Multiplexed T cell receptor compositions, combination therapies thereof, and uses

Abstract

Provided herein are multiplexed T cell receptor compositions, combination therapies thereof, and uses. The present invention is, for example, 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 derived from the same target protein or a target protein different from i) as a pMHC complex, and optionally, 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, providing the composition.

Description

Technical Field

[0001] Cross - Reference to Related Applications

Background Art

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

Summary of the Invention

Means for Solving the Problems

[0003] The present invention is at least partially based on the discovery of certain binding proteins that include a T cell receptor (TCR), which, when combined, recognize multiple antigens (e.g., multiple antigens on the same target and / or multiple antigens on different targets), and engineered cells containing the same can overcome antigen heterogeneity and / or loss of human leukocyte antigen (HLA) heterozygosity to treat cancers including solid tumors. For example, adoptive cell transfer (ACT) using genetically engineered T cells has great promise for the treatment of cancers such as solid tumors, but complete responses are rare because only one antigen is targeted at a time, and it is often short-lived due to the heterogeneous expression of cancer-related antigens and loss of HLA heterozygosity. Multiplexed T cell receptor-T cell (TCR-T) therapies that span multiple target antigens and / or HLA molecules mimic the natural oligoclonal T cell response 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 to target a mixed tumor cell culture with heterogeneous antigen expression. The presence of one TCR-T / target cell pair enhanced the activity of the other TCR-T against its target. This effect was mediated through secreted soluble factors. These results suggest that the use of multiplexed T cell receptors and related compositions (e.g., multiplexed TCR-T) can overcome antigen heterogeneity not only through independent targeting of different target cells within the same tumor but also through cytokine-mediated enhancement of the response of each T cell. Surprisingly, these results further demonstrate an unexpected synergistic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0004]

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Mode for Carrying Out the Invention

[0005] The present invention is based, at least in part, on the discovery of certain binding proteins that include a T cell receptor (TCR), which, when combined, recognize multiple antigens (e.g., multiple antigens on the same target and / or multiple antigens on different targets), and engineered cells containing the same can overcome antigen heterogeneity and / or loss of heterozygosity of human leukocyte antigen (HLA) to treat cancers, including solid tumors.

[0006] Accordingly, the present invention relates in part to a specific binding protein (e.g., TCR), a host cell expressing the binding protein (e.g., TCR), a composition comprising the binding protein (e.g., TCR), and a method for diagnosing, prognosticating, and monitoring the T cell response to a cell expressing an antigen and / or target of interest, as well as administering two or more binding proteins directly or administering a composition providing the same, e.g., a single composition comprising two or more binding proteins, a single composition comprising a nucleic acid, and / or a vector encoding two or more binding proteins (e.g., TCR), for preventing and / or treating a non-malignant disorder, a hyperproliferative disorder, or recurrence of a hyperproliferative disorder characterized by the expression of an antigen and / or target of interest, a single composition comprising a host cell type expressing two or more binding proteins (e.g., TCR), 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 a nucleic acid and / or a vector encoding at least one binding protein (e.g., TCR), 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 by a single composition or a combination of compositions, either simultaneously or sequentially. The 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 therebetween (including both ends), e.g., 2 to 5 binding proteins, 2 to 4 binding proteins, 2 to 3 binding proteins, etc. In some embodiments, suitable subjects are selected using an active step of expression analysis of a target gene, loss of HLA heterozygosity (LOH), and / or HLA typing to determine compatibility with TCR binding to a desired MHC:peptide (pHC) complex and the expected therapeutic effect. Numerous representative non-limiting combinations are exemplified herein, and any combination of any of the agents described herein is contemplated as a composition and its use and is encompassed by the present invention.

[0007] Furthermore, as further described below and in the examples, host cells encompassed by the present invention can encode and / or express accessory proteins useful in any of the same polynucleotides or different polynucleotides or components thereof as the binding protein, in addition to the binding proteins described herein. For example, the host cell can encode and / or express TCRα, TCRβ, CD8α, CD8β, DN-TGFβR (e.g., DN-TGFβRII), and / or a selectable protein marker (optionally, this selectable protein marker is DHFR).

[0008] The term "dominant negative TGFβ receptor" or "DN-TGFβR" refers to a variant or mutant of the transforming growth factor (TGF) beta receptor that confers resistance to transforming growth factor (TGFβ) signaling. There are five type II receptors (activating receptors) and seven type I receptors (signaling 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, this DN-TGFβR is DN-TGFβRII (i.e., a variant or mutant of TGF beta receptor II). In some embodiments, the resistance is to the inhibitory effect of TGFβ signaling on immune cells such as T cells, and the TGFβ can be produced by cancer cells or other immune cells in the cellular environment, for example, 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 captures the receptor and thereby inhibits signaling, antibodies that bind to TGFβ and / or TGFβ receptors (e.g., rerdelimumab, metelimumab, fresolimumab, etc.), soluble TGFβ binding proteins such as portions of TGFβ receptors that capture TGFβ (e.g., TGFβRII-Fc fusion protein) or other binding factors, such as beta-glycan. Instead of, or in addition to, the DN-TGFβR (e.g., DN-TGFβRII) described herein, any and all known TGFβ signaling inhibitors may be used. 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 representative non-limiting examples of Brand et al. (1993) J. Biol. Chem. 268:11500-11503; Weiser et al. (1993) Mol. Cell Biol. 13:7239-7247; Bolard et al. (2002) Blood 99::3179-3187; PCT Publication WO2009 / 152610; PCT Publication WO2017 / 156484; Kloss et al. (2018) Mol. Ther. 26:1855-1866; PCT Publication WO.2019 / 089884; PCT Publication WO2020 / 042647; and PCT Publication WO2020 / 042648).

Example

[0009] Example 1: Materials and Methods of Example 2 a. Multiplexing of HPV and MAGE-A1 TCRs (i) HPV16-E7 11-19 Specific or MAGE-A1 290-297 Engineer T cells to express the TCRs

[0010] Primary CD3+ T cells were isolated from Leukopak according to the manufacturer's protocol using the StraightFrom® Leukopak® CD3 MicroBead Kit (Miltenyi Biotec). The isolated cells were frozen in CryoStor® CS10 (Stem Cell Technologies) and stored in liquid nitrogen until use. On day 1, the CD3+ 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, the CD3+ T cells were washed and resuspended in fresh T cell medium and activated using ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (5 μL / 1×10 6 CD3+ T cells, Stem Cell Technologies). On day 1, the cells were washed and resuspended in fresh complete T cell medium and plated at 1×10 6 cells per well. Triplicate wells were transduced with lentiviral particles to express either HPV or MAGE-A1 TCR. On day 2, the cells were washed, the triplicates were combined and resuspended in fresh complete T cell medium, and grown in 1 well of a G-Rex® 6-well plate (Wilson Wolf) until day 5. On day 5, the cells were harvested and the cell concentration was adjusted to 100×10 6Adjusted to CD3+ T cells / mL, washed with EASYSep buffer, and separated using a QuadroMACS® Separator and an LS column (Miltenyi). The isolated cells were washed and resuspended in fresh complete T cell medium and grown in G-Rex® 10 flasks (Wilson Wolf) until day 12, at which point the cells were frozen in CryoStor® CS10 and stored in liquid nitrogen until use. b. Multiplexing of MAGE-C2 and MAGE-A1 TCRs (i) MAGE-C2 184-192 or MAGE-A1 290-297 Engineer T cells to express the TCR

[0011] Primary CD8+ T cells were isolated using the StraightFrom® Leukopak® CD8 MicroBead Kit (Miltenyi Biotec) according to the manufacturer's protocol. The isolated cells were frozen in CryoStor® CS10 (Stem Cell Technologies) and stored in liquid nitrogen until use. On day 1, the 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, the CD8+ T cells were washed and resuspended in fresh T cell medium and activated using ImmunoCult™ Human CD3 / CD28 / CD2 T Cell Activator (5 μL / 1×10 6 CD8+ T cells, Stem Cell Technologies). On day 1, the cells were washed and resuspended in fresh complete T cell medium and seeded at 1×10 per well in 96-well plates. 6Plated with cells. Each well was transduced with lentiviral particles to express MAGE-C2 or MAGE-A1 in triplicate, or maintained as a non-transduced donor control. On day 2, the cells were washed, the triplicates were combined, resuspended in fresh complete T cell medium, and grown in a G-Rex® 6-well plate (Wilson Wolf) until day 5. On day 5, the cells were harvested, the cell concentration was adjusted to 100×10 6 CD8+ T cells / mL in MACS® running buffer (Miltenyi), anti-mTCR biotin antibody (BioLegend) was added at a 1:50 dilution for 10 minutes at room temperature, and then washed with MACS® running buffer. Anti-biotin microbeads (Miltenyi) were added at a 1:5 dilution and incubated for 10 minutes at room temperature. The cells were washed with MACS® running buffer and resuspended in MACS® running buffer for manual magnetic separation using a QuadroMACS® Separator and LS column (Miltenyi). The isolated cells were washed and resuspended in fresh complete T cell medium and grown in a G-Rex® 10 flask (Wilson Wolf) until day 12, at which point the cells were frozen in CryoStor® CS10 and stored in liquid nitrogen until use.

