Multiple t cell receptor compositions, combination therapy

Abstract

Provided herein are multiplex T cell receptor compositions, combination therapies, and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 337,522, filed on May 2, 2022; U.S. Provisional Application Serial No. 63 / 342,451, filed on May 16, 2022; U.S. Provisional Application Serial No. 63 / 413,553, filed on October 5, 2022; and U.S. Provisional Application Serial No. 63 / 423,269, filed on November 7, 2022; the entire contents of each of which are incorporated herein by reference in their entirety. Summary of the invention

[0003] The present invention is based at least in part on the discovery that certain binding proteins (including T cell receptors (TCRs)) that combine to recognize more than one antigen (e.g., more than one antigen on the same target and / or more than one antigen on different targets) and engineered cells containing the binding proteins can overcome antigen heterogeneity and / or loss of human leukocyte antigen (HLA) heterozygosity to treat cancer, including solid tumors. For example, adoptive cell transfer (ACT) in the context of genetically engineered T cells has great promise for treating cancers such as solid tumors, but only one antigen is targeted at a time, complete responses are rare and often short-lived due to heterogeneous expression of cancer-associated antigens and loss of HLA heterozygosity. Multiple TCR-T cell (TCR-T) therapies within multiple target antigens and / or HLA molecules mimic the response of natural oligoclonal T cells to cancer and provide a way to address some of the major challenges associated with resistance to adoptive cell therapy. As a non-limiting representative example, synergistic cytotoxicity was achieved using two TCRs targeting mixed tumor cell cultures with heterogeneous antigen expression. The presence of one TCR-T / target cell pair enhances the activity of another TCR-T against its target; this effect is mediated via secreted soluble factors. The results demonstrate that the use of multiple T cell receptors and related compositions (e.g., multiple TCR-T) can overcome antigen heterogeneity not only by individually targeting different target cells in the same tumor but also by cytokine-mediated enhancement of the responses of each T cell. Surprisingly, these results further unexpectedly demonstrate a synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 A to Figure 1 C shows that inter- and intra-tumor target expression heterogeneity is a clinical challenge for therapeutic TCR-expressing T cell (TCR-T) therapy. An example of variable inter- and intra-tumor antigen expression in human melanoma tumor samples is provided. Immunohistochemical analysis of human melanoma tumor microarrays was performed using PRAME-specific antibodies (pink) and MAGEC2-specific antibodies (blue). Figure 1 Heterogeneous antigen expression within the tumor was observed in multiple sections presented in A and in sections with different expression levels of a single antigen ( Figure 1 B and Figure 1 C).

[0005] Figure 2 A and Figure 2 B shows that HLA loss of heterozygosity (LOH) is common and highlights the need for multiplex TCR-T therapies. Figure 2 A shows the results of a representative non-restricted HLA LOH analysis of non-small cell lung cancer samples, demonstrating the widespread incidence of clonal and partial LOH of the HLA-A*02:01 allele. Figure 2 B shows that monotherapy TCR-T often leads to partial responses and rapid relapse, which is partly attributed to target antigen or HLA heterogeneity. Here, we have developed and described a multiplexed approach to address this issue, thereby improving long-term remission.

[0006] Figure 3 A and Figure 3 B provides a representative non-restrictive multiplexing approach for overcoming target heterogeneity and demonstrates that multiplexing TCR-T cells has synergistic anti-tumor activity. Figure 3 A shows Results of NucLight Red labeled HPV+ (CaSki) and MAGEA1+ (A101D) cell lines grown in the presence of HPV16 E7-TCR-T, MAGEA1-TCR-T, or a combination of both TCR-Ts. Assays were performed to assess cell growth. Figure 3 B also shows the results that synergistic cytotoxicity was observed between the two TCRs as calculated by the percentage of cell viability at 72 hours.

[0007] Figure 4 A to Figure 4 C also showed that TCR-T multiplexing had synergistic antitumor activity due to cytokine-mediated enhancement. Figure 4 A shows a schematic diagram of modeling intratumoral target expression variability using two different cell lines. Figure 4 B shows the result that co-culturing T cells expressing MAGEA1-specific TCR with a target cell line with high MAGEA1 expression (A2058) enhanced the cytotoxicity of T cells expressing MAGEC2 TCR against a cell line with moderate MAGEC2 expression (SKMEL5). Figure 4C demonstrates that the increased cytotoxicity is driven by soluble factors secreted by T cells targeting MAGEA1, which results in increased activation of T cells targeting MAGEC2 as shown in the illustrative Transwell experiments described.

[0008] Figure 5 A and Figure 5 B provides a representative non-restrictive screening strategy for selecting patients and TCR-T and also illustrates an ImmunoBank strategy to achieve customized multiplexing of TCR-T. Figure 5 A shows an illustrative screening strategy for selecting patients and TCR-T for multiple TCR-T therapies. After germline HLA genotyping, the patient's tumor is evaluated for target expression using any of many well-known methods (such as immunohistochemistry (IHC) or RNA in situ hybridization (ISH). Tumor samples can also be evaluated for HLA LOH by genome sequencing. If LOH is observed, TCR-T targeting 2 different HLAs on the complete chromosome arm is selected. If LOH is not observed, TCR-T targeting the HLA on the opposite chromosome is selected. Figure 5 B shows that TCR-T therapy customized for individual cancer patients would benefit from constructing an ImmunoBank of therapeutic TCRs that recognize different targets (in columns) presented on different HLA alleles (in rows). By multiplexing on both targets and HLA, this strategy is designed to prevent resistance from rising due to target loss or HLA LOH.

[0009] Figure 6Provided are summary data of representative examples of multiple TCR-T therapeutic agents for solving intratumor heterogeneity in the case of two TCR-T therapies targeting different antigens on a single HLA. The heterogeneous expression of cancer-related proteins MAGE-A1 and PRAME was evaluated by immunohistochemistry using antibodies specific to MAGE-A1 and PRAME. Leading TCRs specific to MAGE-A1-derived epitopes presented on HLA-A*02:01 and leading TCRs specific to PRAME-derived epitopes presented on HLA-A*02:01 were used. TCRs exhibit high performance in vitro and in vivo and appear to be highly selective for their corresponding peptide / MHC targets. The figure also shows the value of combining MAGE-A1-specific TCRs with PRAME-specific TCRs to solve a tumor model consisting of a mixture of HEK293T cells expressing MAGE-A1 or PRAME that is positive for HLA-A*02:01 in vitro and in vivo. The two target cell subsets are labeled with fluorescent dyes to enable flow cytometry analysis. The HEK293T cells used also contain a granzyme B-activated infrared fluorescent protein (IFP) reporter to allow cells targeted by the TCR to fluoresce. Although the use of a single TCR is suboptimal because it only targets a portion of tumor cells, rather than a subset of cells that cannot be recognized by the TCR, the combination of two TCRs allows the simultaneous killing of two subsets of cancer cells. By accumulating effective and selective therapeutic TCRs that recognize different epitopes from a variety of cancer-related proteins and address different HLA alleles in the ImmunoBank of therapeutic TCRs, it is possible to form a customized TCR combination based on the biology of the patient's tumor to make a treatment decision.

[0010] Figure 7 A representative display of the mechanisms of action of multiple TCR-T therapeutics is shown.

[0011] Figure 8 A to Figure 8 E shows a representative flow cytometry gating strategy for determining TCR-T cell-mediated killing. Three representative batches of single TSC-204-A0201 or TSC-204-C0702 and multiplex T-Plex-204-A0201 / C0702 TCR-T cells were co-cultured with a target cell population consisting of HLA-A*02:01-deleted U266B1 ("C7 target", CFSE-labeled) and HLA-C*07:02-deleted U266B1 ("A2 target", CellTrace TMThe cell viability of the residual cells from the different subsets (effectors, A2 targets, and C7 targets) in the co-culture was analyzed by flow cytometry (representative data obtained using T-Plex-204-A0201 / C0702 TCR-T cells from a representative batch are shown). Cells were gated by FSC-A vs. SSC-A dot plots ( Figure 8 A) and use CellTrace TM CFSE and CellTrace TM Purple plots distinguish subgroups ( Figure 8 B) In comparison to the LIVE / DEAD of the event TM The viable cells of each subpopulation were identified in the histogram ( Figure 8 C to Figure 8 E).

[0012] Fig. 9 A representative display of the viability of target cells in a heterogeneous tumor model (U266B1 HLA-A*02:01KO combined with U266B1 HLA-C*07:02KO) co-cultured with single (TSC-204-A0201 or TSC-204-C0702) or multiple (T-Plex-204-A0201 / 204-C0702) TCR-T cells is shown. U266B1 HLA-C*07:02KO and U266B1 HLA-A*02:01KO target cells were fluorescently barcoded, and aliquots of each were designated as controls, and the remainder was then combined to form a balanced heterogeneous target. Three batches of single TCR-T cell agents ("single weight"), TSC-204-A0201 or TSC-204-C0702 and multiple T-Plex-204-A0201 / C0702 TCR-T cells were co-cultured with heterogeneous target cells or only single target cells (control). The number and viability of barcoded target cells were determined by flow cytometry analysis. The viable cell counts of each target were standardized for absolute counting beads and then mapped. Similarly, the viability percentage of each target was mapped to determine the killing effect mediated by T-Plex. The left frame of each pair of framed data represents the data of U266B1 HLA-C7 KO cells and the right frame of each pair of framed data represents the data of U266B1 HLA-A2 KO cells. DETAILED DESCRIPTION

[0013] The present invention is based at least in part on the discovery that certain binding proteins, including T cell receptors (TCRs), that recognize more than one antigen (e.g., more than one antigen on the same target and / or more than one antigen on different targets) in combination and engineered cells comprising the binding proteins can overcome antigenic heterogeneity and / or loss of human leukocyte antigen (HLA) heterozygosity to treat cancer, including solid tumors.

[0014] Thus, the present invention relates in part to identified binding proteins (e.g., TCRs), host cells expressing binding proteins (e.g., TCRs), compositions comprising binding proteins (e.g., TCRs), and host cells expressing binding proteins (e.g., TCRs), methods of diagnosing, predicting, and monitoring T cell responses to cells expressing antigens and / or targets of interest, and methods for preventing and / or treating non-malignant disorders, hyperproliferative disorders, or recurrence of hyperproliferative disorders characterized by expression of antigens and / or targets of interest, by directly administering two or more binding proteins or administering a composition providing the two or more binding proteins, such as Such as a single composition comprising two or more binding proteins, a single composition comprising nucleic acids and / or vectors encoding two or more binding proteins (e.g., TCRs), a single composition comprising a host cell type expressing two or more binding proteins (e.g., TCRs), a combination of two or more compositions each comprising at least one binding protein (e.g., TCR), a combination of two or more compositions each comprising nucleic acids and / or vectors encoding at least one binding protein (e.g., TCRs), a combination of two or more compositions each comprising a host cell expressing at least one binding protein (e.g., TCR), etc. Administration may be a single composition or a combination of compositions administered in parallel or sequentially. Two or more binding proteins may be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more binding proteins or any range (including endpoints) therebetween, such as 2-5 binding proteins, 2-4 binding proteins, 2-3 binding proteins, etc. In some embodiments, active target gene expression analysis, HLA loss of heterozygosity (LOH) and / or HLA typing steps are used to select suitable subjects to determine compatibility and expected therapeutic efficacy with TCR binding to the desired MHC:peptide (pMHC) complex. Many representative non-limiting combinations are exemplified herein and any combination of any agent described herein is encompassed in the compositions and uses thereof and is encompassed by the present invention.

[0015] In addition, as further described below and in the working examples, in addition to the binding proteins as described herein, the host cells encompassed by the invention may encode and / or express the same polynucleotide as the binding protein or its components or available accessory proteins on different polynucleotides. For example, the host cell may encode and / or express TCRα, TCRβ, CD8α, CD8β, DN-TGFβR (e.g., DN-TGFβRII) and / or an optional protein marker, optionally wherein the optional protein marker is DHFR.

