendix cancer; basal cell carcinoma; bile duct cancer, extrahepatic; bladder cancer; bone cancer, osteosarcoma and malignant fibrous histiocytoma; brain stem glioma, childhood; brain tumor, adult; brain tumor, brain stem glioma, childhood; brain tumor, central nervous system atypical teratoid / rhabdoid tumor, childhood; central nervous system embryonal tumors; cerebellar astrocytoma; cerebral astrocytotna / malignant glioma; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medulloepithelioma; pineal parenchymal tumors of intermediate differentiation; supratentorial primitive neuroectodermal tumors and pineoblastoma; visual pathway and hypothalamic glioma; brain and spinal cord tumors; breast cancer; bronchial tumors; Burkitt lymphoma; carcinoid tumor; carcinoid tumor, gastrointestinal; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; central nervous system lymphoma; cerebellar astrocytoma cerebral astrocytoma / malignant glioma, childhood; cervical cancer; chordoma, childhood; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer, Ewing family of tumors; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer, gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (gist); germ cell tumor, extracranial; germ cell tumor, extragonadal; germ cell tumor, ovarian; gestational trophoblastic tumor; glioma; glioma, childhood brain stem; glioma, childhood cerebral astrocytoma; glioma, childhood visual pathway and hypothalamic; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer; histiocytosis, Langerhans cell; Hodgkin lymphoma; hypopharyngeal cancer, hypothalamic and visual pathway glioma; intraocular melanoma; islet cell tumors; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, chronic lymphocytic; leukemia, chronic myelogenous; leukemia, hairy cell; lip and oral cavity cancer; liver cancer; lung cancer, non-small cell; lung cancer, small cell; lymphoma, aids- related; lymphoma, Burkitt; lymphoma, cutaneous T-cell; lymphoma, non-Hodgkin lymphoma; lymphoma, primary central nervous system; macroglobulinemia, Waldenstrom; malignant fibrous histiocytoma of bone and osteosarcoma; medulloblastoma; melanoma; melanoma, intraocular (eye); Merkel cell carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndrome, (childhood); multiple myeloma / plasma cell neoplasm; mycosis; fungoides; myelodysplastic syndromes; myelodysplastic / myeloproliferative diseases; myelogenous leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple; myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer, pancreatic cancer, islet cell tumors; papillomatosis; parathyroid cancer; penile cancer; pharyngeal cancer, pheochromocytoma; pineal parenchymal tumors of intermediate differentiation; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumor; plasma celt neoplasm / multiple myeloma; pleuropulmonaiy blastoma; primary central nervous system lymphoma; prostate cancer, rectal cancer; renal cell (kidney) cancer; renal pelvis and ureter, transitional cell cancer; respiratory tract carcinoma involving the nut gene on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland cancer, sarcoma, Ewing family of tumors; sarcoma, Kaposi; sarcoma, soft tissue; sarcoma, uterine; Sezary syndrome; skin cancer (nonmelanoma); skin cancer (melanoma); skin carcinoma, Merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma, squamous neck cancer with occult primary, metastatic; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T- cell lymphoma, cutaneous; testicular cancer; throat cancer, thymoma and thymic carcinoma; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor, gestational; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer, vulvar cancer; Waldenstrom macroglobulinemia; and Wilms tumor.

[0420] In some embodiments, the present invention provides a method of inducing an immune response against LOXHD1 in a subject. For example, administration of a composition described herein can be used to induce a specific immune response, including a T-cell-mediated immune response, against LOXHD1 and cancer cells expressing a HLA:LOXHD1 -peptide complex. In certain instances, induction of an immune response directed against LOXHD1 results in inhibition of tumor growth and / or tumor cell death.

[0421] In one embodiment, the present invention provides a method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof. In one embodiment, the method comprises administering to the subject with one or more compositions of the invention. For example, in some embodiments, the method comprises administering to the subject a composition comprising at least one TCR as described elsewhere herein. In some embodiments, the method comprises administering to the subject a composition comprising at least one modified T cell as described elsewhere herein. In some embodiments, the method comprises administering to the subject a composition comprising at least one antigen binding molecule as described elsewhere herein. In some embodiments, the method comprises administering to the subject a composition comprising at least one modified T cell modified to express a CAR as described elsewhere herein.

[0422] In some embodiments, the subject is identified as having an HLA type associated with the LOXHD1 peptide to which the TCR binds. For example, as described herein, in certain instances the TCR binds to a HLA:LOXHD1 -peptide complex wherein the HLA is HLA-A*02:01 and / or HLA-B *07:02:LOXHD1. Thus, in some embodiments, the method comprises identifying a subject as having a specific HLA molecule and then administering to the subject a composition comprising or encoding a TCR described elsewhere herein. For example, in one embodiment, the method comprises identifying a subject as having HLA-A*02:01 molecule and administering to the subject a composition comprising a TCR, a composition comprising at least one modified cell modified to express a TCR, a composition comprising at least one antibody, and / or a composition comprising at least one modified cell modified to express a CAR, where the TCR specifically binds to a HLA-A*02:01:LOXHD1 peptide -complex wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 1-27 and 30-61, or a variant or a fragment thereof.