[0012] (ii) Cell line The epidermal cancer 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 American Type Culture (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].

[0013] (iii) Generation of stable cell lines expressing Incucyte® Nuclight Red CaSki, A101D, and SK-MEL-5 cells were transduced with Incucyte® NucLight Red Lentivirus Reagent (EF-1α promoter, puromycin selection) (Sartorius). Twenty-four hours after transduction, the cells were washed and resuspended in their respective cell line media and cultured at 37°C, 5% CO 2 . Two to three days after transduction, puromycin (Gibco, Waltham, MA) was added to the cultures at a predetermined concentration (in the range of 0.5 μg / mL to 1 μg / mL) to select the transduced cells. The cultures were grown under puromycin selection until at least 90% of the cells were positive for Incucyte® Nuclight Red as determined by flow cytometry analysis.

[0014] (iv) In vitro cytotoxicity assay The in vitro cytotoxicity assay was performed in a 96-well flat-bottom tissue culture plate not coated with poly-L-ornithine. Here, adherent cells were plated and allowed to attach one day prior to the addition of T cells. When indicated, T cells were co-cultured with the indicated E:T ratio with Incucyte® Nuclight Red-expressing CaSki, A101D, or SK-MEL-5 cells. Data was acquired with an Incucyte® S3 instrument (Sartorius), and target cell proliferation was quantified with the Incucyte® S3 as a readout of T cell cytotoxicity.

[0015] (v) Transwell T cell activation assay A Corning® HTS Transwell®-96 permeable support with a 1.0 μm pore polycarbonate membrane insert (Sigma-Aldrich #CLS3392) was used according to the manufacturer's instructions. A101D melanoma cells were seeded in the upper chamber, and SK-MEL-5 melanoma cells were seeded in the lower chamber, and both strains were allowed to adhere overnight. The next day, CD8+ T cells engineered with MAGEA1 TCR were co-cultured with A101D cells in the upper chamber, while CD8+ T cells engineered with MAGEC2 TCR were co-cultured with SK-MEL-5 cells in the lower chamber at a 1:2 E:T ratio and incubated at 37 °C for 48 h in 5% CO 2 After incubation, cells were harvested 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 analyzed for CD137 and CD69 double-positive cells with a CytoFLEX flow cytometer (Beckman Coulter).

[0016] Example 2: Examples of representative non-limiting combination therapies Adoptive cell transfer using genetically engineered T cells holds great promise for the treatment of solid tumors. So far, in clinical trials of T cell therapy by TCR engineering, one antigen has been targeted at a time, generating encouraging response rates in the range of 30-50%. Unfortunately, complete responses are rare, and responses are mostly short-lived. There are thought to be two main challenges associated with TCR-T cell therapy targeting a single antigen.

[0017] First, the expression of most cancer-related antigens is heterogeneous. In one representative and non-limiting example, multiplex immunohistochemistry was performed using two cancer germline antigens, MAGE-C2 and PRAME, and significant heterogeneity was observed across samples from different solid tumor types (Figures 1A-1C). Furthermore, heterogeneous antigen expression was observed at the single cell level - not all cancer cells within a given tumor express each antigen (Figures 1A-1C). This indicates that a single TCR may not be sufficient to eliminate all cancer cells within a given tumor, thereby allowing tumor cells lacking the treated antigen to escape and promoting recurrence.

[0018] Second, single-agent TCR-T cell therapy targets only a single HLA allele, which is affected by the loss via the commonly observed mechanism of HLA heterozygosity loss (LOH) (Figures 2A and 2B). 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 higher percentage of tumors.

[0019] Multiplex TCR-T cell therapy mimics the natural oligoclonal T cell response to cancer and provides a way to address both challenges associated with the treatment of solid tumors.

[0020] Using its proprietary ReceptorScan and TargetScan platforms, various TCRs, such as HPV16 E7-specific, MAGEA1-specific, and MAGEC2-specific TCRs, have been discovered for TCR-T cell therapy.

[0021] In representative non-limiting examples, two lead TCRs (MAGE-A1 and HPV), a low-affinity TCR (MAGE-C2), and target cell lines expressing their cognate antigens were multiplexed using direct and indirect co-culture experiments to evaluate the potential synergistic effects of using multiple TCRs to target tumors, as well as to understand the biological mechanisms underlying such synergistic effects. The materials and methods, as well as the results, are shown in FIGS. 1A - 5B. Briefly, using two different TCR:antigen pairs, multiplexed T cell-mediated cancer killing and heterogeneity were modeled in vitro. Additionally, the vector used to generate the data shown in FIG. 3 expresses the CD8 co-receptor, while the vector used to generate the data shown in FIG. 4 has the murine TCR constant region but does not express the CD8 co-receptor.

[0022] In one representative case, the multiplexing of two high-affinity TCR-Ts was tested, namely, one named TCR E7-11-28 (also named 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 named TCR32-41, TCR-204-C7, and TCR-204-C0702, see Table 1) targeting the HLA-C*07:02-restricted epitope of MAGE-A1. 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. The CaSki cervical cancer cells are A*02:01+ and HPV+. The A101D melanoma cells are C*07:02+ and MAGE-A1+. Both cell lines were engineered to express Incucyte® NucLight Red and mixed together to mimic tumor heterogeneity. Engineered T cells or non-engineered donor control T cells (control TCR-T) were co-cultured with the Incucyte® NucLight Red-labeled target cell lines at the indicated effector cell to target cell (E:T) ratios, and their viability was quantified with IncuCyte® as a readout of T cell cytotoxicity. Individual TCR-Ts caused approximately 50 - 60% cell killing at 72 hours, but a 1:1 mixture of the two TCR-Ts resulted in approximately 80% cell killing at the same overall effector to target (E:T) ratio, showing a synergistic effect (Figures 3A and 3B).

[0023] In another representative case, multiplexing of a high-affinity TCR-T for MAGE-A1 and a low-affinity TCR-T for MAGE-C2 was tested. TCR-204-C07 (also known as TCR32-41, TCR-204-C7, and TCR-204-C0702; see Table 1) is a high-affinity TCR of natural origin that recognizes the HLA-C*07:02-restricted epitope of MAGEA1 and exhibits potent 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 TCR8-3; see Table 1) (Figure 4A). CD8+ T cells were transduced using a lentiviral vector encoding an HM codon-optimized TCR for better expression. The transduced cells were selected based on the expression of the relevant TCR (MAGE-A1 or MAGE-C2). The target cells were a mixture of MAGE-A1-expressing cells or MAGE-C2-expressing cells. A2058 and SK-MEL-5 cells are both melanoma cell lines that are C*07:02+ and B*07:02+, but A2058 cells only highly express MAGE-A1, and SK-MEL-5 cells only moderately express MAGE-C2. It was previously found that MAGE-C2 TCR-T cells effectively kill A101D cells that highly express MAGEC2, but are not effective in killing SK-MEL-5 cells with a low expression level of MAGEC2. Co-culture experiments were performed to determine whether the cytotoxic activity of MAGE-C2 TCR-T cells could be enhanced when multiplexed with a more cytotoxic TCR. SK-MEL-5 cells engineered to express IncuCyte® NucLight Red were mixed with unlabeled A2058 cells, such that the activity quantified by IncuCyte® reflects only the cytotoxicity against SK-MEL-5 cells. Engineered T cells or non-engineered donor control T cells (non-transduced T cells) were co-cultured with the target cell line at the indicated effector cell to target cell (E:T) ratio, and their survival was quantified by IncuCyte® as a readout of T cell cytotoxicity.Low-level killing of SK-MEL-5 was observed by single MAGE-C2 T cells, while combining MAGE-C2 T cells and MAGE-A1 T cells synergistically enhanced the cytotoxic activity of MAGE-C2 TCR. Therefore, although partial killing of MAGE-C2 positive cells was shown by MAGE-C2 TCR-T alone, the addition of MAGE-A1 TCR-T synergistically enhanced the activity of MAGE-C2 TCR-T (Figure 4B).

[0024] To investigate the mechanism of this synergistic activity, the Corning® HTS Transwell® system was used. The HTS Transwell-96 1.0 μm pore polyester membrane transwell system with a permeable support was selected to allow the diffusion of soluble factors rather than cells between two cell compartments (upper chamber and lower chamber). A101D melanoma cells were seeded in the upper chamber and SK-MEL-5 melanoma cells were seeded in the lower chamber, and both strains were allowed to adhere overnight. The next day, CD8+ T cells engineered with MAGEA1 TCR were co-cultured with A101D cells in the upper chamber, while CD8+ T cells engineered with MAGEC2 TCR were co-cultured with SK-MEL-5 cells in the lower chamber at a 1:2 E:T ratio. After 48 hours, cells were harvested from either chamber 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 analyzed for CD137 and CD69 double-positive cells using a CytoFLEX flow cytometer (Beckman Coulter). Using the transwell culture system, it was found that cytokines secreted by MAGE-A1 TCR-T potently enhanced the T cell activation of MAGE-C2 TCR-T cells upon antigen binding (Figure 4C). These findings indicate that multiplexed TCR-T can overcome antigen heterogeneity not only by independent targeting of different cancer cell populations but also by cytokine-mediated T cell enhancement. Surprisingly, these results demonstrate an unexpected synergistic effect.