[0016] The term "dominant negative TGFβ receptor" or "DN-TGFβR" refers to a transforming growth factor (TGF) β receptor variant or mutant that provides resistance to TGFβ signaling. There are five type II receptors (activating receptors) and seven type I receptors (signal propagation receptors). The active TGFβ receptor is a heterotetramer consisting of two TGFβ receptor I (TGFβRI) and two TGFβ receptor II (TGFβRII). In some embodiments, the DN-TGFβR is DN-TGFβRII (i.e., a T GFβ receptor II variant or mutant). In some embodiments, resistance is an inhibitory effect on TGFβ signaling on immune cells (such as T cells), which may be produced by cancer cells or by other immune cells within the cellular environment, such as by stromal cells, macrophages, myeloid cells, epithelial cells, natural killer cells, etc. TGFβ signaling inhibitors are well known in the art and include, but are not limited to, mutant TGFβ that sequesters receptors and thereby inhibits signaling, antibodies that bind to TGFβ and / or TGFβ receptors (e.g., lerdelimumab, metlimumab, fressolim umab, etc.), soluble TGFβ binding proteins, such as portions of TGFβ receptors that sequester TGFβ (e.g., TGFβRII-Fc fusion proteins), or other binding agents, such as β-glycans. Any and all known TGFβ signaling inhibitors may be used alternatively or in addition to the DN-TGFβRs (e.g., DN-TGFβRII) described herein. In some embodiments, the DN-TGFβR lacks the intracellular portion required for TGFβ-mediated signaling, such as the entire intracellular domain, the kinase signaling domain, etc. DN-TGFβR constructs are well known in the art (see Brand et al. (1993) J. Biol. Chem. 268: 11500-11503; Weiser et al. (1993) Mol. Cell Biol. 13: 7239-7247; Bollard et al. (2002) Blood 99: 3179-3187; PCT Publication WO 2009 / 152610; PCT Publication WO 2017 / 156484; Kloss et al. (2018) Mol. Ther. 26: 1855-1866; PCT Publication WO. 2019 / 089884; PCT Publication WO 2020 / 042647; and representative, non-limiting embodiments of PCT Publication WO 2020 / 042648)

[0017] Example

[0018] Example 1: Materials and Methods for Example 2

[0019] a. Multiplexing of HPV and MAGE-A1 TCRs

[0020] (i) Engineering T cells to express HPV16-E7 11-19 Specific or MAGE-A1 290-297 TCR

[0021] use Primary CD3+ T cells were isolated from Leukopak using the CD3 microbead kit (Miltenyi Biotec) according to the manufacturer’s protocol. The isolated cells were frozen at CS10 (StemCell Technologies) and stored in liquid nitrogen until use. On day -1, CD3+ T cells were thawed, washed with complete T cell medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU / mL penicillin, 100 μg / mL streptomycin, recombinant human IL-2 [50 U / mL, PeproTech, Cranbury, NJ], recombinant human IL-15 [5 ng / mL, R&D Systems], and recombinant human IL-7 [5 ng / mL, (R&D Systems]). On day 0, CD3+ T cells were washed and resuspended in fresh T cell medium and cultured using ImmunoCult TM Human CD3 / CD28 / CD2 T cell activator (1x10 6 CD3+ T cells (5 μL, Stem Cell Technologies) were activated. On day 1, cells were washed and resuspended in fresh complete T cell medium and 1×10 6 Three replicate wells were transduced with lentiviral particles to express HPV or MAGE-A1 TCR. On day 2, the cells were washed, the triplicates were resuspended and combined in fresh complete T cell medium and The cells were expanded in 1 well of a 6-well plate (Wilson Wolf) until day 5. On day 5, the cells were harvested and the cell concentration was adjusted to 100 x 10 cells / ml in EasySep buffer (StemCell Inc) and FcBlock solution using CD34 magnetic beads (Miltenyi) at 4°C for 30 minutes. 6 CD3+ T cells were washed with EasySep buffer and used Separator and LS column (Miltenyi). The separated cells were washed and resuspended in fresh complete T cell culture medium and 10 flasks (Wilson Wolf) until day 12, when the cells were CS10 and stored under liquid nitrogen until use.

[0022] b. Multiplexing of MAGE-C2 and MAGE-A1 TCRs

[0023] (i) Engineering T cells to express MAGE-C2 184-192 or MAGE-A1 290-297 TCR

[0024] use Primary CD8+ T cells were isolated using the CD8 microbead kit (Miltenyi Biotec) according to the manufacturer’s protocol. CS10 (Stem Cell Technologies) and stored in liquid nitrogen until use. On day -1, CD8+ T cells were thawed and washed with complete T cell medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU / mL penicillin, 100 μg / mL streptomycin, recombinant human IL-2 [50 U / mL, PeproTech, Cranbury, NJ], recombinant human IL-15 [5 ng / mL, R&D Systems], and recombinant human IL-7 [5 ng / mL, (R&D Systems]). On day 0, CD8+ T cells were washed and resuspended in fresh T cell medium and cultured using ImmunoCult TM Human CD3 / CD28 / CD2 T cell activator (1x10 6 On day 1, cells were washed and resuspended in fresh complete T cell culture medium and 1×10 6 Each well was transduced in triplicate with lentiviral particles to express MAGE-C2 or MAGE-A1 or maintained as a non-transduced donor control. On day 2, the cells were washed, the triplicates combined, and resuspended in fresh complete T cell culture medium and incubated for 1 hr. 6-well plates (Wilson Wolf) until day 5. On day 5, cells were harvested and The cell concentration was adjusted to 100 x 10 cells / mL in the operating buffer (Miltenyi). 6 CD8+ T cells were added and anti-mTCR biotin antibody (BioLegend) was added at a dilution of 1:50 for 10 minutes at room temperature, and then Anti-biotin microbeads (Miltenyi) were added at a 1:5 dilution and incubated at room temperature for 10 minutes. Wash with running buffer and resuspend in In operating buffer for use Separator and LS column (Miltenyi) were used for manual magnetic separation. The separated cells were washed and resuspended in fresh complete T cell culture medium and 10 flasks (Wilson Wolf) until day 12, when the cells were CS10 and stored under liquid nitrogen until use.

[0025] (ii) Cell lines

[0026] The epidermoid carcinoma cell line CaSki (ATCC CRL-1550) and melanoma cell lines A101D (ATCC CRL-7898), SK-MEL-5 (ATCC HTB-70), and A2058 (ATCC CRL-11147) were purchased from the American Type Culture Collection (ATCC, Manassas, VA). CaSki cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS and 1% penicillin-streptomycin [Thermo Fisher Scientific]. A101D and A2058 cells were maintained in DMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin [Thermo Fisher Scientific] and SK-MEL-5 cells were cultured in EMEM containing 10% heat-inactivated FBS and 1% penicillin-streptomycin [Thermo Fisher Scientific].

[0027] (iii) Produce expression Nuclight Red Stable Cell Lines

[0028] CaSki, A101D and SK-MEL-5 cells were NucLight Red Lentivirus Reagent (EF-1α promoter, puromycin selection) (Sartorius) transduction. 24 hours after transduction, cells were washed and resuspended in their corresponding cell line culture medium and cultured at 37°C, 5% CO2. 2-3 days after transduction, puromycin (Gibco, Waltham, MA) was added to the culture at a predetermined concentration (ranging from 0.5 ug / mL to 1 ug / mL) to select for transduced cells. The culture was expanded under puromycin selection until it was at least 90% as determined by flow cytometry analysis. Nuclight red positive.

[0029] (iv) In vitro cytotoxicity analysis

[0030] In vitro cytotoxicity assays were performed in 96-well flat-bottom tissue culture plates without poly-L-ornithine coating; adherent cells were plated here and allowed to attach one day prior to the addition of T cells. Where indicated, T cells were incubated with cells expressing Nuclight red CaSki, A101D or SK-MEL-5 cells were co-cultured. Data were acquired on an S3 instrument (Sartorius) and Target cell growth was quantified on S3 as a readout of T cell cytotoxicity.

[0031] (v) Transwell T cell activation assay

[0032] A 1.0-μm pore polycarbonate membrane insert (Sigma-Aldrich #CLS3392) was used according to the manufacturer's instructions. HTS -96 permeable support. A101D melanoma cells were seeded in the upper chamber, while SK-MEL-5 melanoma cells were seeded in the lower chamber and both cell lines were allowed to adhere overnight. The next day, MAGEA1 TCR-engineered CD8+ T cells were co-cultured with A101D cells in the upper chamber, while MAGEC2 TCR-engineered CD8+ T cells were co-cultured with SK-MEL-5 cells in the lower chamber at a 1:2E:T ratio and incubated at 37°C for 48 hours under 5% CO2. After incubation, cells were collected for evaluation by staining with antibodies against T cell activation markers. Briefly, T cells were stained with PE-labeled anti-CD137 and AF647-labeled anti-CD69 (BioLegend), washed, and then CD137 and CD69 double positive cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter).

[0033] Example 2: Representative non-limiting combination therapy examples

[0034] Adoptive cell transfer in the context of genetically engineered T cells holds great promise for the treatment of solid tumors. To date, clinical studies of TCR engineered T cell therapies have targeted one antigen at a time and have produced encouraging response rates in the range of 30-50%. Unfortunately, complete responses are rare, and responses are often short-lived. It is believed that there are two major challenges associated with single antigen targeted TCR-T cell therapies.

[0035] First, the expression of most cancer-associated antigens is heterogeneous. In a representative, non-limiting example, multiple immunohistochemical analysis was performed using two cancer germline antigens, MAGE-C2 and PRAME, and considerable heterogeneity was observed between samples from different solid tumor types ( Figure 1 A to Figure 1 C). In addition, heterogeneous antigen expression was observed at the single-cell level (not every cancer cell within a given tumor expressing each antigen). Figure 1 A to Figure 1 C) This suggests that a single TCR may not be sufficient to eliminate all cancer cells within a given tumor, allowing tumor cells lacking processed antigen to escape and drive relapse.

[0036] Second, single-agent TCR-T cell therapy targets only a single HLA allele, which undergoes loss through the commonly observed HLA loss of heterozygosity (LOH) mechanism ( Figure 2 A and Figure 2 B). Clonal HLA class I LOH has been observed in 17% of all solid tumors (Montesion et al., Cancer Discovery, 2021) and subclonal HLA LOH occurs in an even larger percentage of tumors.

[0037] Multiplex TCR-T cell therapy mimics natural oligoclonal T cell responses to cancer and offers a way to address two challenges associated with treating solid tumors.

[0038] Using TScan's proprietary ReceptorScan and TargetScan platforms, multiple TCRs for TCR-T cell therapy were discovered, such as HPV16 E7-specific, MAGEA1-specific, and MAGEC2-specific TCRs.

[0039] In one representative, non-limiting example, two lead TCRs (MAGE-A1 and HPV), a lower affinity TCR (MAGE-C2), and a target cell line expressing their cognate antigens were multiplexed using direct and indirect co-culture experiments to evaluate the potential synergy of using more than one TCR to target tumors and to understand the biological mechanisms behind such synergy. Figure 1 A to Figure 5 Materials and methods and results are shown in B. Briefly, multiple T cell-mediated cancer killing and heterogeneity were modeled in vitro using two unique TCR:antigen pairs. Figure 3 The vectors used to generate the data shown in Figure 4 The vector for the data shown in does not express the CD8 co-receptor, but it has the mouse TCR constant region.

[0040] In one representative case, a multiplex of two high-affinity TCR-Ts was tested (i.e., one named TCR E7-11-28 (also known as TCR28 or 28; see Table 1), targeting the HLA-A*02:01 restricted epitope of HPV16-E7; and a second named TCR-204-C07 (also known as TCR 32-41, TCR-204-C7, and TCR-204-C0702; see Table 1), targeting the HLA-C*07:02 restricted epitope of MAGE-A1. Whole T cells were transduced and selected to express the relevant TCR (HPV or MAGE-A1) and, in some cases, the CD8 co-receptor. The target cells were a mixture of two cell lines, each expressing only one of the two antigens. CaSki cervical cancer cells were A*02:01+ and HPV+. A101D melanoma cells were C*07:02+ and MAGE-A1+. Both cell lines were engineered to express NucLight Red and mixed together to simulate tumor heterogeneity. Engineered T cells or non-engineered donor control T cells (control TCR-T) were mixed with NucLight Red-labeled target cell lines were co-cultured at the indicated effector to target (E:T) ratios and Their survival was quantified as a readout of T cell cytotoxicity. Although individual TCR-Ts caused approximately 50-60% cell killing at 72 h, a 1:1 mixture of two TCR-Ts at the same total effector to target (E:T) ratio produced approximately 80% cell killing, indicating a synergistic effect ( Figure 3 A and Figure 3 B).

[0041] In another representative case, the test multiplexes a high affinity TCR-T for MAGE-A1 with a low affinity TCR-T for MAGE-C2. TCR-204-C07 (also known as TCR 32-41, TCR-204-C7, and TCR-204-C0702; see Table 1) is a naturally occurring high affinity TCR that recognizes the HLA-C*07:02 restricted epitope of MAGEA1 and exhibits robust killing of cell lines expressing MAGEA1. TCR-LD8-3 is a low affinity TCR that recognizes the HLA-B*07:02 restricted epitope of MAGEC2 (also known as TCR 8-3; see Table 1) ( Figure 4 A). CD8+T cells were transduced using a lentiviral vector encoding a TCR that was optimized for better expression. Transduced cells were selected based on the expression of the relevant TCR (MAGE-A1 or MAGE-C2). Target cells were a mixture of cells expressing MAGE-A1 or expressing MAGE-C2. Both A2058 and SK-MEL-5 cells are melanoma cell lines that are C*07:02+ and B*07:02+, however A2058 cells only highly express MAGE-A1 and SK-MEL-5 cells only moderately express MAGE-C2. It was previously discovered that while MAGE-C2 TCR-T cells effectively kill A101D cells that express MAGEC2 at high levels, it is ineffective against SK-MEL-5 cells that express MAGEC2 at lower levels. To determine whether the cytotoxic activity of MAGE-C2 TCR-T cells would be enhanced when multiplexed with a more cytotoxic TCR, a co-culture experiment was performed. will be engineered to express NucLight Red-labeled SK-MEL-5 cells were mixed with unlabeled A2058 cells to make The activity quantified above will reflect cytotoxicity against SK-MEL-5 cells only. Engineered T cells or non-engineered donor control T cells (untransduced T cells) were co-cultured with target cell lines at the indicated effector cell to target cell (E:T) ratios and The survival rate was quantified as a readout of T cell cytotoxicity. Although low-level killing of SK-MEL-5 was observed by single MAGE-C2 T cells, the cytotoxic activity of MAGE-C2 TCR was synergistically enhanced when MAGE-C2 was combined with MAGE-A1 T cells. Thus, although MAGE-C2 TCR-T alone exhibited partial killing of MAGE-C2 positive cells, the addition of MAGE-A1 TCR-T synergistically enhanced the activity of MAGE-C2 TCR-T ( Figure 4 B).

