HK40060048B - T cell receptors specific for mesothelin and their use in immunotherapy - Google Patents

Description

Mesothelin-specific T-cell receptor and its application in immunotherapy

Related applications

The sequence listing associated with this application is provided in text format instead of a paper copy, and is incorporated herein by reference. The text file containing the sequence listing is named 360056_475WO_SEQUENCE_LISTING.txt. The text file is 115KB in size, created on November 8, 2019, and submitted electronically via EFS-Web.

Technical Field

This disclosure relates to the biomedical field, and more specifically, to methods and compositions for treating diseases or conditions in which cells express mesothelin, such as in cancer treatment. In particular, embodiments of this disclosure relate to methods and compositions for TCRs with high affinity for the tumor-associated antigen mesothelin, T cells expressing such high-affinity antigen-specific TCRs, nucleic acids encoding them, and methods of use for cell immunotherapy including engineered T cells.

Background Technology

Adoptive transfer of tumor-specific T cells is an attractive strategy for eliminating existing tumors and requires the establishment of a robust antigen-specific T cell population in vivo to eliminate existing tumors and prevent recurrence (see Stromnes, et al., Immunol. Rev. 257:145, 2014). Although metastatic tumor-specific CD8 ⁺ cytotoxic T lymphocytes (CTLs) are safe and can mediate direct antitumor activity in specific patients (see Chapuis et al., Cancer Res. 72:LB-136, 2012; Chapuis et al., Sci. Transl. Med. 5:174ral27, 2013; and Chapuis et al., Proc. Natl. Acad. Sci. USA 09:4592, 2012), the variability in the affinity of CTLs isolated from each patient or donor limits their antitumor efficacy in clinical trials (see Chapuis et al., 2013). Since TCR affinity is a crucial determinant of CTL affinity (see Zoete et al., Frontiers Immunol. 4:26%, 2013), strategies have been developed to isolate T cells from well-characterized T cell clones specific to tumor-specific antigens using high-affinity TCRα/β gene retargeting donor or patient T cell antigen specificity (see Stromnes et al., Immunol. Rev. 257:145, 2014 and Robbins et al., J. Clin. Oncol. 29:917, 2011).

Such high-affinity self/tumor-responsive T cells are rare because T cells expressing self/tumor-responsive TCRs are subject to central and peripheral tolerance (see Stone and Kranz, Frontiers Immunol. 4:244, 2013), and there is significant variation in relative TCR affinity among donors. Therefore, screening numerous matched donors is necessary to identify sufficiently high-affinity tumor-specific T cell clones from which TCRα/β gene therapy constructs can be generated. For example, isolating naturally induced Wilms tumor antigen 1 (WT1)-specific TCRs with high functional affinity for a single HLA allele requires screening hundreds of WT-specific T cell lines from the periphery, which represent a library of tens of thousands of individual T cell clones from more than 75 normal donors. This is a very time-consuming and labor-intensive process (see Chapuis et al., 2013; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; and Ho et al., J. Immunol. Methods 310:40, 2006).

There is a need for alternative antigen-specific immunotherapies for various cancers, such as solid tumors. The currently disclosed embodiments address these needs and provide other related advantages.

Attached Figure Description

The embodiments disclosed herein will become clearer from the following description and the appended claims, taken in conjunction with the accompanying drawings.

Figure 1A illustrates the identification and selection of Msln ₂₀ (SEQ ID NO:31)-specific TCRs based on fold enrichment of TCRs for peptide:HLA tetramer binding. TCRs selected for further research are circled.

Figure 1B depicts the identification and selection of TCRs specific to Msln ₅₃₀ (SEQ ID NO:32) based on the multi-enrichment of tetramers from the TCR pool that binds to the Msln ₅₃₀ :HLA tetramer. TCRs selected for further research are circled.

Figure 2 illustrates whether measuring cell-expressed TCRs (ranked by affinity, based on tetramer binding) in the presence or absence of the CD8 co-receptor can detect the binding of Msln ₅₃₀ -specific TCRs to the tetramer in the assay of the Msln ₅₃₀ peptide:HLA complex. CD8-independent binding is associated with high affinity for individual T cell clones.

Figures 3A-3C show further functional assays of Msln-specific TCRs. (A) Representative data of antigen-driven activated T cell clones, measured by flow cytometry using reporter cell lines expressing the Nur77-tdTomato transgene. Nur77 is an indicator of antigen receptor signal transduction in human T cells (see, for example, Ashouri and Weiss, J. Immunol. 198(2):657-658(2017)). In this assay, as shown, T cell clones were incubated with T2 target cells treated with pulses of peptide at increased concentrations. (B) Nur77 reporter gene activity of TCRs, ranked according to sensitivity to T2 cells treated with peptide pulses at specified concentrations. (C) Ranking of affinity for TCRs based on EC50 of the Nur77 reporter gene active peptide. Among the TCR clones tested, “B11” (also referred to herein as 11B) had the lowest EC50.