[0025] These results have been clinically applied. For example, to address the heterogeneity of solid tumors in the clinic, an exemplary screening strategy was designed to examine patients' tumors for antigen positivity and HLA LOH (Figure 5A). Furthermore, an ImmunoBank of therapeutic TCRs that recognize different targets presented by different HLA alleles. Selecting multiplexed TCR-Ts that target intact antigens and HLA alleles in patient tumors is thought to synergistically overcome the heterogeneity of solid tumors. Additional in vivo studies have been conducted to further confirm the synergistic effect of multiplexed TCR-T cell therapy, and clinical trials have been designed to further confirm the synergistic effect clinically.

[0026] Accordingly, this example provides compositions and methods useful for multiplexed TCR-T cell therapy, including combinations of anti-MAGE-A1 and anti-HPV TCRs, or combinations of anti-MAGE-A1 and anti-MAGE-C2 TCRs, and engineered cells expressing the same. While not wishing to be bound by any particular scientific theory, this example further includes that multiplexed TCR-T cell therapy mimics the natural oligoclonal T cell response to cancer. Multiplexed TCR-T cell therapy, such as the combinations described above, provides methods and compositions for addressing the particular challenges associated with the treatment of solid tumors.

[0027] To confirm the utility of multiplexed TCR-T cell therapy, such as the combinations described above, various assays can be used. In a representative non-limiting example, combinations of anti-MAGE-A1 and anti-HPV TCRs, or combinations of anti-MAGE-A1 and anti-MAGE-C2 TCRs, and one or more target cell lines expressing their cognate antigens are multiplexed using direct and indirect co-culture experiments to evaluate the potential synergistic effect of using multiple TCRs to target tumors and to understand the biological mechanisms underlying such synergistic effects. Using this assay, multiplexed T cell-mediated cancer killing by the combination of TCRs of interest and multiplexed TCR-T cell therapy, including heterogeneity, can be modeled in vitro.

[0028] In one representative case, the multiplexing of (i) an anti-MAGE-A1 TCR targeting the HLA-C*07 serotype-restricted epitope of MAGE-A1 (Table 3A), and (ii) an anti-HPV16 E7 TCR targeting the HLA-C*02 serotype-restricted epitope of HPV16 E7 (Table 3C) can be used and / or tested, for example, by using engineered cells expressing such TCRs.

[0029] In another representative case, the multiplexing of (i) an anti-MAGE-A1 TCR targeting the HLA-C*07 serotype-restricted epitope of MAGE-A1 (Table 3A) and (ii) an anti-MAGE-C3 TCR targeting the HLA-C*07 serotype-restricted epitope of MAGE-C3 (Table 3B) can be used and / or tested, for example, by using engineered cells expressing such TCRs.

[0030] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention, either alone or in any combination thereof, has 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 to a TCR alpha chain sequence selected from the group consisting of the TCR alpha sequences listed in the tables provided herein, and / or b) 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 to a TCR beta chain sequence selected from the group consisting of the TCR beta sequences listed in the tables provided herein, and may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) such TCR alpha and / or beta chain sequences.

[0031] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be, either alone or in any combination thereof: a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in the table provided herein; and / or, b) a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences described in the table provided herein, and include (e.g., include, consist essentially of, or consist of) a TCR.

[0032] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be, either alone or in any combination thereof: a) a TCR alpha 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 more identity to a TCR alpha chain variable (V α ) domain sequence selected from the group consisting of the TCR V α ) domain sequences listed in the table provided herein; and / or b) a TCR beta 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 more identity to a TCR beta chain variable (V β ) domain sequence selected from the group consisting of the TCR V β ) domain sequences listed in the table provided herein, and include (e.g., include, consist essentially of, or consist of) a TCR.

[0033] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention, either alone or in any combination thereof, :a) a TCR alpha chain variable (V α ) domain sequence selected from the group consisting of the TCR V α domain sequences provided in the table herein; and / or b) a TCR beta chain variable (V β ) domain sequence selected from the group consisting of the TCR V β domain sequences described in the table provided herein, and may be a TCR (e.g., comprising, consisting essentially of, or consisting of).

[0034] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention, either alone or in any combination thereof, has 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 to a TCR alpha chain complementarity determining region (CDR) sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in the table provided herein, and may be a TCR comprising at least one (e.g., 1, 2, or 3, e.g., CDR alone or in combination with CDR1 and CDR2) (e.g., comprising, consisting essentially of, or consisting of). CDR3 is considered the major CDR involved in the recognition of processed antigen, and since CDR1 and CDR2 mainly interact with MHC, in some embodiments, a binding protein comprising only CDR3 from the TCR alpha chain and / or only CDR3 from the TCR beta (each CDR3 having the sequence homology described in this example) is provided.

[0035] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be a TCR that includes (e.g., consists of, consists essentially of, or comprises) a TCR beta-chain complementarity determining region (CDR) 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 more identity to a TCR beta-chain CDR sequence selected from the group consisting of the TCR beta-chain sequences listed in the table provided herein, alone or in any combination thereof. As noted above, CDR3 is thought to be the major CDR involved in the recognition of processed antigen, and CDR1 and CDR2 primarily interact with MHC, so in some embodiments, binding proteins are provided that include only CDR3 from the TCR beta-chain and / or only CDR3 from the TCR alpha-chain (each CDR3 having the sequence homology described in this example) listed in the table provided herein.

[0036] The TCRs encompassed by the present invention, the anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR (alone or in any combination thereof), may be a TCR that includes (e.g., consists of, consists essentially of, or comprises) at least one (e.g., one, two, or three) TCR alpha-chain complementarity determining regions (CDRs) described in the table provided herein.

[0037] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be a TCR that includes (e.g., consists of, consists essentially of, or comprises) the complementarity determining region (CDR) of at least one (e.g., one, two, or three) TCR beta chains listed in the table provided herein, either alone or in any combination thereof.

[0038] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be a TCR that includes (e.g., consists of, consists essentially of, or comprises) the constant region (C α ) sequence of a TCR alpha chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR Cα sequences listed in the table provided by the present invention, either alone or in any combination thereof.

[0039] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be a TCR that includes (e.g., consists of, consists essentially of, or comprises) the constant region (C β ) sequence of a TCR beta chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR C β ) sequences listed in the table provided herein, either alone or in any combination thereof.

[0040] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be TCRs that are either alone or in any combination thereof and that comprise (e.g., consist of, consist essentially of, or consist of) a TCR alpha chain constant region (C α ) selected from the group consisting of the TCR C α sequences provided in the table herein.

[0041] The anti-MAGE-A1 TCR, anti-HPV TCR, anti-MAGE-C2 TCR, and / or anti-PRAME TCR encompassed by the present invention may be TCRs that are either alone or in any combination thereof and that comprise (e.g., consist of, consist essentially of, or consist of) a TCR beta chain constant region (C β ) sequence selected from the group consisting of the TCR C β sequences provided in the table herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

[0042] Table 1 is grouped according to the presentation of MHC serotypes and partially shows representative TCR sequences that are grouped according to the different peptides presented by the MHC serotypes and to which the subgrouped TCRs bind. Individual TCRs such as those typically exemplified in the table, as well as the genus of binding proteins that bind to the peptide epitope sequences described herein, either alone or in complex with MHC, such as those grouped in the tables provided herein, are described and claimed. Further provided are the TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein. The sequences of each TCR described herein are provided as pairs of homologous alpha and beta chains for each designated TCR. The TCR sequences described herein are annotated. Variable domain sequences are represented in capital letters. Constant domain sequences are in lower case. CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined strings. CDR1, CDR2, and CDR3 are shown in their standard order of appearance from left (N-terminus) to right (C-terminus). The TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein, are annotated according to the well-known IMGT nomenclature described herein. Similarly, CDR1 and CDR2 of TRAV and TRBV are well-known in the art as they are based on annotated TRAV and TRBV sequences (e.g., annotated in databases such as IMGT available at imt.org and IEDB available at iedb.org).

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

[0043] *For the vectors in Table 2, the MSCV promoter is in bold. The beta chain is annotated using bold and italic strings. The alpha chain is annotated using bold and underlined strings. The CD34 enrichment tag (e.g., Q tag) is annotated using italic and underlined strings. CD8-alpha is italic. CD8-beta is underlined.

[0044] In Tables 1 and 2 are included RNA nucleic acid molecules (e.g., where thymidine is replaced by uridine), nucleic acid molecules encoding orthologs of the encoded proteins, and nucleic acid sequences of any of the sequences listed in Table 1 or 2, or a part thereof, and DNA or RNA nucleic acid sequences 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 more identity over their full length. Such nucleic acid molecules can have the function of the full-length nucleic acid as further described herein.

Table 3A

Table 3B

Table 3C

Table 3D

[0045] In Table 3, for example, Tables 3A, 3B, 3C, and 3D are included peptide epitopes, and polypeptide molecules containing amino acid sequences of any of the sequences listed in Table 3, for example, Tables 3A, 3B, 3C, and 3D, and amino acid sequences 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 more identity over their full length. Such polypeptides can have the function of the full-length peptide or polypeptide as further described herein.