[0042] To explore the mechanism of this synergistic activity, we used HTS System. The HTS Transwell-96 permeable support with a 1.0 μm pore polyester membrane transwell system was selected to allow soluble factors to diffuse between the two cell compartments (upper and lower chambers), but the cells did not diffuse. A101D melanoma cells were inoculated in the upper chamber, while SK-MEL-5 melanoma cells were inoculated in the lower chamber and both cell lines were allowed to attach overnight. The next day, MAGEA1 TCR-engineered CD8+ T cells were co-cultured with A101D cells in the upper chamber, while MAGEC2 TCR-engineered CD8+ T cells were co-cultured with SK-MEL-5 cells in the lower chamber at a 1:2E:T ratio. After 48 hours, cells from either chamber were collected for evaluation by staining with antibodies against T cell activation markers. Briefly, T cells were stained with PE-labeled anti-CD137 and AF647-labeled anti-CD69 (BioLegend), washed, and then CD137 and CD69 double-positive cells were analyzed on a CytoFLEX flow cytometer (Beckman Coulter). Using a transwell culture system, it was found that cytokines secreted by MAGE-A1 TCR-T cells greatly enhanced T cell activation of MAGE-C2 TCR-T cells after antigen engagement ( Figure 4 C). These findings suggest that multiple TCR-T can overcome antigen heterogeneity not only by independently targeting different cancer cell populations but also through cytokine-mediated T cell enhancement. Surprisingly, these results unexpectedly demonstrated a synergistic effect.

[0043] These results are suitable for clinical application. For example, to address the heterogeneity of solid tumors in the clinic, an illustrative screening strategy was designed to test patient tumors for antigen positivity and HLA LOH ( Figure 5 A). In addition, an ImmunoBank of therapeutic TCRs that identify different targets presented on different HLA alleles is identified. It is believed that multiple TCR-Ts that target intact antigens and HLA alleles in patient tumors are selected to synergistically overcome solid tumor heterogeneity. Additional in vivo studies are ongoing to further confirm the synergistic effects of multiple TCR-T cell therapies and clinical trials are being designed to further confirm clinical synergistic effects.

[0044] Therefore, this embodiment provides compositions and methods that can be used for multiple TCR-T cell therapy, including a combination of anti-MAGE-A1 and anti-HPV TCR or a combination of anti-MAGE-A1 and anti-MAGE-C2 TCR, and engineered cells expressing the combination. Without wishing to be bound by any particular scientific theory, this embodiment also includes multiple TCR-T cell therapy that simulates the response of natural oligoclonal T cells to cancer. Multiple TCR-T cell therapy (such as the combination described above) provides methods and compositions that address certain challenges associated with treating solid tumors.

[0045] A variety of analyses can be used to confirm the efficacy of multiple TCR-T cell therapies (such as the combinations described above). In a representative non-limiting example, direct and indirect co-culture experiments are used to multiplex the combination of anti-MAGE-A1 and anti-HPV TCRs or the combination of anti-MAGE-A1 and anti-MAGE-C2 TCRs and one or more target cell lines expressing their cognate antigens to evaluate the potential synergy of using more than one TCR to target tumors, and to understand the biological mechanisms behind such synergy. This analysis can be used to model the multiple T cell-mediated in vitro cancer killing and heterogeneity of multiple TCR-T cell therapies including the TCR combination of interest.

[0046] In one representative instance, multiplexing of such TCRs can be used and / or tested, such as by using engineered cells expressing (i) an anti-MAGE-A1 TCR targeting an HLA-C*07 serotype restricted epitope of MAGE-A1 (Table 3A) and (ii) an anti-HPV16 E7 TCR targeting an HLA-A*02 serotype restricted epitope of HPV16 E7 (Table 3C).

[0047] In another representative instance, multiplexing of such TCRs can be used and / or tested, such as by using engineered cells expressing (i) an anti-MAGE-A1 TCR targeting an HLA-C*07 serotype restricted epitope of MAGE-A1 (Table 3A) and (ii) an anti-MAGE-C3 TCR targeting an HLA-B*07 serotype restricted epitope of MAGE-C3 (Table 3B).

[0048] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR encompassed by the present invention, alone or in any combination thereof, can be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of): a) a TCR alpha chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, and / or b) a TCR alpha chain sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical to a TCR alpha chain sequence selected from the group consisting of the TCR β chain sequences listed in the tables provided herein.

[0049] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR alone or any combination thereof encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of): a) a TCR α chain sequence selected from the group consisting of the TCR α chain sequences listed in the tables provided herein; and / or b) a TCR β chain sequence selected from the group consisting of the TCR β chain sequences listed in the tables provided herein.

[0050] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR alone or in any combination thereof encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of): a) a TCR V selected from the group consisting of the TCRs listed in the tables provided herein; α The TCRα chain variable (V α ) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. α ) domain sequence; and / or b) a TCR V domain sequence selected from the group consisting of those listed in the tables provided herein β The domain sequence of the group consisting of TCRβ chain variable (V β) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. β ) domain sequence.

[0051] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR alone or in any combination thereof encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of): a) a TCR V selected from the group consisting of the TCRs listed in the tables provided herein; α The TCRα chain variable (V α ) domain sequence; and / or b) a TCR V domain sequence selected from the group consisting of the TCR V domains listed in the tables provided herein. β The domain sequence of the group consisting of TCRβ chain variable (V β ) domain sequence.

[0052] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR encompassed by the present invention, alone or in any combination, may be a TCR that comprises (e.g., includes, consists essentially of, or consists of) the following: at least one (e.g., one, two or three, such as CDR3 alone or in combination with CDR1 and CDR2) TCR alpha chain complementary determining region (CDR) sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in the tables provided herein. It is believed that CDR3 is the primary CDR responsible for recognizing processed antigens and that CDR1 and CDR2 primarily interact with MHC, therefore, in some embodiments, binding proteins are provided comprising a single CDR3 from a TCRα chain and / or a single CDR3 from a TCRβ chain listed in the table provided herein, each CDR3 having a sequence homology as described in this example.

[0053] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR encompassed by the present invention, alone or in any combination thereof, can be a TCR that comprises (e.g., includes, consists essentially of, or consists of) at least one (e.g., one, two, or three, such as CDR3, alone or in combination with CDR1 and CDR2) TCR β chain complementary determining region (CDR) sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β chain CDR sequence selected from the group consisting of the TCR β chain CDR sequences listed in the tables provided herein. As described above, it is believed that CDR3 is the primary CDR responsible for recognizing processed antigens and that CDR1 and CDR2 primarily interact with MHC, therefore, in some embodiments, binding proteins are provided comprising a single CDR3 from a TCRβ chain and / or a single CDR3 from a TCRα chain listed in the tables provided herein, each CDR3 having a sequence homology as described in this example.

[0054] The TCRs encompassed by the present invention (anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAME TCR alone or any combination thereof) can be TCRs that include (e.g., comprise, consist essentially of, or consist of) at least one (e.g., one, two, or three) TCR alpha chain complementarity determining regions (CDRs) listed in the tables provided herein.

[0055] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR alone or any combination thereof encompassed by the present invention can be a TCR that includes (e.g., comprises, consists essentially of, or consists of) at least one (e.g., one, two, or three) TCR β chain complementarity determining region (CDR) listed in the table provided herein.

[0056] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAME TCR, alone or in any combination thereof, encompassed by the present invention may be a TCR alpha chain constant region (C alpha ) that comprises (e.g., includes, consists essentially of, or consists of) at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the TCR Ca sequences listed in the tables provided herein. α) sequence of TCR.

[0057] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAME TCR alone or in any combination thereof encompassed by the present invention may be a TCR CCR listed in the table provided herein (e.g., comprising, consisting essentially of, or consisting of). β The present invention relates to a TCR beta chain constant region (C) having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence. β ) sequence of TCR.

[0058] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR alone or in any combination thereof encompassed by the present invention may be a TCR C selected from the group consisting of (e.g., comprising, consisting essentially of, or consisting of) a TCR C listed in the table provided herein. α The TCRα chain constant region (C α ) sequence of TCR.

[0059] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR and / or anti-PRAMETCR alone or in any combination thereof encompassed by the present invention may be a TCR C selected from the group consisting of (e.g., comprising, consisting essentially of, or consisting of) a TCR C listed in the table provided herein. β The TCRβ chain constant region (C β ) sequence of TCR.

[0060] Table 1: Representative TCR sequences

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] * Table 1 partly provides the representative TCR sequence of the subgrouping according to the MHC serotype presentation grouping and according to the different peptides presented by the MHC serotype and by the TCR binding of the subgrouping.Describe and require those individual TCRs such as those representatively illustrated in the table, and the associated protein species of the peptide epitope sequence described herein in combination with the individual or compound peptide epitope sequence described herein with MHC, such as those associated proteins grouped in the table provided by this paper.In addition, TRAV, TRAJ and TRAC genes of each TCR alpha chain described herein and TRBV, TRBJ and TRBC genes of each TCR beta chain described herein are provided.The sequence of each TCR described herein is provided in the form of each pair of homologous alpha chain and beta chain of each named TCR.TCR sequences described herein are marked.Variable domain sequences use capital letters.Constant domain sequences are lowercase letters.CDR1, CDR2 and CDR3 sequences are marked with bold and underlined text.CDR1, CDR2 and CDR3 are displayed in the standard appearance order from left (N end) to right (C end). The TRAV, TRAJ and TRAC genes of each TCR alpha chain described herein and the TRBV, TRBJ and TRBC genes of each TCR beta chain described herein are annotated according to the well-known IMGT nomenclature described herein. Similarly, the CDR1 and CDR2 of TRAV and TRBV are well known in the art because they are based on the well-known and annotated TRAV and TRBV sequences (e.g., as annotated in the databases of IMGT available at imt.org and IEDB available at iedb.org).

[0077] Table 2

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] *For the vectors in Table 2, the MSCV promoter is in bold. The β chain is annotated with bold and italic text. The α chain is annotated with bold and underlined text. The CD34 enrichment tag (e.g., Q tag) is annotated with italic and underlined text. CD8-α is in italics. CD8-β is underlined.

[0105] * Tables 1 and 2 include RNA nucleic acid molecules (e.g., thymine is replaced with uridine), nucleic acid molecules encoding heterologous homologs of the encoded proteins, and DNA or RNA nucleic acid sequences comprising nucleic acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over the entire length to any sequence listed in Table 1 or Table 2, or portions thereof. Such nucleic acid molecules may function as full-length nucleic acids as further described herein.

[0106] Table 3: Epitopes

[0107] Table 3A: MAGEA1 epitopes presented by HLA serotype HLA-C*07

[0108] Peptide epitope VRFFFPSL (SEQ ID NO:52) FFFPSLREA (SEQ ID NO:53) ARVRFFFPSL (SEQ ID NO:54) VRFFFPSLR (SEQ ID NO:55) RVRFFFPSL (SEQ ID NO:56) FFPSLREA (SEQ ID NO:57) ARVRFFFPSLR (SEQ ID NO:58) SARVRFFF (SEQ ID NO:59) VRFFFPSLREA (SEQ ID NO:60) RFFFPSLREA (SEQ ID NO:61)

[0109] Table 3B: MAGEC2 epitopes presented by HLA serotype HLA-B*07

[0110] Peptide epitope RAREFMEL (SEQ ID NO:62) RAREFMELL (SEQ ID NO:63) RAREFMELLF (SEQ ID NO:64) LKRAREFMEL (SEQ ID NO:65) VILKRAREF (SEQ ID NO:66) FPVILKRAR (SEQ ID NO:67) KRAREFMEL (SEQ ID NO:68) KRAREFMELL (SEQ ID NO:69) LKRAREFMELL (SEQ ID NO:70) RAREFMELLFG (SEQ ID NO:71)

[0111] Table 3C: HPV16 E7 epitopes presented by HLA serotype HLA-A*02

[0112] Peptide epitope YMLDLQPET (SEQ ID NO: 72) YMLDLQPETT (SEQ ID NO:72a)

[0113] Table 3D: MAGEA1 epitopes presented by HLA serotype HLA-A*02 (eg HLA-A*02:01)

[0114] Peptide epitope KVLEYVIKV (SEQ ID NO:83) VLEYVIKV (SEQ ID NO:83a) KVLEYVIK (SEQ ID NO:83b)

[0115] * Table 3 (such as Table 3A, Table 3B, Table 3C and Table 3D) includes peptide epitopes, and polypeptide molecules comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater identity over the entire length to the amino acid sequence of any sequence listed in Table 3 (such as Table 3A, Table 3B, Table 3C and Table 3D), or a portion thereof. Such polypeptides may have the function of the full-length peptide or polypeptide as further described herein.

[0116] Example 3: Representative non-limiting combination therapy examples

[0117] This embodiment provides compositions and methods that can be used for multiple TCR-T cell therapies including anti-MAGE-A1 TCR and anti-PRAME TCR. Without wishing to be bound by any particular scientific theory, this embodiment also includes multiple TCR-T cell therapies that mimic the response of natural oligoclonal T cells to cancer. Multiple TCR-T cell therapies (e.g., including anti-MAGE-A1 TCR and anti-PRAME TCR) provide methods and compositions that address certain challenges associated with treating solid tumors.

[0118] A variety of assays can be used to demonstrate the efficacy of multiplexed TCR-T cell therapies, such as those comprising anti-MAGE-A1 TCRs (such as "TCR1479", also referred to as "MAGE-A1-1479", "1479", "TSC-204-A02", and "TSC-204-A0201") and anti-PRAME TCRs (such as "TCR 366", also referred to as "366" and "TSC-203-A02" (also referred to as "TSC-203-A0201") and / or "TCR 358", also referred to as "358")). In one representative, non-limiting example, direct and indirect co-culture experiments are used to multiplex anti-MAGE-A1 TCRs, anti-PRAME TCRs, and one or more target cell lines expressing their cognate antigens to assess the potential synergy of using more than one TCR to target a tumor, and to understand the biological mechanisms behind such synergy. This assay can be used to model the multiplex T cell-mediated cancer killing in vitro and the heterogeneity of multiplex TCR-T cell therapies including anti-MAGE-A1 TCR and anti-PRAME TCR.