Figure 4 shows the functional assessment of TCR clonal response peptides based on Nur77 tomato reporter gene activity. Despite exhibiting low tetramer binding, several TCRs, including “B9” and “A11” (also referred to as “11A” in this paper), still possess high antigen specificity. The TCRs are sorted according to tetramer binding (from left to right, top to bottom).

Figures 5A-5C show the functional assessment of heterologously expressed TCRs in primary CD8+ T cells. CD8+ T cells were purified from donor PBMCs and lentivirally transduced with each TCR. After 8 days, highly tetramer-positive cells were further sorted and expanded for 8-10 days.

Figures 6A-6C show the characteristics of primary CD8+ T cells transduced with indicated Msln-specific TCRs and assess functional activity, as measured by interferon-γ production (A, B), after incubation with peptide-pulsed T2 cells. TCRs were ranked by EC50 based on the amount of peptide entering T2 cells (C).

Figures 7A-7D show the specific lysis of two representative Msln-positive tumor cell lines ((A,B)MDA-MB-468 and (C,D)MDA-MB- ₂₃₁ ) by CD8+ T cells transduced with indicated Msln ₅₃₀ -specific TCRs or Msln 20-specific TCRs in the presence or absence of exogenous IFN-γ.

Figure 8 illustrates an alanine mutagenesis scan to assess which amino acids of the target MsIn ₂₀ peptide (SEQ ID NO:31) are essential for efficient binding and killing by the exemplary MsIn ₂₀ -specific TCR. A series of variant peptides were generated in which alanine was replaced at each consecutive position along the peptide in SEQ ID NO:31, and IFN-γ production of each variant peptide was assessed by CD8+ T cells expressing the specified MsIn _20- specific TCR. These data indicate that positions 3–6 of SEQ ID NO:31 are essential for TCR binding. In the concordant sequence SEQ ID NO:60, “X” indicates that substitution mutations of the residues shown in SEQ ID NO:31 to alanine do not affect or substantially do not affect the functional binding of the TCR to its homologous peptide target.

Figures 9A-9D show the results of alanine mutagenesis scanning experiments using TCRs specific to Msln ₂₀ (SEQ ID NO:31) or Msln ₅₃₀ (SEQ ID NO:32). The x-axis shows the percentage of IFN-γ+ T cells responding to each alanine-substituted peptide. The y-axis shows the sequence of the peptide tested, where X indicates that the residue is not essential for TCR specificity, as indicated by near-normal functional activity compared to wild-type peptides.

Figure 10 shows the human peptide (SEQ ID NO: 63-77), which showed potential cross-reactivity with the target Msln ₅₃₀ peptide (SEQ ID NO: 32) based on the specificity of the TCR determined by the alanine scan experiment. The genes encoding the peptides shown are shown on the left side of the table. As described in Example 8, a common sequence containing the essential residues identified by the alanine scan was input into the prediction algorithm.

Figures 11A and 11B depict the analysis of synthetic peptides with potential homology to Msln ₅₃₀ in the human proteome. As described in Example 9, two Msln ₅₃₀ -specific TCRs were measured by IFN-γ (Figures 11A and 11B) (functional activity after incubation with T2 cells pulsed with a high dose (10 μM) of peptide). (B) For peptides showing cross-reactivity with the tested TCRs, dose-dependent titration was performed to determine the EC50. TCRs were ranked by EC50 based on the amount of peptide pulsed into T2 cells. Peptide #10 originates from a gene called EHF. This gene encodes a protein belonging to the ETS transcription factor subfamily characterized by epithelial-specific expression. ETS acts as a transcriptional repressor and may be involved in epithelial differentiation and oncogenesis.

Figures 12A-12I depict experiments that investigate the allogeneic potential of T cells (Meso20-3B, Meso530-11A, or Meso530-11B) expressing the exemplary Msln-specific TCR of this disclosure by targeting multiple donor-derived lymphoblast-like cell lines (LCLs) in the absence of wild-type peptides.

Figures 13A-13H depict additional analyses of homoreactivity by targeting LCLs from different donor sources to assess homoreactivity without cross-reactivity with other HLA subtypes, as described in Example 10. As shown for cell lines FAH and GIM, Meso530-11B indicates peptides of potentially reactive non-HLA-A2 alleles in the presence of these alleles (as highlighted in the table).

Detailed Implementation

In some aspects, this disclosure provides binding proteins comprising a TCRα chain variable domain (Vα) and a TCRβ chain variable domain (Vβ), and capable of specifically binding Msln _20-28 or Msln _530-538 epitopes and/or peptides (Msln _20-28 (SLLFLLFSL; SEQ ID NO: 31)) and Msln _530-538 (SEQ ID NO: 32 (VLPLTVAEV)), also referred to herein as Msln ₂₀ and M ₅₃₀ , respectively); for example, in peptide:HLA complexes. In any of the embodiments currently disclosed, the Msln-specific binding protein is capable of binding Msln peptide:HLA complexes, wherein the Msln peptide comprises the amino acid sequence shown in SEQ ID NO: 31 or 32, and wherein HLA is or comprises HLA-A2, such as HLA-A*02:01.