[0046] Example 3: Examples of Representative Non-Limiting Combination Therapies This example provides compositions and methods useful for multiplexed TCR-T cell therapy, including anti-MAGE-A1 TCR and anti-PRAME TCR. Without wishing to be bound by any particular scientific theory, this example further includes that multiplexed TCR-T cell therapy mimics the natural oligoclonal T cell response to cancer. Multiplexed TCR-T cell therapy (e.g., including anti-MAGE-A1 TCR and anti-PRAME TCR) provides methods and compositions for addressing specific challenges associated with the treatment of solid tumors.

[0047] To confirm the utility of multiplexed TCR-T cell therapy (e.g., multiplexed TCR-T cell therapy including anti-MAGE-A1 TCR (e.g., “TCR 1479” also known as “MAGE-A1-1479”, “1479”, “TSC-204-A02”, and “TSC-204-A0201”), and anti-PRAME TCR (e.g., “TCR 366” also known as “366” and “TSC-203-A02” (also known as “TSC-203-A0201”), and / or “TCR358” also known as “358”)), various assays can be used. In a representative non-limiting example, anti-MAGE-A1 TCR, anti-PRAME TCR, and one or more target cell lines expressing their cognate antigens are multiplexed using direct and indirect co-culture experiments to evaluate the potential synergistic effect of using multiple TCRs to target tumors and to understand the biological mechanisms underlying such synergistic effect. Using this assay, multiplexed T cell-mediated cancer killing by anti-MAGE-A1 TCR and anti-PRAME TCR, as well as multiplexed TCR-T cell therapy including heterogeneity, can be modeled in vitro.

[0048] In one representative case, for example, (i) an anti-MAGE-A1 TCR targeting the HLA-A*02 serotype-restricted epitope of MAGE-A1 (Table 5, e.g., SEQ ID NO: 83) and (ii) an anti-PRAME TCR targeting the HLA-A*02 serotype-restricted epitope of PRAME (Table 7, e.g., SEQ ID NO: 104) can be used and / or tested, for example, by using engineered cells that express such TCRs. In some embodiments, naïve 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 expressing 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 Incucyte® NucLight Red and mixed together to mimic tumor heterogeneity. Engineered T cells or unengineered donor control T cells (control TCR-T) are co-cultured with the Incucyte® NucLight Red-labeled target cell lines at the indicated effector cell to target cell (E:T) ratio, and their survival can be quantified with IncuCyte® as a readout of T cell cytotoxicity.

[0049] The anti-MAGE-A1 TCRs encompassed by the present invention are TCRs that include (e.g., include, consist essentially of, or consist 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%, or more identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 4; and / or b) a TCR beta 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%, or more identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 4.

[0050] The anti-MAGE-A1 TCRs encompassed by the present invention may be TCRs that include (e.g., include, consist essentially of, or consist of) a) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 4 and a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 4.

[0051] The anti-MAGE-A1 TCRs encompassed by the present invention are a) the variable (V α ) domain sequences of the TCR alpha chain selected from the group consisting of the TCR V α ) domain sequences 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; and / or b) the variable (V α ) domain sequences of the TCR beta chain selected from the group consisting of the TCR V β ) domain sequences 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; and / or b) the variable (V β) A TCR beta 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 more identity to the domain array, may be included (e.g., including, consisting essentially of, or consisting of).

[0052] The anti-MAGE-A1 TCRs encompassed by the present invention are a) TCR alpha chain variable (V α ) domain sequences selected from the group consisting of the TCR V α ) domain sequences; and / or TCR beta chain variable (V β ) domain sequences selected from the group consisting of the TCR V β ) domain sequences, may be included (e.g., including, consisting essentially of, or consisting of).

[0053] The anti-MAGE-A1 TCRs encompassed by the present invention have at least about 80%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identity to at least one (e.g., 1, 2, or 3, e.g., CDR alone or in combination with CDR1 and CDR2) TCR alpha chain complementarity determining region (CDR) sequences selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 4, may be included (e.g., including, consisting essentially of, or consisting of). CDR3 is considered to be the major CDR involved in the recognition of processed antigens, and since CDR1 and CDR2 mainly interact with MHC, in some embodiments, binding proteins containing only CDR3 from the TCR alpha chain and / or only CDR3 from the TCR beta (each CDR3 having the sequence homology described in this paragraph) are provided.

[0054] An anti-MAGE-A1 TCR encompassed by the present invention is at least about 80%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identical to a TCR beta-chain CDR sequence selected from the group consisting of the TCR beta-chain CDR sequences listed in Table 4, and includes at least one (e.g., 1, 2, or 3, e.g., CDR alone or in combination with CDR1 and CDR2) TCR beta-chain complementarity determining region (CDR) sequence, and may be a TCR (e.g., including, consisting essentially of, or consisting of). As described above, CDR3 is considered the major CDR involved in the recognition of processed antigen, and CDR1 and CDR2 mainly interact with MHC. Thus, in some embodiments, binding proteins are provided that include only CDR3 from the TCR beta-chain and / or only CDR3 from the TCR alpha-chain listed in Table 4 (each CDR3 having the sequence homology described in this paragraph).

[0055] An anti-MAGE-A1 TCR encompassed by the present invention may be a TCR that includes at least one (e.g., 1, 2, or 3) complementarity determining region (CDR) of a TCR alpha-chain listed in Table 4 (e.g., including, consisting essentially of, or consisting of).

[0056] An anti-MAGE-A1 TCR encompassed by the present invention may be a TCR that includes at least one (e.g., 1, 2, or 3) complementarity determining region (CDR) of a TCR beta-chain listed in Table 4 (e.g., including, consisting essentially of, or consisting of).

[0057] The anti-MAGE-A1 TCRs encompassed by the present invention may be TCRs that include (e.g., that include, consist essentially of, or consist of) a constant region (C α ) sequence of a TCR alpha chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR C alpha sequences listed in Table 4.

[0058] The anti-MAGE-A1 TCRs encompassed by the present invention may be TCRs that include (e.g., that include, consist essentially of, or consist of) a constant region (C β ) sequence of a TCR beta chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR C sequences listed in Table 4. β ) sequence of a TCR beta chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR C sequences listed in Table 4.

[0059] The anti-MAGE-A1 TCRs encompassed by the present invention may be TCRs that include (e.g., that include, consist essentially of, or consist of) a TCR alpha chain constant region (C α ) sequence selected from the group consisting of the TCR C sequences listed in Table 4. α ) sequence selected from the group consisting of the TCR C sequences listed in Table 4.

[0060] The anti-MAGE-A1 TCRs encompassed by the present invention may be TCRs that include (e.g., that include, consist essentially of, or consist of) a TCR beta chain constant region (C β ) sequence selected from the group consisting of the TCR C sequences listed in Table 4. β ) sequence selected from the group consisting of the TCR C sequences listed in Table 4. [Table 4-1] [Table 4-2] [Table 4-3]

Table 4-4

Table 4-5

Table 4-6

[0061] Table 4 is grouped according to the presentation of MHC serotypes and partially shows representative TCR sequences subgrouped according to the different peptides presented by the MHC serotypes and bound by the subgrouped TCRs. Individual TCRs such as those typically exemplified in the table, as well as the genus of binding proteins that bind to the peptide epitope sequences described herein, either alone or in complex with MHC, such as those grouped in the tables provided herein, are described and claimed. Further provided are the TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein. The sequences of each TCR described herein are provided as pairs of homologous alpha and beta chains for each designated TCR. The TCR sequences described herein are annotated. The variable domain sequences are represented in capital letters. The constant domain sequences are in lower case. The CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined strings. CDR1, CDR2, and CDR3 are shown in their standard order of appearance from left (N-terminus) to right (C-terminus). The TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein, are annotated according to the well-known IMGT nomenclature described herein. Similarly, CDR1 and CDR2 of TRAV and TRBV are well-known in the art as they are based on well-known and annotated TRAV and TRBV sequences (e.g., annotated in databases such as IMGT available at imt.org and IEDB available at iedb.org).

[0062] *In certain illustrated vectors, the MSCV promoter is in bold. The beta chain is annotated using bold and italicized text. The alpha chain is annotated using bold and underlined text. The CD34 enrichment tag (e.g., Q tag) is annotated using italicized and underlined text. CD8-alpha is italicized. CD8-beta is underlined. *Included in Table 4 are polypeptide sequences, as well as amino acid sequences or portions thereof of any of the sequences recited herein, and polypeptide molecules comprising amino acid sequences 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 more identity over their full length. Such polypeptides can have the function of a full-length peptide or polypeptide, as further described herein.

[0063] *Included in Table 4 are RNA nucleic acid molecules (e.g., where thymidine is replaced by uridine), nucleic acid molecules encoding orthologs of the encoded proteins, as well as nucleic acid sequences or portions thereof of any of the sequences recited herein, and DNA or RNA nucleic acid sequences comprising nucleic acid sequences 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 more identity over their full length. Such nucleic acid molecules can have the function of a full-length nucleic acid, as further described herein.

Table 5

[0064] As noted above, any combination of TCRs described herein is contemplated for use.

[0065] For example, an anti-MAGE-A1 TCR encompassed by the present invention is a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) 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%, or more identity to a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 6; and / or a TCR beta 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%, or more identity to a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 6.