[0119] In a representative case, for example, multiplexing of such TCRs can be used and / or tested, such as by using engineered cells expressing (i) an anti-MAGE-A1 TCR targeting an HLA-A*02 serotype restricted epitope of MAGE-A1 (Table 5, e.g., SEQ ID NO: 83) and (ii) an anti-PRAME TCR targeting an HLA-A*02 serotype restricted epitope of PRAME (Table 7, e.g., SEQ ID NO: 104). In some embodiments, whole T cells are transduced and selected to express the relevant TCR (anti-MAGE-A1 or anti-PRAME). The target cells are a mixture of two cell lines, each of which expresses only one of the two antigens. U266B1 cells are HLA-A*02:01+ and MAGE-A1+. Hs695T, A375, and NCI-H1563 cells are HLA-A*02:01+ and PRAME+. Both cell lines are engineered to express NucLight Red and mixed together to simulate tumor heterogeneity. Engineered T cells or non-engineered donor control T cells (control TCR-T) were mixed with NucLight Red-labeled target cell lines were co-cultured at the indicated effector to target (E:T) ratios and were available in Their survival rate was quantified as a readout of T cell cytotoxicity.

[0120] Anti-MAGE-A1 TCRs encompassed by the invention may be TCRs that include (e.g., comprise, consist essentially of, or consist of) a) have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% affinity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha sequences listed in Table 4. or greater identity to a TCR alpha chain sequence; and / or b) a TCR beta chain sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identical to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 4.

[0121] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (e.g., includes, consists essentially of, or consists of) a) a TCR α chain sequence selected from the group consisting of the TCR α chain sequences listed in Table 4, ) a TCR β chain sequence selected from the group consisting of the TCR β chain sequences listed in Table 4.

[0122] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) the following: a) a TCR V selected from the group consisting of α The TCRα chain variable (V α ) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. α ) domain sequence; and / or b) a TCR V domain sequence selected from those listed in Table 4 β The domain sequence of the group consisting of TCRβ chain variable (V β) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. β ) domain sequence.

[0123] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) the following: a) a TCR V selected from the group consisting of the TCRs listed in Table 4; α The TCRα chain variable (V α ) domain sequence; and / or b) selected from the TCR V β The domain sequence of the group consisting of TCRβ chain variable (V β ) domain sequence.

[0124] The anti-MAGE-A1 TCR encompassed by the invention may be a TCR that comprises (e.g., includes, consists essentially of, or consists of) at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2) TCR alpha chain complementary determining region (CDR) sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 4. It is believed that CDR3 is the primary CDR responsible for recognizing processed antigens and that CDR1 and CDR2 primarily interact with MHC, therefore, in some embodiments, a binding protein is provided comprising a single CDR3 from a TCRα chain and / or a single CDR3 from a TCRβ chain listed in Table 4, each CDR3 having a sequence homology as described in this paragraph.

[0125] The anti-MAGE-A1 TCR encompassed by the invention can be a TCR that includes (e.g., comprises, consists essentially of, or consists of) at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2) TCR β chain complementary determining region (CDR) sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β chain CDR sequence selected from the group consisting of the TCR β chain CDR sequences listed in Table 4. As described above, it is believed that CDR3 is the primary CDR responsible for recognizing processed antigens and that CDR1 and CDR2 primarily interact with MHC, therefore, in some embodiments, a binding protein is provided comprising a single CDR3 from a TCRβ chain and / or a single CDR3 from a TCRα chain listed in Table 4, each CDR3 having a sequence homology as described in this paragraph.

[0126] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (eg, includes, consists essentially of, or consists of) at least one (eg, one, two, or three)) TCR alpha chain complementarity determining region (CDR) listed in Table 4.

[0127] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (eg, includes, consists essentially of, or consists of) at least one (eg, one, two, or three)) TCR β chain complementarity determining region (CDR) listed in Table 4.

[0128] Anti-MAGE-A1 TCRs encompassed by the invention may be TCR alpha chain constant regions (C alphas) ​​that are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the TCR Ca sequences listed in Table 4. α ) sequence of TCR.

[0129] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR C that includes (e.g., comprises, consists essentially of, or consists of) the TCR C listed in Table 4. β The sequences are at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR beta chain constant region (C β) sequence of TCR.

[0130] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (e.g., including, consisting essentially of, or consisting of) a TCR C selected from the group consisting of those listed in Table 4. α The TCRα chain constant region (C α ) sequence of TCR.

[0131] The anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (e.g., including, consisting essentially of, or consisting of) a TCR C selected from the group consisting of those listed in Table 4. β The TCRβ chain constant region (C β ) sequence of TCR.

[0132] Table 4: TCR sequences recognizing the MAGEA1 antigen presented by HLA serotype HLA-A*02

[0133]

[0134]

[0135]

[0136]

[0137] * Table 4 partly provides the representative TCR sequence of the subgrouping according to the MHC serotype presentation grouping and according to the different peptides presented by the MHC serotype and by the TCR binding of the subgrouping.Describe and require those individual TCRs such as those representatively illustrated in the table, and the associated protein species of the peptide epitope sequence described herein in combination with the individual or compound peptide epitope sequence described herein with MHC, such as those associated proteins grouped in the table provided by this paper.In addition, TRAV, TRAJ and TRAC genes of each TCR alpha chain described herein and TRBV, TRBJ and TRBC genes of each TCR β chain described herein are provided.The sequence of each TCR described herein is provided in the form of each pair of homologous alpha chain and β chain of each named TCR.TCR sequences described herein are marked.Variable domain sequences use capital letters.Constant domain sequences are lowercase letters.CDR1, CDR2 and CDR3 sequences are marked with bold and underlined text.CDR1, CDR2 and CDR3 are displayed in the standard appearance order from left (N end) to right (C end). The TRAV, TRAJ and TRAC genes of each TCR alpha chain described herein and the TRBV, TRBJ and TRBC genes of each TCR beta chain described herein are annotated according to the well-known IMGT nomenclature described herein. Similarly, the CDR1 and CDR2 of TRAV and TRBV are well known in the art because they are based on the well-known and annotated TRAV and TRBV sequences (e.g., as annotated in the databases of IMGT available at imt.org and IEDB available at iedb.org).

[0138] *For some of the displayed vectors, the MSCV promoter is in bold. The β chain is noted using bold and italicized text. The α chain is noted using bold and underlined text. The CD34 enrichment tag (Q tag) is noted using italic and underlined text. CD8-α is in italics. CD8-β is underlined.

[0139] * Table 4 includes polypeptide sequences and polypeptide molecules comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the amino acid sequence of any sequence listed therein over its entire length, or a portion thereof. Such polypeptides may have the function of a full-length peptide or polypeptide as further described herein.

[0140] * Table 4 includes RNA nucleic acid molecules (e.g., thymine replaced with uridine), nucleic acid molecules encoding heterologous homologs of the encoded proteins, and DNA or RNA nucleic acid sequences comprising nucleic acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over the entire length to any of the sequences listed therein, or portions thereof. Such nucleic acid molecules may function as the full-length nucleic acids further described herein.

[0141] Table 5: MAGEA1 epitopes presented by HLA serotype HLA-A*02 (eg HLA-A*02:01)

[0142] Peptide epitope KVLEYVIKV (SEQ ID NO:83) VLEYVIKV (SEQ ID NO:83a) KVLEYVIK (SEQ ID NO:83b)

[0143] As described above, any TCR combination described herein is contemplated for use.

[0144] For example, the anti-PRAME TCR encompassed by the invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) a) a TCR alpha chain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% affinity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha sequences listed in Table 6. or greater identity to a TCR alpha chain sequence; and / or b) a TCR beta chain sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 6.

[0145] The anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., includes, consists essentially of, or consists of) the following: a) a TCR α chain sequence selected from the group consisting of the TCR α chain sequences listed in Table 6, ) a TCR β chain sequence selected from the group consisting of the TCR β chain sequences listed in Table 6.

[0146] The anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) the following: a) a TCR V selected from the group consisting of α The TCRα chain variable (V α) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. α ) domain sequence; and / or b) a TCR V domain sequence selected from those listed in Table 6 β The domain sequence of the group consisting of TCRβ chain variable (V β ) domain sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity. β ) domain sequence.

[0147] The anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of): a) a TCR V selected from the group consisting of the TCRs listed in Table 6 α The TCRα chain variable (V α ) domain sequence; and / or b) selected from the TCR V β The domain sequence of the group consisting of TCRβ chain variable (V β ) domain sequence.

[0148] An anti-PRAME TCR encompassed by the invention may be a TCR that comprises (e.g., includes, consists essentially of, or consists of) at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2) TCR alpha chain complementary determining region (CDR) sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR alpha chain CDR sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 6. It is believed that CDR3 is the primary CDR responsible for recognizing processed antigens and that CDR1 and CDR2 primarily interact with MHC, therefore, in some embodiments, a binding protein is provided comprising a single CDR3 from a TCRα chain and / or a single CDR3 from a TCRβ chain listed in Table 6, each CDR3 having a sequence homology as described in this paragraph.

[0149] An anti-PRAME TCR encompassed by the invention may be a TCR that includes (e.g., comprises, consists essentially of, or consists of) at least one (e.g., one, two, or three, such as CDR3 alone or in combination with CDR1 and CDR2) TCR β chain complementary determining region (CDR) sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a TCR β chain CDR sequence selected from the group consisting of the TCR β chain CDR sequences listed in Table 6. As described above, it is believed that CDR3 is the primary CDR responsible for recognizing processed antigens and that CDR1 and CDR2 primarily interact with MHC, therefore, in some embodiments, a binding protein is provided comprising a single CDR3 from a TCRβ chain and / or a single CDR3 from a TCRα chain listed in Table 6, each CDR3 having a sequence homology as described in this paragraph.

[0150] The anti-PRAME TCR encompassed by the invention may be a TCR comprising (eg, includes, consists essentially of, or consists of) at least one (eg, one, two, or three)) TCR alpha chain complementarity determining region (CDR) listed in Table 6.

[0151] The anti-PRAME TCR encompassed by the present invention may be a TCR that comprises (eg, includes, consists essentially of, or consists of) at least one (eg, one, two, or three)) TCR β chain complementarity determining region (CDR) listed in Table 6.

[0152] Anti-PRAME TCRs encompassed by the invention may be TCR Cα chain constant regions (Cα) that include (e.g., comprise, consist essentially of, or consist of) at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the TCR Cα sequences listed in Table 6. α )sequence.

[0153] The anti-PRAME TCR encompassed by the present invention may be a TCR C comprising (e.g., including, consisting essentially of, or consisting of) a TCR C listed in Table 6. β The present invention relates to a TCR beta chain constant region (C) having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity with the sequence. β ) sequence of TCR.

[0154] The anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., including, consisting essentially of, or consisting of) selected from the group consisting of the TCR C listed in Table 6. α The TCRα chain constant region (C α ) sequence of TCR.

[0155] The anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., including, consisting essentially of, or consisting of) selected from the group consisting of the TCR C listed in Table 6. β The TCRβ chain constant region (C β ) sequence of TCR.

[0156] Table 6: TCR sequences recognizing the PRAME antigen presented by HLA serotype HLA-A*02

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] * Table 6 partly provides the representative TCR sequence of the subgrouping according to the MHC serotype presentation grouping and according to the different peptides presented by the MHC serotype and by the TCR binding of the subgrouping.Describe and require those individual TCRs such as those representatively illustrated in the table, and the associated protein species of the peptide epitope sequence described herein in combination with the individual or compound peptide epitope sequence described herein with MHC, such as those associated proteins grouped in the table provided by this paper.In addition, TRAV, TRAJ and TRAC genes of each TCR alpha chain described herein and TRBV, TRBJ and TRBC genes of each TCR β chain described herein are provided.The sequence of each TCR described herein is provided in the form of each pair of homologous alpha chain and β chain of each named TCR.TCR sequences described herein are marked.Variable domain sequences use capital letters.Constant domain sequences are lowercase letters.CDR1, CDR2 and CDR3 sequences are marked with bold and underlined text.CDR1, CDR2 and CDR3 are displayed in the standard appearance order from left (N end) to right (C end). The TRAV, TRAJ and TRAC genes of each TCR alpha chain described herein and the TRBV, TRBJ and TRBC genes of each TCR beta chain described herein are annotated according to the well-known IMGT nomenclature described herein. Similarly, the CDR1 and CDR2 of TRAV and TRBV are well known in the art because they are based on the well-known and annotated TRAV and TRBV sequences (e.g., as annotated in the databases of IMGT available at imt.org and IEDB available at iedb.org).

[0165] *For some of the displayed vectors, the MSCV promoter is in bold. The β chain is noted using bold and italicized text. The α chain is noted using bold and underlined text. The CD34 enrichment tag (Q tag) is noted using italic and underlined text. CD8-α is in italics. CD8-β is underlined.

[0166] * Table 6 includes polypeptide sequences and polypeptide molecules comprising an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the amino acid sequence of any sequence listed therein over the entire length, or a portion thereof. Such polypeptides may have the function of a full-length peptide or polypeptide as further described herein.

[0167] * Table 6 includes RNA nucleic acid molecules (e.g., thymine replaced with uridine), nucleic acid molecules encoding heterologous homologs of the encoded proteins, and DNA or RNA nucleic acid sequences comprising nucleic acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical over the entire length to any of the sequences listed therein, or portions thereof. Such nucleic acid molecules may function as the full-length nucleic acids further described herein.