[0423] In one embodiment, the method the method comprises identifying a subject as having a specific HLA molecule and then administering to the subject a composition comprising or encoding a TCR described elsewhere herein. For example, in one embodiment, the method comprises identifying a subject as having HLA-B *07:02:LOXHDlmolecule and administering to the subject a composition comprising a TCR, a composition comprising at least one modified cell modified to express a TCR, a composition comprising at least one antibody, and / or a composition comprising at least one modified cell modified to express a CAR, where the TCR specifically binds to a HLA-B *07:02:LOXHDl-peptide complex- wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the amino acid sequence selected from SEQ ID NOs: 29 and 62-73, or a variant or a fragment thereof. The subject may be identified as being of a particular HL A type using any method known in the art, including, but not limited to, DNA sequence, RNA sequencing, nextgen sequencing, PCR, immunoassays, or the like.

[0424] The compositions of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention may comprise a composition as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0425] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[0426] When “an immunologically effective amount,” “an anti-tumor effective amount,”

[0427] 6« a, n tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

[0428] The administration of the subject compositions (e.g., compositions comprising at least one TCR, compositions comprising at least one modified T cell, compositions comprising at least one CAR, compositions comprising at least one T cell modified to express at least one TCR, compositions comprising at least one antibody, compositions comprising at least one T cell modified to express a CAR, etc., as described elsewhere herein) may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the compositions of the present invention are administered by i.v. injection. In some embodiments, the compositions of be injected directly into a tumor or lymph node.

[0429] In one embodiment, the invention provides a method to treat cancer comprising treating the subject prior to, concurrently with, or subsequently to the administration of the composition of the invention, with a complementary therapy for the cancer, such as surgeiy, chemotherapy, chemotherapeutic agent, radiation therapy, or hormonal therapy or a combination thereof.

[0430] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramucine phosphate sodium, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon alfa-2a recombinant, paclitaxel, teniposide, and streptozoci), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethylesulfonic acid), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulphan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatinum, clomesone, cyanomo...

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a T cell receptor (TCR), wherein the TCR specifically binds to a human leukocyte antigen (HLA): lipoxygenase homology PLAT (polycystin / lipoxygenase / alpha-toxin) domains 1 (LOXHD1)- peptide complex.

2. The composition of claim 1, wherein the TCR comprises a TCR a chain and a TCR 0 chain, wherein the TCR a chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof, and wherein the TCR 0 chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

3. The composition of claim 1, wherein the HLA comprises HLA-A *02:01, HLA-B *07:02, or a combination thereof.

4. The composition of claim 1, wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof.

5. A modified T cell modified to express a TCR, wherein the TCR specifically binds to a HLA:LOXHD1 -peptide complex.

6. The modified T cell of claim 5, wherein the TCR comprises a TCRa chain and a TCR 0 chain, wherein the TCR a chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof, and wherein the TCR 0 chain comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

7. The modified T cell of claim 5, wherein the HLA comprises HLA- A *02:01, HLA-B *07:02, or a combination thereof.

8. The modified T cell of claim 5, wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof.

9. A composition connprising an antibody, wherein the antibody specifically binds to a HLA:LOXHD1 -peptide complex.

10. The composition of claim 9, wherein the antibody comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof, and at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

11. The composition of claim 9, wherein the HLA comprises HLA- A*02:01, HLA-B *07:02, or a combination thereof.

12. The composition of claim 9, wherein the L0XHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof.

13. A modified T cell modified to express a chimeric antigen receptor (CAR), wherein the CAR specifically binds to a HLA:LOXHDl-peptide complex.

14. The modified T cell of claim 13, wherein the CAR comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 78-80, 90, and 94-96, or a variant or a fragment thereof, and at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 84-86, 92, and 100-102, or a variant or a fragment thereof.

15. The modified T cell of claim 13, wherein the HL A comprises HLA-A *02:01, HLA-B *07:02, or a combination thereof.

16. The modified T cell of claim 13, wherein the LOXHD1 peptide comprises at least one amino acid sequence that is at least about 70% identical to the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-27 and 29-73, or a variant or a fragment thereof.

17. A method of preventing or treating a LOXHD1 -associated disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject a composition that specifically binds to a HLAzLOXHDl -peptide complex.

18. A method of administering a composition that specifically binds to a HLA:LOXHD1 -peptide complex to a subject in need thereof, wherein the subject has a L0XHD1 -associated disease or disorder.

19. The method of claim 17 or 18, wherein the composition comprises the composition of claim 1.

20. The method of claim 17 or 18, wherein the composition comprises the modified T cell of claim 5.

21. The method of claim 17 or 18, wherein the composition comprises composition of claim 9.

22. The method of claim 17 or 18, wherein the composition comprises the modified T cell of claim 13.

23. The method of claim 17 or 18, wherein the LOXHD1 -associated disease or disorder is a L0XHD1 -associated cancer.

24. The method of claim 23, wherein the LOXHD1 -associated cancer is an Ewing Sarcoma.

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