[0066] An anti-MAGE-A1 TCR encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) a TCR alpha chain sequence selected from the group consisting of the TCR alpha chain sequences listed in Table 6 and a TCR beta chain sequence selected from the group consisting of the TCR beta chain sequences listed in Table 6.

[0067] An anti-PRAME TCR encompassed by the present invention is a) a TCR alpha 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 more identity to a TCR alpha chain variable (V α ) domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 6; and / or b) a TCR beta 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 more identity to a TCR beta chain variable (V β ) domain sequence selected from the group consisting of the TCR V domain sequences listed in Table 6; and / or c) a TCR beta chain variable (V β)A TCR beta 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 more identity to the domain sequence, may be included (e.g., included, consisting essentially of, or consisting of).

[0068] An anti-PRAME TCR encompassed by the present invention is a TCR alpha chain variable (V α ) domain sequence selected from the group consisting of the TCR alpha chain variable (V α ) domain sequences listed in Table 6; and / or a TCR beta chain variable (V β ) domain sequence selected from the group consisting of the TCR beta chain variable (V β ) domain sequences listed in Table 6, may be included (e.g., included, consisting essentially of, or consisting of).

[0069] An anti-PRAME TCR encompassed by the present invention may be a TCR having at least about 80%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identity to at least one (e.g., 1, 2, or 3, e.g., CDR alone or in combination with CDR1 and CDR2) TCR alpha chain complementarity determining region (CDR) sequence selected from the group consisting of the TCR alpha chain CDR sequences listed in Table 6, may be included (e.g., included, consisting essentially of, or consisting of). CDR3 is considered the major CDR involved in the recognition of processed antigen, and CDR1 and CDR2 mainly interact with MHC, so in some embodiments, binding proteins containing only CDR3 from the TCR alpha chain and / or only CDR3 from the TCR beta (each CDR3 having the sequence homology described in this paragraph) are provided.

[0070] An anti-PRAME TCR encompassed by the present invention is a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) at least one (e.g., 1, 2, or 3, e.g., CDR alone or in combination with CDR1 and CDR2) TCR beta-chain complementarity-determining region (CDR) sequence having at least about 80%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more identity to a TCR beta-chain CDR sequence selected from the group consisting of the TCR beta-chain CDR sequences listed in Table 6. As noted above, CDR3 is thought to be the major CDR involved in the recognition of processed antigen, and since CDR1 and CDR2 mainly interact with MHC, in some embodiments, binding proteins comprising only CDR3 from the TCR beta-chain and / or only CDR3 from the TCR alpha-chain (each CDR3 having the sequence homology described in this paragraph) are provided.

[0071] An anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) at least one (e.g., 1, 2, or 3) complementarity-determining region (CDR) of a TCR alpha-chain listed in Table 6.

[0072] An anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., comprising, consisting essentially of, or consisting of) at least one (e.g., 1, 2, or 3) complementarity-determining region (CDR) of a TCR beta-chain listed in Table 6.

[0073] An anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., consisting of, consisting essentially of, or comprising) a constant region (C α ) sequence of a TCR alpha chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR Cα sequences listed in Table 6.

[0074] An anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., consisting of, consisting essentially of, or comprising) a constant region (C β ) sequence of a TCR beta chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR Cβ sequences listed in Table 6. β ) sequence of a TCR beta chain having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity to the TCR Cβ sequences listed in Table 6.

[0075] An anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., consisting of, consisting essentially of, or comprising) a TCR alpha chain constant region (C α ) sequence selected from the group consisting of the TCR C sequences listed in Table 6. α ) sequence selected from the group consisting of the TCR C sequences listed in Table 6.

[0076] An anti-PRAME TCR encompassed by the present invention may be a TCR comprising (e.g., consisting of, consisting essentially of, or comprising) a TCR beta chain constant region (C β ) sequence selected from the group consisting of the TCR C sequences listed in Table 6. β ) sequence selected from the group consisting of the TCR C sequences listed in Table 6.

Table 6-1

Table 6-2

Table 6-3

Table 6-4

Table 6-5

Table 6-6

Table 6-7

Table 6-8

Table 6-9

Table 6-10

Table 6-11

Table 6-12

Table 6-13

[0077] Table 6 is grouped according to the presentation of MHC serotypes and partially shows representative TCR sequences that are grouped according to different peptides presented by MHC serotypes and to which the subgrouped TCRs bind. Individual TCRs such as those typically exemplified in the table, as well as the genus of binding proteins that bind to the peptide epitope sequences described herein, either alone or in complex with MHC, such as those grouped in the tables provided herein, are described and claimed. Further provided are the TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein. The sequences of each TCR described herein are provided as pairs of homologous alpha and beta chains for each designated TCR. The TCR sequences described herein are annotated. The variable domain sequences are represented in uppercase letters. The constant domain sequences are in lowercase. The CDR1, CDR2, and CDR3 sequences are annotated using bold and underlined strings. CDR1, CDR2, and CDR3 are shown in their standard order of appearance from left (N-terminus) to right (C-terminus). The TRAV, TRAJ, and TRAC genes for each TCR alpha chain described herein, and the TRBV, TRBJ, and TRBC genes for each TCR beta chain described herein, are annotated according to the well-known IMGT nomenclature described herein. Similarly, CDR1 and CDR2 of TRAV and TRBV are well-known in the art as they are based on well-known and annotated TRAV and TRBV sequences (e.g., those annotated in databases such as IMGT available at imt.org and IEDB available at iedb.org).

[0078] *In certain illustrated vectors, the MSCV promoter is in bold. The beta chain is annotated using bold and italicized text. The alpha chain is annotated using bold and underlined text. CD34 enrichment tags (e.g., Q tags) are annotated using italicized and underlined text. CD8-alpha is italicized. CD8-beta is underlined.

[0079] *Included in Table 6 are polypeptide sequences, as well as amino acid sequences or portions thereof of any of the sequences listed herein, and polypeptide molecules comprising amino acid sequences 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 more identity over their full length. Such polypeptides can have the function of the full-length peptide or polypeptide, as further described herein.

[0080] *Included in Table 6 are RNA nucleic acid molecules (e.g., where thymidine is replaced by uridine), nucleic acid molecules encoding orthologs of the encoded proteins, as well as nucleic acid sequences or portions thereof of any of the sequences listed herein, and DNA or RNA nucleic acid sequences comprising nucleic acid sequences 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 more identity over their full length. Such nucleic acid molecules can have the function of the full-length nucleic acid, as further described herein. [Table 7]

[0081] Example 4: Multiplexed TCR-T cell therapy targeting MAGEA1 and PRAME enhances the activity of adoptive T cell therapy in preclinical models This example is based in part on the recognition that adoptive cell transfer using genetically engineered T cells holds great promise for the treatment of solid tumors. In certain previous clinical trials of TCR-engineered T cell therapy (TCR-T), one antigen was targeted at a time, generating response rates in the range of 30-50% in various examples. Complete responses to such therapies are rare, and responses have been observed to be often short-lived. Without wishing to be bound by any particular scientific theory, one possible reason for the rapid recurrence of patients after responding to such therapies is that those tumors exhibit substantial heterogeneity in antigen expression: not every cancer cell within the tumor expresses the target of the single-agent TCR therapy, and even if they do, the target is expressed at variable levels between individual tumor cells. This suggests that TCR-T targeting one antigen may avoid cells lacking the treated antigen and promote recurrence.

[0082] This example presents the development of a multiplexed TCR-T cell therapy in which a patient is treated with multiple TCR-T cell products selected from a pre-screened set of TCRs that are compatible with the patient's tumor antigens and HLA type, as a solution to address antigen heterogeneity. As a proof of concept, two different cancer / testis antigens targeted by two different TCRs were selected. One of these antigens, MAGEA1, was identified as a target for expanded tumor-infiltrating T cells derived from head and neck cancer patients using the TScan TRBV2 / TRBJ2-7 / MGTM-modified TRBC screening technology as described in Luomo et al. (2022) Cell. S0092-8674(22)00723-1. The other antigen, PRAME, is highly expressed in various cancers. This example includes the development of two high-affinity TCRs that recognize HLA-A*02:01-restricted epitopes derived from MAGEA1 and PRAME (see Tables 5 and 7, respectively). The advantage of combining these two TCR-T cell products, which have sequences according to Tables 4 and 6, respectively, was evaluated using various preclinical models. For example, the TSC-203-A0201 and TSC-204-A0201 TCR-T cell products, which express the MGTM TCR and are codon-optimized, may be used.

[0083] Individually, both TCRs (i.e., the TCRs that recognize MAGEA1 and PRAME, respectively) are thought to exhibit potent cytotoxic activity in vitro when co-cultured with HLA-compatible cancer cell lines that express endogenous MAGEA1 and PRAME. Furthermore, in xenograft mouse models, each TCR is thought to be able to control the growth of tumors that express their cognate antigens and HLA.