[0168] Table 7: PRAME epitopes presented by HLA serotype HLA-A*02 (eg HLA-A*02:01)

[0169] Peptide epitope SLLQHLIGL (SEQ ID NO: 104)

[0170] Example 4: Multiplex TCR-T cell therapy targeting MAGEA1 and PRAME enhances the activity of adoptive T cell therapy in preclinical models

[0171] This example is based in part on the recognition that adoptive cell transfer in the context of genetically engineered T cells holds great promise for the treatment of solid tumors. Certain TCR engineered T cell therapies (TCR-T) have previously been clinically studied targeting one antigen at a time, and in various cases, have produced response rates in the range of 30-50%. It has been observed that complete responses to such therapies are rare, and responses are often short-lived. Without wishing to be bound by any particular scientific theory, one can reasonably conclude that the reason patients relapse quickly after responding to such therapies is that their tumors exhibit gross heterogeneity in antigen expression: not every cancer cell within a tumor expresses the target of a single TCR therapy, and even if they do, the target is expressed at variable levels in individual tumor cells. This suggests that TCR-Ts targeting one antigen may allow cells lacking the processed antigen to escape and drive relapse.

[0172] This embodiment presents the development of multiple TCR-T cell therapies, in which patients are treated using a variety of TCR-T cell products as a solution to antigen heterogeneity, and the multiple TCR-T cell products are selected from a collection of pre-examined TCRs that match the patient's tumor antigen and HLA type. As a proof of concept, two different cancer / testis antigens targeted by two different TCRs were selected. As described in Luomo et al. (2022) Cell.S0092-8674 (22) 00723-1, using the screening technology of TScan, one of these antigens, MAGEA1, was identified as a target for amplified tumor-infiltrating T cells from head and neck cancer patients. Another of the antigens, PRAME, is highly expressed in a variety of cancers. This embodiment includes the development of two high-affinity TCRs that recognize HLA-A*02:01 restricted epitopes from MAGEA1 and PRAME (see Tables 5 and 7, respectively). The benefits of combining these two TCR-T cell products with sequences according to Table 4 and Table 6, respectively, are evaluated using a variety of preclinical models. For example, TSC-203-A0201 and TSC-204-A0201 TCR-T cell products, such as those expressing the MGTM TCR and codon optimized, can be used.

[0173] It is believed that both TCRs (i.e., TCRs that recognize MAGEA1 and PRAME, respectively) exhibit potent in vitro cytotoxic activity when co-cultured with HLA-matched cancer cell lines expressing endogenous MAGEA1 and PRAME, respectively. In addition, in a xenograft mouse model, it is believed that each TCR is able to control the growth of tumors expressing the cognate antigen and HLA.

[0174] Mixtures of two different cell lines expressing MAGEA1 or PRAME and HLA-A*02:01 were tested in vitro or grown as xenograft tumors in mice and treated with either TCR-T tested individually or with a mixture of both TCR-Ts. Notably, the MAGE-specific TCR-Ts and PRAME-specific TCR-Ts were designed to selectively target their respective target cell subsets, and the multiplexed MAGEA1 / PRAME TCR-Ts were designed to target both cancer cell subsets simultaneously. Mice were designed to achieve longer sustained tumor control when treated with multiplexed MAGEA1 / PRAME TCR-Ts compared to TCR-Ts targeting a single antigen.

[0175] Reported in this example and further summarized in Figure 6The discovery in is expected to confirm that multiple TCR-T is an effective means for targeting cancers with heterogeneous target antigen expression, and therefore, is a favorable treatment method. Without wishing to be bound by any particular scientific theory, this embodiment confirms that multiple TCR-T simulates the response of natural oligoclonal T cells to cancer and has the potential to overcome antigen heterogeneity, which can make it possible to observe a lack of durability in a monotherapy TCR-T clinical trial. The duration of the co-culture analysis described is relatively short-term, and it is believed that further synergistic activity (such as from the cytokine-dependent phenomenon described herein) can be observed in other analyses, such as reducing the effector and target ratio of one or more TCRs of the TCR combination in a longer duration study to show the support of other TCRs to the reduced TCRs, etc.

[0176] Figure 6 Representative examples of variable antigen expression in human non-small cell lung (NSCLC) tumor samples are shown. Immunohistochemical analysis of human NSCLC tumor microarrays was performed using MAGE-A1 specific antibodies (clone SPM282; Abcam catalog number ab25834) or PRAME specific antibodies (clone EPR20330; Abcam catalog number ab219650). As presented for MAGE-A1 and PRAME with variable expression levels, heterogeneous antigen expression within the tumor was observed in multiple sections. Unstained tumor controls are also shown.

[0177] Figure 6 Also shown is the characterization of the TCR that recognizes the MAGE-A1 derived epitope presented on HLA-A*02:01 and the TCR that recognizes the PRAME derived epitope presented on HLA-A*02:01. The TCR-T cells expressing MAGE-A1 specific TCR and presenting a series of MAGE-A1 (i.e., MCI0H1703, HJS936T and A375) of a group of HLA-A*02:01 positive cancer cell lines or PRAME specific TCR and presenting a series of PRAME expression (i.e., HS695T, A375 and NCI-H15632) cells were co-cultured. HLA-A*02:01 is positive but the cell lines of MAGE-A1 (A2-HEK293T) or PRAME (647-V) negative are tested as negative controls.

[0178] The TCR-T cells used in these experiments were whole T cells engineered by lentiviral transduction. The vector contained the MGTM modification and the co-receptors CD8α and CD8β were co-delivered with the recombinant TCR, ensuring recognition of the TCR primarily on the CD4+ fraction of whole T cells.

[0179] The data demonstrate that each TCR-T exhibits high performance and selectivity for cells presenting the cognate peptide / MHC (pMHC), thereby killing relevant target cells while sparing cell lines that are negative for the targeted protein.

[0180] In vivo efficacy studies were also performed to further test the performance of individual TCR-T cells. em26Cd52 Il2 gem26Cd22 NjuCrl (NCG) mice were subcutaneously implanted with U266B1 cancer cells (HLA-A*02:01 positive cells expressing MAGE-A1). Growing tumors (average tumor volume of about 100 mm) were confirmed. 3 Animals with 100% leukemia (20E6 per group; injected on the second day after randomization and again one week after inoculation) were randomized to different experimental groups and received two intravenous injections of MAGE-A1 TCR-T cells (20E6 per group; injected on the second day after randomization and again one week after inoculation) or donor-matched non-engineered control T cells (20E6 per group; injected on the second day after randomization and again one week after inoculation). Tumor volume was measured twice a week. Although animals from the control group presented an average tumor volume of more than 800 mm at day 42, the tumor volume was significantly increased compared to the control group. 3 However, mice treated with MAGE-A1-specific TCR-T cells showed robust anti-tumor responses despite growing tumors.

[0181] In a similar experiment, female NCG animals were implanted with Hs695T cells (HLA-A*02:01 positive cells expressing PRAME) and received a single dose of PRAME-specific TCR-T cells or donor-matched non-engineered control T cells (20E6 T cells, one day after randomization). When compared to animals treated with control T cells, animals injected with TCR-T cells exhibited anti-tumor responses.

[0182] These studies demonstrated that intravenously injected TCR-T cells could successfully control the growth of subcutaneously inoculated pMHC-positive tumors in mice, confirming the performance of individual TCR-T cells.

[0183] In vitro experiments were performed to demonstrate the value of combining TCR-T cells to treat heterogeneous tumors. Reporter HEK293T cells that exclusively express HLA-A*02:01 and express granzyme B-activated infrared fluorescent protein (IFP) were further engineered to express MAGE-A1 or PRAME. MAGE-A1-expressing cells were GFP-tagged, and PRAME-positive cells were GFP-tagged with CellTrace TMPurple labeling is to enable tracking blood cell count readings in downstream logistics.When TCR-T cell identification target cells, TCR-T cells secrete cytotoxic granules into target cells, so that target cells become fluorescent (IFP positive).Two target cells (that is, PRAME positive or MAGE-A1 positive) are mixed with MAGE-A1 TCR-T cells, PRAME TCR-T cells or the multiple products of two TCR-T cells of combination with balanced ratio and co-culture.PRAME TCR-T cells and MAGE-A1 TCR-T cell lines are obtained from T cell engineering from the same donor.TCR-T cells correspond to the delivery vector lentiviral transduction modified by using MGTM and co-deliver CD8 α and CD8 β co-receptors and engineered full T cells (such as table 1) as described above.Also include the non-engineered T cells (NTC) of donor matching as a control. The experiment then measures the ratio of each subset of targets (GFP-positive MAGE-A1 targets; GFP / CTV-positive PRAME targets) recognized and targeted by TCR-T cells as measured by the ratio of GFP or GFP / CTV that becomes IFP-positive. NTC does not induce any IFP positivity in any target subset. When MAGE-A1 TCR-T cells are co-cultured with mixed target cells, the ratio of IFP positive in GFP-positive increases, but does not increase in GFP / CTV-positive cells. These results confirm that only MAGE-A1-positive subsets are recognized under this co-culture condition. On the contrary, when PRAME TCR-T cells are co-cultured with a target cell mixture, the ratio of IFP positive in GFP / CTV-positive targets also increases, but does not increase in GFP-positive target cells. These results confirm that only PRAME-positive target cell subsets are targeted under this co-culture condition. Finally, after co-culturing a mixture of PRAME TCR-T cells and MAGE-A1 TCR-T cells with mixed target cells, both GFP-positive and GFP / CTV-positive cell subsets showed IFP-positive signals, indicating that both target cell subsets were effectively recognized. The proportion of each subset that became IFP-positive in co-culture with multiple products was similar to that observed when co-cultured with each individual TCR-T cell product.

[0184] Overall, the data demonstrate that while each individual TCR-T cell product is capable of targeting cells that are positive for the relevant pMHC, multiple products are required to broadly target a heterogeneous mixture of cancer cells.

[0185] Female NCG mice were also inoculated subcutaneously with a mixture of HEK293T cells expressing MAGE-A1 or PRAME and HLA-A*02:01. 3Afterwards, the animals were randomized and received a single intravenous injection of 20E6 MAGE-A1 TCR-T cells, 20E6 PRAME TCR-T cells, or a multiple product consisting of 10E6 MAGE-A1 TCR-T cells and 10E6 PRAME TCR-T cells, or a multiple product consisting of 20E6 MAGE-A1 TCR-T cells and 20E6 PRAME TCR-T cells. One group of animals received an intravenous injection of 20E6 donor-matched non-engineered T cells. The same effector T cells as those described above were used in these experiments. Tumor volume was then measured every two weeks. Animals in the control group showed rapidly growing tumors; tumor volumes reached more than 1000mm at the end of the study. 3 (On day 24 after inoculation). On the other hand, animals given each individual TCR-T cell subset presented tumors that grew at a slower rate, reaching only about 600-750 mm at the end of the study. 3 When with a diameter of about 500 mm 3 (10E6 each TCR-T) or about 300mm 3 Animals receiving multiple TCR-T cell products achieved broader and more durable responses when compared to animals receiving individual TCR-T cell products with an average tumor volume of 20E6 each TCR-T. Figure 6 Together, the in vitro data presented in and described above demonstrate that each TCR-T cell targets only a subset of a heterogeneous tumor, thereby achieving a partial response: a subset of cancer cells remains resistant to a single dose of TCR-T cells and drives relapse. On the other hand, by broadly targeting both cell subsets of a heterogeneous tumor, multiple TCR-T products prevent the selection of resistant cells and achieve a stronger anti-tumor response. In addition, Figure 6 The concept of an ImmunoBank-based method for therapeutic TCR therapy is described, wherein several therapeutic TCRs that address multiple cancer-related proteins in combination with HLA restrictions are used to achieve a TCR therapy combination customized for each patient based on tumor biology. For each patient, (a) using immunohistochemistry (IHC) or reverse transcription polymerase chain reaction (RT-PCR) to determine which cancer-related proteins are expressed in their tumors (rows of ImmunoBank), and (b) by genomic sequencing measurements to determine which HLA genes (columns of ImmunoBank) in their tumors are complete (i.e., HLA loci have not yet experienced heterozygosity loss [LOH]) to make treatment decisions. After determining the patient's tumor target and HLA pattern, multiple TCRs (e.g., 2 TCRs, 3 TCRs, etc.) are selected from ImmunoBank to prepare customized multiple TCR-T cell drug products.

[0186] Example 5: Multiple TCR-T cell therapy targeting the same target using different TCRs recognizing epitopes presented on unique HLAs enhances the activity of adoptive T cell therapy in preclinical models

[0187] This example is based in part on the recognition that adoptive cell transfer in the context of genetically engineered T cells holds great promise for the treatment of solid tumors.

[0188] Patients who are positive for specific HLA alleles of interest (such as HLA-A*02:01 and HLA-C*07:02) are suitable for treatment with TCRs that recognize epitopes of a given target presented by such HLAs, such as TSC-204-A0201 and TSC-204-C0702, respectively (e.g., by simultaneous or sequential infusion of TCRs). Patients in which specific HLA alleles appear on separate chromosomes (separate haplotypes) are more likely to be resistant to HLA loss because natural killer (NK) cells target tumor cells that have lost both class I HLA haplotypes (O'Connor et al. (2006) Immunol. 117: 1-10). Other TCR-T components that address different targets and a wider range of HLA types can be used to further enhance the combination of TCRs and enable a wider range of patients to be treated with multiple TCR-Ts.