[0084] A mixture of two different cell lines expressing either MAGEA1 or PRAME together with HLA-A*02:01 was tested in vitro or grown as xenograft tumors in mice and treated with either an individually tested TCR-T or a mixture of two TCR-Ts. Notably, the MAGE-specific TCR-T and the PRAME-specific TCR-T are designed to selectively target their respective target cell subsets, and the multiplexed MAGEA1 / PRAME TCR-T is designed to target both cancer cell subsets simultaneously. Treatment with the multiplexed MAGEA1 / PRAME TCR-T was designed to achieve longer-term tumor control in mice compared to TCR-T targeting a single antigen.

[0085] Based on the findings reported in this example and summarized further in Figure 6, it is expected that multiplexed TCR-T will be demonstrated to be a powerful means of targeting cancer with heterogeneous target antigen expression and thus an advantageous approach to therapy. While not wishing to be bound by any particular scientific theory, this example demonstrates that multiplexed TCR-T mimics the natural oligoclonal T cell response to cancer and has the potential to overcome antigen heterogeneity, which can contribute to the lack of persistence observed in single-agent TCR-T clinical trials. The co-culture assay described is a relatively short-term treatment, and in other assays, such as longer-term tests, additional synergistic activity (from cytokine-dependent phenomena described herein, etc.) may be observed, such as a decrease in the effector-to-target ratio of one or more TCRs in a TCR combination that shows a supportive effect of other TCR(s) on reduced TCR(s).

[0086] Figure 6 shows representative examples of variable antigen expression in human non-small cell lung (NSCLC) tumor samples. Immunohistochemistry was performed on human NSCLC tumor microarrays using an MAGE-A1-specific antibody (clone SPM282; Abcam catalog number ab25834) or a PRAME-specific antibody (clone EPR20330; Abcam catalog number ab219650). Heterogeneous antigen expression within tumors was observed in multiple sections at varying degrees of expression, as represented by MAGE-A1 and PRAME. Non-stained tumor controls are also shown.

[0087] Figure 6 further shows the characterization of TCRs that recognize epitopes derived from MAGE-A1 presented by HLA-A*02:01 and TCRs that recognize epitopes derived from PRAME presented by HLA-A*02:01. TCR-T cell co-cultures were performed with either a panel of HLA-A*02:01-positive cancer cell lines that present a range of MAGE-A1 (i.e., MCI0H1703, HJS936T, and A375) and TCR-T cells expressing a MAGE-A1-specific TCR, or with cells that present a range of PRAME expression (i.e., HS695T, A375, and NCI-H15632) and TCR-T cells expressing a PRAME-specific TCR. Cell lines that are positive for HLA-A*02:01 but negative for MAGE-A1 (A2-HEK293T) or PRAME (647-V) were tested as negative controls.

[0088] The TCR-T cells used in these experiments were polyclonal T cells engineered by lentiviral transduction. This vector included MGTM modifications, and the co-receptors CD8α and CD8β were co-delivered with the recombinant TCRs, mainly to ensure recognition of the TCR on the CD4+ fraction of these polyclonal T cells.

[0089] This data demonstrates that each TCR-T exhibits high potency and selectivity against cells presenting the cognate peptide / MHC (pMHC), killing the relevant target cells while sparing cell lines negative for the target protein.

[0090] To further test the efficacy of individual TCR-T cells, in vivo efficacy tests were also conducted. Female NOD-Prkdc em26Cd52 Il2r gem26Cd22 / NjuCrl (NCG) mice were subcutaneously implanted with U266B1 cancer cells (HLA-A*02:01-positive cells expressing MAGE-A1). Animals in which tumor growth was confirmed (average tumor volume of approximately 100 mm 3 ; 21 days after inoculation) were randomized into different experimental groups and received two intravenous injections of MAGE-A1 TCR-T cells (20E6 each; injected on the day after randomization and again 1 week later), or donor-matched, unmanipulated control T cells (20E6 each; injected on the day after randomization and again 1 week later). Tumor volume was measured twice a week. Animals in the control group presented with proliferative tumors reaching an average of over 800 mm 3 by day 42, while mice treated with MAGE-A1-specific TCR-T cells showed a strong anti-tumor response.

[0091] In a similar experiment, female NCG animals were transplanted with Hs695T cells (HLA-A*02:01-positive cells expressing PRAME), and a single dose of PRAME-specific TCR-T cells or donor-matched, unmanipulated control T cells was administered (20E6 T cells, 1 day after randomization). Animals injected with TCR-T cells showed an anti-tumor response when compared to animals treated with control T cells.

[0092] These tests confirmed that intravenous injection of TCR-T cells could successfully control the growth of pMHC-positive tumors subcutaneously inoculated in mice, and the efficacy of individual TCR-T cells was confirmed.

[0093] In vitro experiments were conducted 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 either MAGE-A1 or PRAME. MAGE-A1-expressing cells were labeled with GFP, and PRAME-positive cells were labeled with both GFP and CellTrace™ Violet to enable tracking in downstream flow cytometry readouts. TCR-T cells, when recognizing target cells, secrete cytotoxic granules into those target cells, inducing them to fluoresce (IFP-positive). Two target cells (i.e., PRAME- or MAGE-A1-positive) were mixed at an equilibrated ratio and co-cultured with MAGE-A1 TCR-T cells, PRAME TCR-T cells, or a multiplex product combining the two TCR-T cells. PRAME TCR-T cells and MAGE-A1 TCR-T cells were engineered from T cells from the same donor. These TCR-T cells corresponded to polyclonal T cells engineered by lentiviral transduction using a delivery vector that employs MGTM modification and co-delivers the above CD8α and CD8β coreceptors (e.g., Table 1). Non-engineered T cells (NTC) with a compatible donor were also included as a control. Next, in this experiment, the percentage of each subset of targets (GFP-positive, MAGE-A1 target, GFP / CTV-positive, PRAME target), measured by the percentage of GFP or GFP / CTV (becoming IFP-positive), recognized and targeted by TCR-T cells was measured. NTC did not induce IFP positivity in any of the target subsets. When MAGE-A1 TCR-T cells were co-cultured with the mixed target cells, the percentage of GFP-positive cells that were positive for IFP increased, but the percentage of GFP / CTV-positive cells did not increase. These results demonstrate that only the MAGE-A1-positive subset was recognized under these co-culture conditions. Conversely, when PRAME TCR-T cells were co-cultured with the target cell mixture, the percentage of GFP / CTV-positive targets and the percentage positive for IFP both increased, but the percentage of GFP-positive target cells did not increase.These results confirm that only the PRAME-positive target cell subset was targeted under this co-culture condition. Finally, when a mixture of PRAME TCR-T cells and MAGE-A1 TCR-T cells was co-cultured with the mixed target cells, an IFP-positive signal was shown in both the GFP-positive and GFP / CTV-positive cell subsets, revealing that both target cell subsets were effectively recognized. The proportion of each subset that became IFP-positive in co-culture with the multiplexed product was comparable to the proportion observed when co-cultured with each individual TCR-T cell product.

[0094] Overall, this data demonstrates that each individual TCR-T cell product was able to target cells positive for the relevant pMHC, but a multiplexed product is required to broadly target heterogeneous mixtures of cancer cells.

[0095] A mixture of HEK293T cells expressing either MAGE-A1 or PRAME together with HLA-A*02:01 was also subcutaneously inoculated into female NCG mice. Once the tumors reached an average of 100 mm 3 in size, the animals were randomized and given a single intravenous injection of 20E6 MAGE-A1 TCR-T cells, 20E6 PRAME TCR-T cells, or a multiplexed product consisting of 10E6 MAGE-A1 TCR-T cells and 10E6 PRAME TCR-T cells, or a multiplexed product consisting of 20E6 MAGE-A1 TCR-T cells and 20E6 PRAME TCR-T cells. A group of animals was given an intravenous injection of 20E6 donor-matched non-manipulated T cells. The same effector T cells as above were used in these experiments. Tumor volumes were then measured once every two weeks. Animals in the control group showed rapidly growing tumors; the tumor volume exceeded 1000 mm 3 at the end of the study (day 24 post-inoculation). On the other hand, in animals administered with each individual TCR-T cell subset, tumor growth was slower and only reached approximately 600 - 750 mm 3 at the end of the study. Animals administered with the multiplexed TCR-T cell product had an average tumor volume of approximately 500 mm 3(Each TCR-T is 10E6) or approximately 300 mm 3 When compared to animals administered with individual TCR-T cell products (each TCR-T is 20E6), a broader and more sustained response was achieved. Together with the in vitro data shown in Figure 6 and the above data, these data confirm that each TCR-T cell targets only a subset of heterogeneous tumors and partial efficacy is achieved, i.e., a subset of cancer cells maintains resistance to single-agent TCR-T cells and drives recurrence. On the other hand, multiplex TCR-T products achieved a more potent anti-tumor response by broadly targeting two cell subsets of heterogeneous tumors, preventing the selection of resistant cells.

[0096] Furthermore, Figure 6 shows the concept of an ImmunoBank-based approach to therapeutic TCR therapy, where several therapeutic TCRs that address multiple cancer-related proteins in combination with HLA restriction are utilized to enable a customized combination of TCR therapies based on the tumor biology of each patient. For each patient, the treatment decision is made by determining (a) which cancer-related proteins (ImmunoBank columns) are expressed in the tumor(s) using immunohistochemistry (IHC) or reverse transcription polymerase chain reaction (RT-PCR), and (b) which HLA genes (ImmunoBank rows), measured by genomic sequencing, are intact in those tumor(s) (i.e., have not experienced loss of heterozygosity [LOH] at the HLA locus). Once the patient's tumor targets and HLA profile are determined, multiple TCRs (e.g., two TCRs, three TCRs, etc.) are selected from the ImmunoBank to prepare a customized multiplex TCR-T cell pharmaceutical.