[0189] This example presents the development of a multiplex TCR-T cell therapy in which a patient is treated using multiple TCR-T cell products selected from a collection of pre-screened TCRs that match the patient's tumor antigen and HLA type as a solution to address antigen heterogeneity. As a representative, non-limiting example, two different TCRs are selected for multiplex TCR-T therapy, each of which targets a different epitope belonging to the same target but presented by a different HLA allele. Whole T cells (comprising CD4 + With CD8 + The T cell composition format uses TSC-204-A0201 and TSC-204-C0702 to express (1) the corresponding recombinant TCR, (2) recombinant CD8α and CD8β co-receptors to maximize the efficacy of the therapeutic product, (3) a CD34-derived epitope tag fused to the N-terminus of CD8α to facilitate tracking of engineered cells in vitro and in vivo, (4) a dominant negative type II TGFβ receptor (DN-TGFβRII) to address tumor microenvironment-mediated immunosuppression, and (5) a mutant form of the dihydrofolate reductase (DHFRdm) protein to facilitate enrichment of engineered cells during the manufacturing process. However, it is believed that the results shown in the data provided herein can be attributed to the function of the TCR itself.

[0190] In vitro characterization of TSC-204-A0201 and TSC-204-C0702 materials confirmed that TCR-T cells participated in target-dependent responses, resulting in the secretion of inflammatory cytokines, amplification of effector T cells, and ultimate killing of target cells ( Figure 7 Since the MAGE-A1-derived epitopes targeted by TSC-204-A0201 or TSC-204-C0702 are presented on class I MHC HLA-A*02:01 and HLA-C*07:02, respectively, the recombinant TCR uses the CD8αβ co-receptor to engage pMH C. Helper (CD4 + ) T cells do not naturally express CD8αβ co-receptors. Exogenous CD8α and CD8β co-receptors are co-delivered to engineered T cells along with therapeutic TCRs to promote CD4 + Helper T cells are able to recognize class I restricted epitopes. The engineered CD4 + T cells and engineered CD8 + Cytotoxic T cells underwent proliferation together, demonstrating functional engagement of helper T cells. In addition, because the engineered T cells expressed DN-TGFβRII, TSC-204-A0201 and TSC-204-C0702 TCR-T cells were active even in the presence of the immunosuppressive cytokine TGFβ, which can be observed in the microenvironment of solid tumors.

[0191] Figure 8 and Fig. 9 Results from three independent batches of TSC-204-A0201 and TSC-204-C0702 TCR-T cells applied to a heterogeneous target cancer cell population generated to simulate a MAGE-A1 positive tumor that undergoes LOH are shown. Briefly, the U266B1 (i.e., cell line TIB-196 available from ATCC) cell line, a MAG E-A1 positive, HLA-A*02:01 positive, and HLA-C*07:02 positive cancer cell line, was used. The cells were engineered by CRISPR deletion to form two versions of the cell line, in which only one of the two HLAs of interest was intact (elimination of HLA-A*02:01 or HLA-C*07:02). The U266B1 HLA-C*07:02KO, "A2 target" and U266B1 HLA-A*02:01KO, "C7 target" target cells were each stained with CellTrace TM Purple and CFSE barcoding. After co-culture using single (TSC-204-C0702 or TSC-204-A0201) or multiple (T-Plex-204-A0201 / 204-C0702) conditions, cell suspensions were washed with LIVE / DEADTM Viability dye labeling to determine target cell viability. Each target cell subset is labeled with a unique fluorescent dye to track cytometry readings in downstream logistics and then mixed in a 1:1 ratio.

[0192] Preparation of effector T cells. One day before the performance analysis, effector TCR-T cells were thawed in a 37°C water bath and washed with cytokine-free T cell medium. Cell concentration and viability (CCV) were determined, and live TCR-T cells were plated at a concentration of 1E6 cells per ml in complete T cell medium. 6M well plates. TCR-T cells were recovered in a humidified incubator at 37°C and 5% CO2 for 16-24 hours and then cultured. After thawing and overnight recovery, effector TCR-T cells were harvested, washed with cytokine-free T cell culture medium and resuspended in cytokine-free T cell culture medium with 2E6 live cells per ml to prepare for single and multiple conditions. Each TCR-T cell suspension was made into aliquots for the coating of positive control single conditions. The remaining TCR-T cell suspensions of all three batches were combined in a 1:1 ratio to form a test sample "T-Plex" condition (2E6 live cells per ml).

[0193] Similarly, target cells were prepared. Target cells were thawed, expanded, and maintained in culture for no more than 20 generations before being discarded. One day before co-culture was started, target cells were harvested and CCV was measured and recorded. Target cells were then inoculated at 4E5 viable cells per ml to synchronize cell cycle phases. On the day of co-culture, target cells were harvested and CCV was determined. The harvested cells were washed and the cell density was adjusted to 1E6 cells per ml in protein-free PBS. Target U266B1 HLA-C*07:02KO cells were labeled with cell trace purple and target U266B1 HLA-A*02:01KO cells were labeled with CellTrace TM CFSE labeling (both performed at 1:2000 according to the manufacturer's instructions) and finally resuspended in RPMI-based medium at 5E5 viable cells per ml. TM After labeling, each target cell suspension (5E5 viable cells per ml) was aliquoted for plating negative controls. Heterogeneous target cell preparations were made from the remaining target cell suspension (5E5 viable cells per ml), which was combined in a 1:1 ratio for co-culture with positive controls, singleplex and T-Plex test samples.

[0194] In addition, co-cultures are prepared. The heterogeneous target cells are then co-cultured with different TCR-T cell mixtures made exclusively of TSC-204-A0201, TSC-204-C0702 (corresponding to monotherapy or "single" TCR-T cell products) or by a balanced mixture of TSC-204-A0201 and TSC-204-C0702 (i.e., "multiple" TCR-T cell products). In simple terms, the target cells are spread in the sample wells (U-bottom 96-well plates), and then single or multiple condition effector cell suspensions are added on top of the target cells. The final volume is 200 μL per well, consisting of a 50 / 50 mixture of target cells (100 μL) and effector cells (100 μL) in target cell RPMI culture medium and cytokine-free T cells and target cell culture medium, respectively. The cells are returned to the incubator and co-cultured for 20-24 hours. Each positive control or test sample well contained a combined target suspension of 5E4 total viable cells consisting of 50% CTV-labeled U266B1 HLA-C*07:02KO (2.5E4 cells total) and 50% CTCFSE-labeled U266B1 HLA-A*02:01KO (2.5E4 cells total). Each single-plex condition sample well contained a combination of 2E5 total viable cells of the effector cell suspension TSC-204-A0201 or TSC-204-C0702 and 5E4 total viable cells of the combined target cell suspension. This represents a total effector to target (E:T) ratio of 4:1 and an effector to specific target ratio of 8:1. Each T-Plex condition sample well contained 2E5 total cells of the combined cell suspension T-Plex-204-A0201 / 204-C0702 consisting of 50% TSC-204-A0201 (1E5 cells total) and 50% TSC-204-C0702 (1E5 cells total). In addition, this well was combined with 5E4 total cells of the combined target cell suspension. This represents a 4:1 E:T ratio and a 4:1 effector to specific target ratio.

[0195] The cytotoxic activity of TCR-T cells against target cells was assessed by flow cytometry by evaluating the relative composition of each target cell population in the residual cells. At the end of the co-culture, the cells were pelleted by centrifugation and then resuspended in LIVE / DEAD TM Viability was determined by incubating cells in 4% paraformaldehyde (P < 0.05) for 20 minutes at 4°C in the dark. After washing once, cells were resuspended in EasySep TM CountBright was added according to the manufacturer's instructions. TMAbsolute counting beads. Immediately after adding counting beads, data acquisition was performed on the cell counter to the analysis plate. Data acquisition was performed on the CytoFLEX S flow cytometer following the machine's SOP-PC-0001-Instrument SOP-Use and CytoFLEX Maintenance. Compensation was automatically performed with CytExpert software. Flow cytometry analysis was performed using FlowJo 7.6.5 version, and statistical data was exported to Microsoft Excel 2010 and analyzed. Selected analysis data was plotted in GraphPad Prism (version 5.02).

[0196] Figure 8 A to Figure 8 The gating strategy is illustrated in E. Briefly, cells were gated by FSC vs. SSC dot plots. TM CFSE vs. CellTrace TM Purple map to distinguish subpopulations; C0702 target (CellTrace TM CFSE + / CellTrace TM purple - )、A0201 target (CellTrace TM CFSE - / CellTrace TM purple + ) and effectors (CellTrace TM CFSE - / CellTrace TM purple - ). Using LIVE / DEAD TM Histograms are used to identify live cells in each subpopulation. Dead cells have high fluorescence intensity because LIVE / DEAD TM The dye reacts with free intracellular and extracellular amines of damaged cell membranes. Live cells can be distinguished because they exhibit lower fluorescence intensity since the dye is restricted only to extracellular amines.

[0197] Killing of target cells is defined by percent killing, which is determined by subtracting the percent viability of the test sample from the negative control viability and then dividing by the negative control. When the percent viability of the test sample increases above the negative control viability value, the baseline percent killing value is reported as 0% killing.

[0198] To quantify the absolute count of live target cells obtained from the sample wells, add 20 μL of CountBright TMAbsolute counting beads (20,400 beads per 20 microliters) were added to a 120 μL volume of the cell suspension. The volume of the obtained cell sample was multiplied by the absolute cell count concentration to determine the total viable cell count obtained.

[0199] Negative controls were used to determine the baseline viability of target cells. Briefly, for HLA-C*0702 and MAGE-A1 proteins, CellTrace TM CFSE-labeled U266B1HLA-A*02:01KO targets (i.e., "C7 targets") are intact. These targets constitute the target cells for TS C-204-C0702 TCR-T cells. CellTrace TM The purple-labeled U266B1 HLA-C*07:02KO target (i.e., "A2 target") expresses HLA-A*02:01 and MAGE-A1 proteins. These targets constitute the target cells for TSC-204-A0201 TCR-T cells. To determine the baseline viability of each individual target after overnight culture, negative controls were formed; TSC-204-C0702 was co-cultured with U266B1 HLA-C*07:02KO target cells ("A2 target") only, and TSC-204-A0201 was co-cultured with U266B1-A*02:01KO target cells ("C7 target") only. The mean baseline viability of U266B1 HLA-A*02:01KO (C7 target) was 67.57% (n=3) and that of U266B1 HLA-C*07:02KO (A2 target) was 61.53% (n=3). Total viable cell counts and viability percentages were as follows: Fig. 9 These baseline values ​​were used to calculate specific TCR-T cell-mediated killing.

[0200] Similarly, TCR-T cell-mediated killing of target cells was observed using a positive control. Briefly, to determine the benefit of T-Plex-204-A0201 / 204-C0702-mediated killing, a cell suspension consisting of 50% CTV-labeled U266B1 HLA-C*07:02KO and 50% CTCFSE-labeled U266B1 HLA-A*02:01KO was formed. The resulting target was heterogeneous because one subset of cells expressed HLA-A*02:01 but not HLA-C*07:02, while another subset expressed HLA-C*07:02 but lost HLA-A*02:01. This target combination "A2+C7" was co-cultured with TCR-T cell products consisting of TSC-204-A0201 or TSC-204-C0702 as monotherapy ("monoplex") to confirm the killing capacity of each individual component of the T-Plex. These conditions served as positive controls.

[0201] Fig. 9 The percent killing results related to total live cell counts and percent viability (calculated as the decrease in viability relative to the baseline as described above) are shown in . Specifically, TSC-204-A0201 TCR-T cells from three independent batches exhibited specific cell-mediated killing of U266B1 HLA-C*07:02KO target cells (58.88%, 69.01%, and 64.30%, respectively), but did not kill U266B1 HLA-A*02:01KO target cells (specific killing was 0% in all 3 batches tested). Similarly, TSC-204-C0702 TCR-T cells from three independent batches exhibited cell-mediated killing of U266B1 HLA-A*02:01KO target cells (48.84%, 59.84%, and 47.66%, respectively), but systematically did not kill U266B1 HLA-C*07:02KO (specific killing was 0% in all 3 batches tested). These data confirm that each individual TCR-T cell component of the T-Plex independently and selectively kills target cells that are intact for the relevant HLA.

[0202] To determine the tumor killing ability of T-Plex-204-A0201 / 204-C0702, donor-matched individual TCR-T cell components from three independent batches of process representative material were combined and co-cultured with the heterogeneous target combination "A2+C7". Fig. 9 As shown in, all three batches showed similar trends, confirming that the viability of heterogeneous target cell mixtures in all three batches of T-Plex-204-A0201 / 204-C0702 was reduced. The two target cell populations were barcoded to enable the analysis of the viability of the A2 and C7 target subsets specifically. When co-cultured with T-Plex-204-A0201 / C0702 or with isolated TSC-204-A0201 TCR-T cells, the viability of U266B1 HLA-C*07:02KO target cells was reduced. Similarly, when co-cultured with T-Plex-204-A0201 / C0702 or with isolated TSC-204-C0702 TCR-T cells, the viability of U266B1 HLA-A*02:01KO target cells was reduced.

[0203] When compared to the baseline viability noted above, specific killing (calculated as the decrease in viability relative to the baseline described above) demonstrated that the T-Plex-204-A0201 / 204-C0702 product from three independent batches had 55.31%, 71.18% and 60.89% specific tumor killing activity in the case of U266B1 HLA-C*07:02KO target cells, respectively, and 1.3% specific tumor killing activity in the case of U266B1 HLA-C*07:02KO target cells, respectively. In the case of HLA-A*02:01KO target cells, it is 53.38%, 61.77% and 48.45% respectively. It is worth noting that the individual killing activity of TSC-204-C0702 and TSC-204-A0201TCR-T cell products is similar to the combined killing activity of T-Plex-204-A0201 / 204-C0702, indicating the effective function of the two TCR-T cell components combined. As described above, the duration of the analysis is relatively short-term, and it is believed that further synergistic activity (such as from the cytokine dependency phenomenon described herein) can be observed in other analyses, such as reducing the effector and target ratio of one or more TCRs of the TCR combination in a longer duration study to show the supporting effect of other TCRs on the reduced TCRs, etc.