[0097] Example 5: Multiplexed TCR-T cell therapy targeting the same target using different TCRs that recognize epitopes presented by distinct HLAs enhances the activity of adoptive T cell therapy in preclinical models This example is based in part on the recognition that adoptive cell transfer using genetically engineered T cells holds great promise for the treatment of solid tumors.

[0098] 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 a given target presented by such HLA, for example, epitopes such as TSC-204-A0201 and TSC-204-C0702, respectively (e.g., by combined or sequential infusion of TCRs). Patients in whom specific HLA alleles occur on separate chromosomes (separate haplotypes) are likely to be resistant to HLA loss because tumor cells that have lost both class I HLA haplotypes become targets for natural killer (NK) cells (O’Connor et al. (2006) Immunol. 117:1-10). The use of additional TCR-T components that address different targets and broader HLA types makes it possible to further enhance TCR combinations and treat a broader range of patients with multiplexed TCR-T.

[0099] This example presents the development of multiplexed TCR-T cell therapy in which a patient is treated with multiple TCR-T cell products selected from a pre-considered set of TCRs that are matched to the patient's tumor antigens and HLA type as a solution to address antigen heterogeneity. As a representative non-limiting example, two different TCRs are selected for multiplexed TCR-T treatment, each of which targets different epitopes of the same target but is presented by different HLA alleles. TSC-204-A0201 and TSC-204-C0702 were engineered into pan T cells (CD4 + and CD8 +used in a form consisting of both T cells to express (1) each recombinant TCR, (2) recombinant CD8α and CD8β coreceptors to maximize the effectiveness of the therapeutic agent, (3) a CD34-derived epitope tag fused at the N-terminus of CD8α to facilitate the tracking of cells engineered 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 dihydrofolate reductase (DHFRdm) protein to facilitate the enrichment of engineered cells during the manufacturing process. Nevertheless, the results shown in the data provided herein are thought to be due to the function of the TCR itself.

[0100] In vitro characterization of the TSC-204-A0201 and TSC-204-C0702 materials demonstrated that TCR-T cells are associated with target-dependent responses leading to the secretion of inflammatory cytokines, the proliferation of effector T cells, and ultimately the killing of target cells (Figure 7). Since the MAGE-A1-derived epitopes targeted by TSC-204-A0201 or TSC-204-C0702 are presented by class I MHC HLA-A*02:01 and HLA-C*07:02, respectively, the recombinant TCRs engage pMHC using the CD8αβ coreceptor. Helper (CD4 + ) T cells do not naturally express the CD8αβ coreceptor. Exogenous CD8α and CD8β coreceptors were co-delivered to T cells engineered with the therapeutic TCR to enhance the ability of CD4 + helper T cells to recognize class I-restricted epitopes. The engineered CD4 + T cells included in TSC-204-A0201 and TSC-204-C0702 proliferated with the engineered CD8 + cytotoxic T cells, demonstrating the functional involvement of helper T cells. Furthermore, since the engineered T cells express DN-TGFβRII, TSC-204-A0201, and TSC-204-C0702, the TCR-T cells are active even in the presence of TGFβ, an immunosuppressive cytokine that can be observed in the microenvironment of solid tumors.

[0101] Figures 8 and 9 show the results of TCR-T cells from three independent batches of TSC-204-A0201 and TSC-204-C0702 applied to a heterogeneous target cancer cell population generated to simulate MAGE-A1-positive tumors with LOH. Briefly, the cell line of the cancer cell line U266B1 (i.e., the cell line TIB-196 available from ATCC) that is positive for MAGE-A1, HLA-A*02:01, and HLA-C*07:02 was used. The cells were engineered by CRISPR knockout to create two versions of the cell line in which only one of the two HLA of interest was intact (knocking out HLA-A*02:01 or HLA-C*07:02). The U266B1 HLA-C*07:02 KO, "A2 target", and U266B1 HLA-A*02:01 KO, "C7 target" target cells were barcoded with CellTrace™ Violet and CFSE, respectively. After co-culture with single (TSC-204-C0702 or TSC-204-A0201) or multiplex (T-Plex-204-A0201 / 204-C0702) conditions, the cell suspension was labeled with LIVE / DEAD™ viability dye to determine the viability of the target cells. Each target cell subset was labeled with a different fluorescent dye and tracked at the downstream flow cytometry readout before mixing at a 1:1 ratio.

[0102] Effector T cells were prepared. One day before performing the assay, the effector TCR-T cells were thawed in a 37°C water bath and washed with cytokine-free T cell medium. The cell concentration and viability (CCV) were determined, and viable TCR-T cells were seeded at a concentration of 1E6 cells / mL in a G-REX® 6M well plate in complete T cell medium. The TCR-T cells were incubated at 37°C and 5% CO 2They were collected within 16 - 24 hours in a humidified incubator and then cultured. After thawing and collecting overnight, effector TCR-T cells were collected, washed with cytokine-free T cell medium, and resuspended in cytokine-free T cell medium at 2E6 viable cells / mL for preparation for single and multiplex conditions. Each TCR-T cell suspension was aliquoted for seeding the positive control single condition. The remaining TCR-T cell suspensions were combined at a 1:1 ratio for all three batches to create the test sample "T-Plex" condition (2E6 viable cells / mL).

[0103] Similarly, target cells were prepared. The target cells were thawed, expanded, maintained during culture without exceeding 20 passages, and then discarded. The day before the start of co-culture, the target cells were harvested and CCV was measured and recorded. Then, the target cells were seeded at 4E5 viable cells / mL to synchronize the cell cycle phase. On the day of co-culture, the target cells were harvested and CCV was determined. The harvested cells were washed and the cell density was adjusted to 1E6 cells / mL with protein-free PBS. Target U266B1 HLA-C*07:02 KO cells were labeled with CellTrace Violet and target U266B1 HLA-A*02:01 KO cells were labeled with CellTrace™ CFSE, both labeled at 1:2000 according to the manufacturer's instructions and finally resuspended in RPMI-based medium at 5E5 viable cells / mL. After CellTrace™ labeling, each target cell suspension (5E5 viable cells / mL) was aliquoted for plating the negative control. A heterogeneous target cell preparation was made from the remaining target cell suspension (5E5 viable cells / mL), which was combined at a 1:1 ratio and co-cultured with the positive control, single, and T-Plex test samples.

[0104] Furthermore, co-cultures were prepared. Next, this heterogeneous population of target cells was co-cultured with various TCR-T cell mixtures consisting of exclusive, TSC-204-A0201, TSC-204-C0702 (corresponding to monotherapy or "single" TCR-T cell products), or an equilibrated mixture of TSC-204-A0201 and TSC-204-C0702 (i.e., "multi" TCR-T cell product). Briefly, target cells were seeded into sample wells (U-bottom 96-well plates), and then effector cell suspensions under single or multi conditions were added on top of the target cells. The final volume was 200 μL / well consisting of a 50 / 50 mixture of target cells (100 μL) and effector cells (100 μL) in target cell RPMI medium as well as cytokine-free T cell and target cell medium. The cells were returned to the incubator and the co-cultures were incubated for 20 - 24 hours. Each positive control or test sample well contained a total target suspension of a total of 5E4 viable cells consisting of 50% CTV-labeled, U266B1 HLA-C*07:02 KO (total 2.5E4 cells) and 50% CTCFSE-labeled, U266B1 HLA-C*07:02 KO (total 2.5E4 cells). Each sample well under single-chain conditions contained a total of 2E5 viable cells of effector cell suspension, TSC-204-A0201, or TSC-204-C0702 combined with a total of 5E4 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 a combined cell suspension T-Plex-204-A0201 / 204-C0702 of a total of 2E5 cells consisting of 50% TSC-204-A0201 (total 1E5 cells) and 50% TSC-204-C0702 (total 1E5 cells). Furthermore, this well was combined with a total of 5E4 cells of the combined target cell suspension. This represents an E:T ratio of 4:1 and an effector-to-specific target ratio of 4:1.

[0105] The cytotoxic activity of TCR-T cells against target cells was evaluated by flow cytometry by assessing the relative composition of each target cell population in the remaining cells. At the end of co-culture, cells were pelleted by centrifugation, then protected from light and resuspended in LIVE / DEAD™ viability dye for 20 minutes at 4°C to confirm cell viability. After a single wash, cells were resuspended in EasySep™ and CountBright™ Absolute Count beads were added according to the manufacturer's instructions. Immediately after adding the Count beads, the assay plate was acquired on the cytometer. Data acquisition was performed on a CytoFLEX S flow cytometer according to the machine's SOP-PC-0001-Instrument SOP-Use and Maintenance of CytoFLEX. Compensation was performed automatically using CytExpert software. Flow cytometry analysis was performed in FlowJo v7.6.5 and statistics were exported to Microsoft Excel 2010 for analysis. The selected analysis data were graphed in GraphPad Prism (v5.02).

[0106] The gating strategy is shown in FIGS. 8A-8E. Briefly, cells were gated from the FSC vs. SSC dot plot. The CellTrace™ CFSE vs. CellTrace™ Violet plot was used to distinguish subpopulations; C0702 target (CellTrace™ CFSE + / CellTrace™ Violet - ), A0201 target (CellTrace™ CFSE- / CellTrace™ Violet + ) and effector (CellTrace™ CFSE− / CellTrace™ )Violet-)。Viable cells for each subpopulation were identified using the LIVE / DEAD™ histogram. Since the LIVE / DEAD™ dye reacts with free intracellular and extracellular amines of damaged cell membranes, dead cells have high fluorescence intensity. Since the dye is limited to extracellular amines only, viable cells exhibit lower fluorescence intensity, allowing viable cells to be limited.

[0107] Killing of target cells was defined by the killing rate determined by subtracting the survival rate of the test sample from the survival rate of the negative control and then dividing by the negative control. When the survival rate of the test sample increased above the baseline negative control survival rate value, the killing rate value was reported as 0% killing.

[0108] To quantify the absolute number of viable target cells obtained from the sample wells, 20 μL of CountBright™ absolute counting beads (20,400 beads / 20 μL) were added to a 120 μL volume of cell suspension. The volume of the obtained cell sample was multiplied by the absolute cell number concentration to determine the total number of viable cells obtained.

[0109] The baseline viability of the target cells was determined using a negative control. Briefly, CellTrace™ CFSE-labeled U266B1 HLA-A*02:01 KO targets (i.e., "C7 targets") are intact with respect to HLA-C*0702 along with the MAGE-A1 protein. These constitute the target cells for the TSC-204-C0702 TCR-T cells. CellTrace™ Violet-labeled U266B1 HLA-C*07:02 KO targets (i.e., "A2 targets") express HLA-A*02:01 and the MAGE-A1 protein. These constitute the target cells for the TSC-204-A0201 TCR-T cells. To determine the baseline viability of the individual targets after overnight culture, negative controls were created; TSC-204-C0702 was co-cultured with only U266B1 HLA-C*07:02 KO target cells ("A2 targets"), and TSC-204-A0201 was co-cultured with only U266B1-A*02:01 KO target cells ("C7 targets"). The mean baseline viability for U266B1 HLA-A*02:01 KO (C7 targets) was 67.57% (n = 3) and 61.53% (n = 3) for U266B1 HLA-C*07:02 KO (A2 targets). The total viable cell count and percent viability are shown in Figure 9 as "control" data. Using these baseline values, specific TCR-T cell-mediated killing was calculated.

[0110] Similarly, TCR-T cell-mediated killing of target cells was observed using a positive control. Briefly, to determine the advantages of T-Plex-204-A0201 / 204-C0702-mediated killing, a cell suspension consisting of 50% CTV-labeled U266B1 HLA-C*07:02 KO and 50% CTCFSE-labeled U266B1 HLA-A*02:01 KO was created. The resulting targets were heterogeneous as a subset of cells that expressed HLA-A*02:01 but not HLA-C*07:02, and another subset that expressed HLA-C*07:02 but had lost HLA-A*02:01. This combination of targets, "A2+C7", was co-cultured with TCR-T cell products consisting of TSC-204-A0201 or TSC-204-C0702 as monotherapy ("single") to show the killing ability of each individual component of T-Plex. These conditions functioned as a positive control.

[0111] The killing result rates related to total viable cell count and percent viability (calculated as the decrease in viability relative to the above baseline) are shown in Figure 9. Specifically, TSC-204-A0201 TCR-T cells from three independent batches showed specific cell-mediated killing of U266B1 HLA-C*07:02 KO target cells (58.88%, 69.01%, and 64.30% respectively), but did not kill U266B1 HLA-A*02:01 KO targets (0% specific killing across all three batches tested). Similarly, TSC-204-C0702 TCR-T cells from three independent batches showed cell-mediated killing of U266B1 HLA-A*02:01 KO target cells (48.84%, 59.84%, and 47.66% respectively), but systematically spared U266B1 HLA-C*07:02 KO (0% specific killing across all three batches tested). These data confirmed that each individual TCR-T cell component of T-Plex, alone, selectively kills target cells intact for the relevant HLA.

[0112] To determine the tumor killing ability of T-Plex-204-A0201 / 204-C0702, individual TCR-T cell components derived from donors from three independent batches of representative materials of the process were combined and co-cultured with a combination of heterogeneous (non-uniform) targets "A2 + C7". As shown in Figure 9, all three batches showed similar trends, confirming that the viability of the non-uniform target cell mixture decreased in all three batches of T-Plex-204-A0201 / 204-C0702. Barcoding of the two target cell populations enabled specific analysis of the viability of the A2 and C7 target subsets. The viability of U266B1 HLA-C*07:02 KO target cells decreased when co-cultured with T-Plex-204-A0201 / C0702 or isolated TSC-204-A0201 TCR-T cells. Similarly, the viability of U266B1 HLA-A*02:01 KO target cells decreased when co-cultured with T-Plex-204-A0201 / C0702 or isolated TSC-204-C0702 TCR-T cells.

[0113] When compared to the baseline survival rate described above, specific killing (calculated as the decrease in survival rate compared to the above baseline) showed that the T-Plex-204-A0201 / 204-C0702 products from three independent batches had specific tumor killing activity, with activities of 55.31%, 71.18%, and 60.89% respectively in U266B1 HLA-C*07:02 KO target cells, and activities of 53.38%, 61.77%, and 48.45% respectively in U266B1 HLA-A*02:01 KO target cells. In particular, the individual killing activities of the TSC-204-C0702 and TSC-204-A0201 TCR-T cell products were comparable to the killing activity of the combination of T-Plex-204-A0201 / 204-C0702, indicating the effective function of the combination of the two TCR-T cell components. As described above, this assay is relatively short-term, and in other assays, such as longer-term tests, additional synergistic activities (from cytokine-dependent phenomena described herein) may be observed, such as a decrease in the effector-to-target ratio of one or more TCRs in a TCR combination that shows a supporting effect of other TCR(s) on reduced TCR(s).

[0114] Therefore, the results provided in Figure 9 demonstrate that in the face of a heterogeneous target cell population, a single TCR-T cell component can effectively address a portion of the tumor cells and spare a subset of cells that cannot be recognized by the TCR-T cells (here, because the cells have lost the relevant HLA). The multiplexed TCR-T therapy (here, the combination of TSC-204-A0201 and TSC-204-C0702) simultaneously resulted in the killing of both subsets of cancer cells. Therefore, this data confirms that combining multiple TCR-T therapies such as TSC-204-A0201 and TSC-204-C0702 can treat tumors with LOH to maximize the chance of achieving complete response.

[0115] Incorporation by reference All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. In case of conflict, this application, including any definitions herein, will control.

[0116] Any polynucleotide and polypeptide sequences having reference numbers related to entries in public databases maintained by, for example, the Institute for Genomic Research (TIGR) at tigr.org on the World Wide Web and / or the National Center for Biotechnology Information (NCBI) at ncbi.nlm.nih.gov on the World Wide Web are also hereby incorporated by reference in their entirety.

[0117] Equivalents and Ranges Details of one or more embodiments encompassed by the present invention are set forth in the foregoing description. Representative, exemplary materials and methods are described above, but any materials and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments encompassed by the present invention. Other features, objects, and advantages of the present invention will be apparent from the description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present description, as above, will control.

[0118] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the descriptions provided herein, and such equivalents are intended to be included within the scope of the appended claims.

[0119] The term "comprising" is intended to be unrestricted and it should be noted that there is no need to include additional elements or steps. Thus, when the term "comprising" is used in this specification, the term "consisting of" is also included and disclosed.

[0120] When ranges are given, the endpoints are included. Further, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges are to be taken as including any specific value or sub-range within the stated range of the lower limit of the unit of the range to one tenth thereof, as appropriate, within the state ranges of different embodiments encompassed by the present invention, unless the context clearly dictates otherwise.

[0121] In addition, it should be understood that any particular embodiment encompassed by the present invention within the prior art scope may be explicitly excluded from any one or more of the claims. Such embodiments are considered to be known to one of ordinary skill in the art and thus may be excluded even if the exclusion is not explicitly described herein. Any particular embodiment of a composition (e.g., any antibiotic, therapeutic or active ingredient, any manufacturing method, any method of use, etc.) encompassed by the present invention may be excluded from any one or more of the claims for any reason, whether or not related to the existence of the prior art.

[0122] The words used are words of description rather than limitation, and it is understood that changes may be made within the scope of the appended claims without departing from the true scope and spirit encompassed by the broader aspects of the present invention.

[0123] The present invention has been described to a certain length and with a certain degree of particularity with respect to some of the described embodiments, but it is not intended that it be limited to any such details or embodiments or specific embodiments, but rather to provide the broadest possible interpretation of the scope of such claims in view of the prior art, and thus the appended claims should be interpreted with reference to the scope intended to be encompassed by the present invention so as to effectively encompass such scope.

Claims

[Claim 1] The invention described in the specification.

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