[0204] therefore, Fig. 9 The results provided in demonstrate that, when faced with a heterogeneous target cell population, a single TCR-T cell component effectively treats a portion of tumor cells, but not a subset of cells that the TCR-T cells cannot recognize (here because the cells have lost the associated HLA). Multiple TCR-T therapies (here combining TSC-204-A0201 and TSC-204-C0702) simultaneously cause killing of two subsets of cancer cells. Therefore, the data confirm that multiple TCR-T therapies (such as TSC-204-A0201 and TSC-204-C0702) can be combined to treat tumors in which LOH occurs, thereby maximizing the chance of achieving a complete response.

[0205] Incorporated by Reference

[0206] All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0207] Any polynucleotide and polypeptide sequences that refer to accession numbers associated with entries in public databases, such as those maintained by The Institute for Genomic Research (TIGR) at the World Wide Web at tigr.org and / or the National Center for Biotechnology Information (NCBI) at the World Wide Web at ncbi.nlm.nih.gov, are also incorporated by reference in their entirety.

[0208] Equivalents and scope

[0209] The above description sets forth the details of one or more embodiments encompassed by the present invention. Although representative exemplary materials and methods have been described above, any materials and methods similar to or equivalent to those described herein can be used to practice or test the embodiments encompassed by the present invention. Other features, objects, and advantages associated with the present invention are apparent from the description. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the description of the present invention provided above shall prevail.

[0210] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments encompassed by the invention described herein. The scope encompassed by the present invention is not intended to be limited to the description provided herein and the appended claims are intended to encompass such equivalents.

[0211] It should also be noted that the term "comprising" is intended to be open ended and allows but does not require the inclusion of other elements or steps. Thus, when the term "comprising" is used herein, the term "consisting of" is also encompassed and disclosed.

[0212] Where ranges are given, the endpoints are included. Further, it should be understood that unless otherwise indicated or otherwise apparent from the context and understanding of one of ordinary skill in the art, the values ​​expressed in ranges may assume any specific value or sub-range within the stated range in the various embodiments encompassed by the present invention, with intervals of one-tenth of the unit of the lower limit of the range unless the context clearly indicates otherwise.

[0213] In addition, it should be understood that any specific embodiment of the invention that is a prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded even if the exclusion is not explicitly stated herein. Any specific embodiment of the composition covered by the invention (e.g., any antibiotic, therapeutic agent, or active ingredient; any method of preparation; any method of use; etc.) may be excluded from any one or more claims for any reason, whether or not related to the existence of prior art.

[0214] It is to be understood that the words which have been used are words of description rather than limitation and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the present invention in its broader aspects.

[0215] While the invention has been described at some length and with a certain specificity with respect to several described embodiments, it is intended that the invention should not be limited to any such specific case or embodiment or any specific embodiment, but rather should be read with reference to the appended claims so as to provide the broadest possible interpretation of such claims in light of the prior art, and therefore, effectively encompass the intended scope encompassed by the invention.

Claims

1. A composition comprising at least two binding proteins, wherein i) at least one of the binding proteins is a T cell receptor (TCR) capable of binding to an immunogenic peptide derived from a target protein as an immunogenic peptide-MHC (pMHC) complex, and ii) at least one of the binding proteins is a TCR capable of binding to a different immunogenic peptide from the same target protein or a different target protein as in i) as a pMHC complex, optionally wherein the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more binding proteins.

2. The composition of claim 1, wherein the binding affinity of the TCR of i) and / or the TCR of ii) is less than or equal to about 5x10 -4 M of K d .

3. The composition of claim 1 or 2, wherein the MHC of i) and the MHC of ii) are the same or different.

4. The composition of any one of claims 1 to 3, wherein the binding protein of i) and / or ii) comprises: a) a T cell receptor (TCR) α chain CDR sequence that has at least about 80% identity to a TCR α chain CDR sequence selected from the group consisting of the TCR α chain CDR sequences listed in Table 1, Table 4 or Table 6; and / or b) a TCR β chain CDR sequence that has at least about 80% identity to a TCR β chain CDR sequence selected from the group consisting of the TCR β chain CDR sequences listed in Table 1, Table 4 or Table 6.

5. The composition of any one of claims 1 to 4, wherein the binding protein of i) and / or ii) comprises: a) a TCR V selected from those listed in Table 1, Table 4 or Table 6 α TCR V domain sequence group α The TCR α chain variable (V α ) domain sequence; and / or b) a TCR V selected from those listed in Table 1, Table 4 or Table 6 β TCR V domain sequence group β The TCR beta chain variable (V β ) domain sequence.

6. The composition of any one of claims 1 to 5, wherein the binding protein of i) and / or ii) comprises: a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 1, Table 4 or Table 6; and / or b) a TCR β chain sequence selected from the group consisting of the TCR β chain sequences listed in Table 1, Table 4 or Table 6.

7. The composition of any one of claims 1 to 6, wherein 1) the TCR α chain CDR, the TCR V α The TCRα chain is encoded by a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group consisting of TRAV, TRAJ and TRAC genes listed in Table 1, Table 2, Table 4 and / or Table 6, the TCRβ chain CDR, the TCR V β The structural domain and / or the TCRβ chain are encoded by TRBV, TRBJ and / or TRBC genes or fragments thereof selected from the group of TRBV, TRBJ and TRBC genes listed in Table 1, Table 2, Table 4 and / or Table 6, and each CDR of the binding protein has up to five amino acid substitutions, insertions, deletions or a combination thereof compared to the homologous reference CDR sequences listed in Table 1, Table 4 or Table 6.

8. The composition of any one of claims 1-7, wherein the immunogenic peptide comprises the amino acid sequence shown in Table 3.

9. The composition of any one of claims 1-8, wherein the binding protein is a chimeric, humanized or human binding protein.

10. A composition as described in any one of claims 1-9, wherein the binding protein is a TCR, an antigen binding fragment of a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or a fusion protein comprising a TCR and an effector domain, optionally wherein the binding domain comprises a transmembrane domain and an intracellular effector domain.

11. The composition of any one of claims 1-10, wherein the TCR alpha chain and the TCR beta chain are covalently linked, optionally wherein the TCR alpha chain and the TCR beta chain are covalently linked via a linker peptide.

12. The composition of any one of claims 1-11, wherein the TCR alpha chain and / or the TCR beta chain is covalently linked to a moiety, optionally wherein the covalently linked moiety comprises an affinity tag or label.

13. The composition of claim 12, wherein the affinity tag is selected from the group consisting of: a CD34 enrichment tag, glutathione-S-transferase (GST), calmodulin binding protein (CBP), a protein C tag, a Myc tag, a Halo tag, a HA tag, a Flag tag, a His tag, a biotin tag, and a V5 tag, and / or wherein the tag is a fluorescent protein.

14. The composition of any one of claims 1-13, wherein the covalently linked moiety is selected from the group consisting of an inflammatory agent, a cytokine, a toxin, a cytotoxic molecule, a radioisotope, or an antibody or antigen-binding fragment thereof.

15. The composition of any one of claims 1-14, wherein the binding protein binds to the pMHC complex on the surface of a cell.

16. The composition of any one of claims 1-15, wherein the MHC is an MHC multimer, optionally wherein the MHC multimer is a tetramer.

17. The composition of any one of claims 1-16, wherein the MHC is an MHC class I molecule.

18. The composition of any one of claims 1-17, wherein the MHC comprises an MHC alpha chain of HLA serotype HLA-A*02.

19. The composition of any one of claims 1-18, wherein the HLA allele is selected from the group consisting of: HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17, and HLA-C*18,Optionally, the HLA allele gene is selected from the group consisting of: HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele, HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 allele, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, HLA-B*0721, HLA-C*0702, HLA-C*0701, HLA-C*0401, HLA-C*0602, HLA-C*0304, HLA-C*0501, HLA-C*1601, HLA-C*0202, HLA-C*0303, HLA-C*1203, HLA-C*0802, HLA-C*0102, HLA-C*1701, HLA-C*1502, HLA-C*1402, HLA-C*1202, HLA-C*0704, HLA-C*0801, HLA-C*0302, HLA-C*1801, HLA-C*1505, HLA-C*1602, HLA-C*0804, HLA-C*0305, and HLA-C*1403 allele., 20. The composition of any one of claims 1-19, wherein binding of the composition to the pMHC complex elicits an immune response greater than that of either TCR alone, optionally wherein the immune response is a T cell response and / or a synergistic response.

21. The composition of any one of claims 1-20, wherein the T cell response is selected from the group consisting of: T cell expansion, cytokine release, and / or cytotoxic killing.

22. The composition of any one of claims 1-21, wherein the binding protein is capable of being expressed in an amount less than or equal to about 1 x 10 -4 M, less than or equal to about 5x10 -5 M, less than or equal to about 1x10 -5 M, less than or equal to about 5x10 -6 M, less than or equal to about 1x10 -6 M, less than or equal to about 5x10 -7 M, less than or equal to about 1x10 -7 M, less than or equal to about 5x10 -8 M, less than or equal to about 1x10 -8 M, less than or equal to about 5x10 -9 M, less than or equal to about 1x10 -9 M, less than or equal to about 5x10 -10 M, less than or equal to about 1x10 -10 M, less than or equal to about 5x10 -11 M, less than or equal to about 1x10 -11 M, less than or equal to about 5x10 -12 M or less than or equal to about 1x10 -12 M of K d Specifically binds to the immunogenic peptide-MHC (pMHC) complex.

23. The composition of any one of claims 1-22, wherein the binding protein has a higher binding affinity for the peptide-MHC (pMHC) than a known T cell receptor.

24. The composition of any one of claims 1-23, wherein the binding protein has at least 1.05-fold higher binding affinity for the peptide-MHC (pMHC) compared to a known T cell receptor.

25. The composition of any one of claims 1-24, wherein the binding protein induces greater T cell expansion, cytokine release and / or cytotoxic killing when contacted with a target cell having heterozygous expression of the target compared to known T cell receptors.

26. The composition of any one of claims 1-25, wherein the binding protein induces at least a 1.05-fold increase in T cell expansion, cytokine release, and / or cytotoxic killing when contacted with a target cell having heterozygous expression of the target compared to a known T cell receptor.

27. The composition of claim 25 or 26, wherein the target cell is a SK-MEL-5, DEL, THP-1 or TF-1 cell line.

28. The composition of claim 25 or 26, wherein the target cell is a cancer cell.

29. The composition of claim 28, wherein the cancer is a hematological malignancy.

30. The composition of claim 28, wherein the cancer is a solid tumor.

31. The composition of claim 30, wherein the cancer is melanoma, head and neck cancer, or lung cancer.

32. An isolated nucleic acid molecule that hybridizes under stringent conditions with the complement of a nucleic acid encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 1, Table 4 or Table 6, or a polypeptide composition as claimed in any one of claims 1-31, or with a sequence encoding a polypeptide selected from the group consisting of polypeptide sequences listed in Table 1, Table 4 or Table 6, or a polypeptide composition as claimed in any one of claims 1-31 having at least about 80% homology to a nucleic acid, optionally wherein the isolated nucleic acid molecule comprises 1) a TRAV, TRAJ and / or TRAC gene or a fragment thereof selected from the group of TRAV, TRAJ and TRAC genes listed in Table 1, Table 2, Table 4 and / or Table 6 and / or 2) a TRBV, TRBJ and / or TRBC gene or a fragment thereof selected from the group of TRBV, TRBJ and TRBC genes listed in Table 1, Table 2, Table 4 and / or Table 6.

33. The isolated nucleic acid of claim 32, wherein the nucleic acid is codon optimized for expression in a host cell.

34. A vector comprising the isolated nucleic acid of claim 32 or 33.

35. The vector of claim 34, wherein the vector is a cloning vector, an expression vector or a viral vector.

36. The vector of claim 34 or 35, wherein the vector further comprises a nucleic acid sequence encoding CD8α, CD8β, dominant negative TGFβ receptor II (DN-TGFβRII), and a selectable protein marker, optionally wherein the selectable protein marker is dihydrofolate reductase (DHFR).

37. The vector of any one of claims 34-36, wherein the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII and / or the selectable protein marker is operably linked to a nucleic acid encoding a tag.

38. The vector of any one of claims 34-37, wherein the nucleic acid encoding the tag is located 5' upstream of the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII and / or the selectable protein marker, such that the tag is fused to the N-terminus of CD8α, CD8β, the DN-TGFβRII and / or the selectable protein marker.

39. The vector of any one of claims 34-38, wherein the tag is a CD34 enrichment tag.

40. The vector of any one of claims 34-39, wherein the isolated nucleic acid of any preceding claim and the nucleic acid sequence encoding CD8α, CD8β, the DN-TGFβRII and / or the selectable protein marker are linked to an internal ribosome entry site or a nucleic acid sequence encoding a self-cleaving peptide.

41. The vector of any one of claims 34-40, wherein the self-cleaving peptide is P2A, E2A, F2A or T2A.

42. A host cell comprising an isolated nucleic acid as described in claim 32 or 33, comprising a vector as described in any one of claims 34-41, and / or expressing a binding protein composition as described in any one of claims 1-31, optionally wherein the cell is genetically engineered, and optionally wherein the host cell is a collection of host cells each comprising an isolated nucleic acid as described in claim 32 or 33, comprising a vector as described in any one of claims 34-41, and / or expressing a binding protein composition as described in any one of claims 1-31.

43. The host cell of claim 42, wherein the host cell comprises a chromosomal knockout of a TCR gene, an HLA gene, or both.

44. The host cell of claim 42 or 43, wherein the host cell comprises a knockout of an HLA gene selected from the group consisting of an α1 macroglobulin gene, an α2 macroglobulin gene, an α3 macroglobulin gene, a β1 microglobulin gene, a β2 microglobulin gene, and combinations thereof.

45. The host cell of any one of claims 42-44, wherein the host cell comprises a knockout of a TCR gene selected from the group consisting of a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and a combination thereof.

46. ​​The host cell of any one of claims 42-45, wherein the host cell expresses CD8α, CD8β, DN-TGFβRII and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR.

47. The host cell of claim 46, wherein the CD8α, the CD8β, the DN-TGFβRII and / or the selectable protein marker are fused to a CD34 enrichment tag.

48. The host cell of claim 47, wherein the host cells are enriched using the CD34 enrichment tag.

49. The host cell of any one of claims 42-48, wherein the host cell is a hematopoietic progenitor cell, a peripheral blood mononuclear cell (PBMC), an umbilical cord blood cell, or an immune cell.

50. The host cell of any one of claims 42-49, wherein the immune cell is a cytotoxic lymphocyte, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte progenitor cell, a cytotoxic lymphocyte stem cell, a CD4 + T cells, CD8 + T cells, CD4 / CD8 double negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells or a combination thereof.

51. The host cell of any one of claims 42-50, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof.

52. The host cell of any one of claims 42-51, wherein the T cell is a primary T cell or a cell of a T cell line.

53. A host cell as described in any one of claims 42-52, wherein the T cell does not express an endogenous TCR or has low surface expression of an endogenous TCR.

54. A host cell as described in any one of claims 42-53, wherein the host cell is capable of producing a cytokine or cytotoxic molecule when contacted with a target cell, the target cell comprising a peptide-MHC (pMHC) complex comprising an HA-2 peptide epitope in the context of an MHC molecule.

55. The host cell of claim 54, wherein the host cell is contacted with the target cell in vitro, ex vivo or in vivo.

56. The host cell of claim 54 or 55, wherein the cytokine is TNF-α, IL-2 and / or IFN-γ.

57. The host cell of any one of claims 54-56, wherein the cytotoxic molecule is perforin and / or a granzyme, optionally wherein the cytotoxic molecule is granzyme B.

58. The host cell of any one of claims 54-57, wherein the host cell is capable of producing higher levels of a cytokine or cytotoxic molecule when contacted with a target cell having heterozygous expression of the target.

59. The host cell of claim 58, wherein the host cell is capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules.

60. The host cell of any one of claims 54-59, wherein the host cell is capable of killing a target cell comprising a peptide-MHC (pMHC) complex comprising a target peptide epitope in the context of an MHC molecule.

61. The host cell of claim 60, wherein the killing is determined by a killing assay.

62. The host cell of claim 60 or 61, wherein the ratio of host cells to target cells in the killing assay is between 20:1 and 0.625:

1.

63. The host cell of any one of claims 60-62, wherein the target cell is a T2 cell pulsed with 1 μg / mL to 50 pg / mL of the immunogenic peptide.

64. The host cell of any one of claims 60-62, wherein the host cell is capable of killing a higher number of target cells when contacted with target cells having heterozygous expression of the target.

65. The host cell of claim 64, wherein the host cell is capable of killing at least 1.05 times more number of target cells.

66. The host cell of any one of claims 60, 61, 64 and 65, wherein the target cell is a SK-MEL-5, DEL, THP-1 or TF-1 cell line.

67. A host cell as described in any one of claims 54-66, wherein the immunogenic peptide comprises the amino acid sequence shown in Table 3.

68. The host cell of any one of claims 54-67, wherein the MHC molecule is an MHC class I molecule.

69. A host cell as described in any of claims 54-68, wherein the MHC molecule comprises an MHC alpha chain of HLA serotype HLA-A*02, HLA-A*03, HLA-A*01, HLA AA*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17 or HLA AC*18.

70. A host cell according to any one of claims 54 - 69, wherein the HLA allele is selected from the group consisting of: HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele, HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 allele, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, HLA-B*0721, HLA-C*0702, HLA-C*0701, HLA-C*0401, HLA-C*0602, HLA-C*0304, HLA-C*0501, HLA-C*1601, HLA-C*0202, HLA-C*0303, HLA-C*1203, HLA-C*0802, HLA-C*0102, HLA-C*1701, HLA-C*1502, HLA-C*1402, HLA-C*1202, HLA-C*0704, HLA-C*0801, HLA-C*0302, HLA-C*1801, HLA-C*1505, HLA-C*1602, HLA-C*0804, HLA-C*0305 and HLA-C*1403 allele.

71. The host cell of any one of claims 54-70, wherein the target cell is a cell line selected from the group consisting of SK-MEL-5, Del, THP-1 and TF-1 cell lines, or a cancer cell expressing the immunogenic peptide.

72. The host cell of claim 71, wherein the cancer cell is a hematological malignancy or a solid tumor.

73. A host cell as described in any one of claims 54-71, wherein the host cell does not express the immunogenic peptide, is not recognized by the binding protein as described in any one of claims 1-30, does not have a serotype listed in claim 69, and / or does not express the HLA allele gene listed in claim 70.

74. A host cell population, wherein the host cells are host cells as claimed in any one of claims 42-73.

75. A method for preventing and / or treating a non-malignant disorder, a hyperproliferative disorder or the recurrence of a hyperproliferative disorder characterized by expression of an immunogenic peptide antigen in a subject, the method comprising administering to the subject a therapeutically effective amount of a combination of a composition according to any one of claims 1-74 or a combination of a binding protein, nucleic acid, vector and / or host cell according to any one of claims 1-74.

76. The method of claim 75, wherein the composition comprises cells, optionally wherein the cells are allogeneic cells, syngeneic cells, or autologous cells.

77. The method of claim 75 or 76, wherein the cell is genetically modified.

78. The method of any one of claims 75-77, wherein the cell comprises a chromosomal knockout of a TCR gene, an HLA gene, or both a TCR gene and an HLA gene.

79. The method of any one of claims 75-78, wherein the cells comprise a knockout of an HLA gene selected from the group consisting of: alpha 1 macroglobulin gene, alpha 2 macroglobulin gene, alpha 3 macroglobulin gene, beta 1 microglobulin gene, beta 2 microglobulin gene, and combinations thereof.

80. The method of any one of claims 75-79, wherein the cell comprises a knockout of a TCR gene selected from the group consisting of a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, and combinations thereof.

81. The method of any one of claims 75-80, wherein the cells express CD8α, CD8β, DN-TGFβRII and / or a selectable protein marker, optionally wherein the selectable protein marker is DHFR and further optionally wherein the CD8α, the CD8β, the DN-TGFβRII and / or the selectable protein marker are fused to a CD34 enrichment tag.

82. The method of claim 81, wherein cells are enriched using the CD34 enrichment tag.

83. The method of any one of claims 75-82, wherein the cell is a hematopoietic progenitor cell, a peripheral blood mononuclear cell (PBMC), an umbilical cord blood cell, or an immune cell.

84. The method of any one of claims 75-83, wherein the immune cell is a cytotoxic lymphocyte, a cytotoxic lymphocyte precursor cell, a cytotoxic lymphocyte progenitor cell, a cytotoxic lymphocyte stem cell, a CD4 + T cells, CD8 + T cells, CD4 / CD8 double negative T cells, gamma delta (γδ) T cells, natural killer (NK) cells, NK-T cells, dendritic cells or a combination thereof.

85. The method of any one of claims 75-84, wherein the T cells are naive T cells, central memory T cells, effector memory T cells, or a combination thereof.

86. The method of any one of claims 75-85, wherein the T cell is a primary T cell or a cell of a T cell line.

87. The method of any one of claims 75-86, wherein the T cells do not express endogenous TCRs or have low surface expression of endogenous TCRs.

88. The method of any one of claims 75-87, wherein the cell is capable of producing a cytokine or cytotoxic molecule when contacted with a target cell comprising a peptide-MHC (pMHC) complex comprising the immunogenic peptide in the context of an MHC molecule.

89. The method of any one of claims 75-89, wherein the cytokine is TNF-α, IL-2 and / or IFN-γ.

90. The method of any one of claims 75-90, wherein the cytotoxic molecule is perforin and / or a granzyme, optionally wherein the cytotoxic molecule is granzyme B.

91. The method of any one of claims 75-90, wherein the cell is capable of producing higher levels of a cytokine or cytotoxic molecule when contacted with a target cell having heterozygous expression of the target.

92. The method of claim 91, wherein the cells are capable of producing at least 1.05-fold higher levels of cytokines or cytotoxic molecules.

93. The method of any one of claims 75-92, wherein the host cell is capable of killing a target cell comprising a peptide-MHC (pMHC) complex comprising the target in the context of an MHC molecule.

94. The method of any one of claims 75-93, wherein the host cell is capable of killing a higher number of target cells when contacted with target cells having heterozygous expression of the target.

95. The method of claim 94, wherein the host cell is capable of killing at least 1.05 times more number of target cells.

96. The method of any one of claims 75-95, wherein the immunogenic peptide comprises the amino acid sequence shown in Table 3.

97. The method of any one of claims 92-96, wherein the MHC molecule is an MHC class I molecule.

98. The method of any one of claims 75-97, wherein the MHC molecule comprises an MHC α chain of an HLA serotype HLA-A*02, HLA-A*03, HLA-A*01, HLA-A*11, HLA-A*24, HLA-B*07, HLA-C*07, HLA-C*01, HLA-C*02, HLA-C*03, HLA-C*04, HLA-C*05, HLA-C*06, HLA-C*08, HLA-C*12, HLA-C*14, HLA-C*15, HLA-C*16, HLA-C*17 or HLA-C*18.

99. The method according to any one of claims 75 - 98, wherein the HLA allele pair is selected from the group consisting of: HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0204, HLA-A*0205, HLA-A*0206, HLA-A*0207, HLA-A*0210, HLA-A*0211, HLA-A*0212, HLA-A*0213, HLA-A*0214, HLA-A*0216, HLA-A*0217, HLA-A*0219, HLA-A*0220, HLA-A*0222, HLA-A*0224, HLA-A*0230, HLA-A*0242, HLA-A*0253, HLA-A*0260, HLA-A*0274 allele pair, HLA-A*0301, HLA-A*0302, HLA-A*0305, HLA-A*0307, HLA-A*0101, HLA-A*0102, HLA-A*0103, HLA-A*0116 allele pair, HLA-A*1101, HLA-A*1102, HLA-A*1103, HLA-A*1104, HLA-A*1105, HLA-A*1119 allele pair, HLA-A*2402, HLA-A*2403, HLA-A*2405, HLA-A*2407, HLA-A*2408, HLA-A*2410, HLA-A*2414, HLA-A*2417, HLA-A*2420, HLA-A*2422, HLA-A*2425, HLA-A*2426, HLA-A*2458 allele pair, HLA-B*0702, HLA-B*0704, HLA-B*0705, HLA-B*0709, HLA-B*0710, HLA-B*0715, HLA-B*0721, HLA-C*0702, HLA-C*0701, HLA-C*0401, HLA-C*0602, HLA-C*0304, HLA-C*0501, HLA-C*1601, HLA-C*0202, HLA-C*0303, HLA-C*1203, HLA-C*0802, HLA-C*0102, HLA-C*1701, HLA-C*1502, HLA-C*1402, HLA-C*1202, HLA-C*0704, HLA-C*0801, HLA-C*0302, HLA-C*1801, HLA-C*1505, HLA-C*1602, HLA-C*0804, HLA-C*0305, and HLA-C*1403 allele pair.

100. The method of any one of claims 75-99, wherein the target cell is a non-malignant cell or a hyperproliferative cell that expresses the antigen in the subject.

101. The method of any one of claims 75-100, wherein the composition further comprises a pharmaceutically acceptable carrier.

102. The method of any one of claims 75-101, wherein the composition induces an immune response against the non-malignant cells or the hyperproliferative cells in the subject expressing the antigen that is greater than any one TCR alone, optionally wherein the immune response is a synergistic response.

103. The method of any one of claims 75-102, wherein the composition induces an antigen-specific T cell immune response against the non-malignant cells or the hyperproliferative cells in the subject that express the antigen.

104. The method of any one of claims 75-103, wherein the antigen-specific T cell immune response comprises CD4 + At least one of a helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response.

105. The method of any one of claims 75-104, wherein the hyperproliferative disorder comprises a hematological malignancy or a solid tumor.

106. The method of any one of claims 75-104, wherein the non-malignant disorder is an autoimmune disorder, optionally wherein the autoimmune disorder is systemic sclerosis or multiple sclerosis.

107. A method as described in any of claims 75-106, wherein the subject is undergoing or has previously undergone hematopoietic cell transplantation (HCT), optionally wherein the HCT comprises cells that do not express the immunogenic antigen, are not recognized by the binding protein as described in any of claims 1-31, do not have the serotype listed in claim 98 and / or do not express the HLA alleles listed in claim 99.

108. The method of claim 107, wherein the HCT comprises donor hematopoietic cells comprising a chromosomal knockout of genes encoding HLA components, a chromosomal knockout of genes encoding TCR components, or both.

109. The method of any one of claims 75-108, further comprising administering to the subject at least one additional treatment for the non-malignant disorder, the hyperproliferative disorder, or a recurrence of the hyperproliferative disorder.

110. The method of any one of claims 75-109, wherein the at least one other treatment for the non-malignant disorder, the hyperproliferative disorder, or a recurrence of the hyperproliferative disorder is administered concurrently or sequentially with the composition.

111. The method of any one of claims 75-110, wherein the subject is an animal model for a disorder characterized by expression of an immunogenic antigen and / or a mammal, optionally wherein the mammal is a human, a primate, or a rodent.

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