In some embodiments, the Msln-specific _20-28 specific binding protein comprises: (a) TCRVα, which comprises the CDR3 amino acid sequence shown in SEQ ID NO: 33 or 35, and TCRVβ, wherein optionally, TCRVβ has at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) the same amino acid sequence as shown in SEQ ID NO: 95 or 97; (b) TCRVβ, which comprises the CDR3 amino acid sequence as shown in SEQ ID NO: 34 or 36, and (b) TCRVα, wherein optionally, TCRVα has the same amino acid sequence as shown in SEQ ID NO: 35 or 36. The amino acid sequence shown in NO:96 or 98 has at least about 85% (i.e., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identity; (c) TCRVα, which comprises the CDR3 amino acid sequence shown in SEQ ID NO:33 or 35, wherein optionally, TCRVα has at least about 85% identity with the amino acid sequence shown in SEQ ID NO:96 or 98; and TCRVβ, which comprises the CDR3 amino acid sequence shown in SEQ ID NO:34 or 36, wherein optionally, TCRVβ has at least about 85% identity with the amino acid sequence shown in SEQ ID NO:95 or 97.

Unless otherwise expressly stated, as used herein, “at least about” indicates a percentage of sequence identity, including the indicated percentage ± 20%, and every integer and non-integer percentage higher than that specific percentage. Thus, “at least about 85%” identity with a reference sequence (e.g., any of SEQ ID NO: 1-123) includes about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the reference sequence, and also includes all non-integer percentages (e.g., 92.5%, 99.1%, etc.) between two integer percentages.

In some embodiments, the Msln _20-28 specific binding protein comprises the CDR3α amino acid sequence shown in SEQ ID NO:33 and the CDR3β amino acid sequence shown in SEQ ID NO:34. In further embodiments, the binding protein comprises the CDR1α amino acid sequence shown in SEQ ID NO:80, the CDR2α amino acid sequence shown in SEQ ID NO:81 or 118, the CDR1β amino acid sequence shown in any one of the sequences in SEQ ID NO:78, 82, 83 or 84, and the CDR2β amino acid sequence shown in SEQ ID NO:79. In some embodiments, _Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:96, and/or _Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:95, wherein optionally CDR1α, CDR2α, CDR1β and/or CDR2β remain unchanged.

In some embodiments, the Msln _20-28 specific binding protein comprises (i) TCRVβ, which comprises (a) an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRBV12-4*01 (e.g., the amino acid sequence encoded by TRBV12-4*01 having a length of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 108 consecutive amino acids); and/or (b) an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRBJ2-7*01 (e.g., the amino acid sequence encoded by TRBJ2-7*01 having a length of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acids). An amino acid sequence with 85% identity; and/or (ii) TCRVα, comprising (a) an amino acid sequence with at least about 85% identity to an amino acid sequence encoded by TRAV1-1*01 (e.g., an amino acid sequence encoded by TRAV1-1*01 having a continuous amino acid length of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 107) and/or (b) an amino acid sequence encoded by TRAJ3*01 (e.g., an amino acid sequence encoded by TRAJ3*01 having a length of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, or 20 amino acids).

In any of the currently disclosed embodiments, TCRVβ may contain at least about 85% of the same amino acid sequence as the amino acid sequence encoded by TRBD1*01 or TRBD2*02.

The amino acid sequences encoded by these and other TCR genes are known and can be found, for example, at imgt.org, a database that provides gene tables and nucleotide and amino acid sequences for human TRAV, TRBV, TRAJ, TRBJ, TRBD, TRAC, and TRBD.

In some embodiments, the Msln _20-28 specific binding protein comprises the CDR3α amino acid sequence shown in SEQ ID NO:36 and the CDR3β amino acid sequence shown in SEQ ID NO:35. In some embodiments, the binding protein further comprises the CDR1α amino acid sequence shown in SEQ ID NO:85, the CDR2α amino acid sequence shown in SEQ ID NO:86 or 119, the CDR1β amino acid sequence shown in any one of SEQ ID NO:82, 83 or 84, and the CDR2β amino acid sequence shown in SEQ ID NO:79. In some embodiments, _Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:98, and/or _Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:97, wherein CDR1α, CDR2α, CDR1β and/or CDR2β optionally remain unchanged.

In some embodiments, the Msln _20-28 specific binding protein comprises TCRVα, which comprises (a) an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRAV12-3*01 (e.g., an amino acid sequence encoded by TRAV12-3*01 of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 108 consecutive amino acids). and/or (b) an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRA29*01 (e.g., an amino acid sequence encoded by TRA29*01 of at least about 5, 6, 7, 8, 9, 10, 11, 12, 12, 14, 15, 16, 17, 18, or 19 consecutive amino acids).

In some embodiments, mutagenesis of any one or more of residues 1, 2, 7, 8, or 9 of SEQ ID NO:31 does not eliminate or substantially impair the binding of the Msln _20-28 specific binding protein. In some embodiments, the Msln _20-28 specific binding protein is capable of binding peptides comprising or consisting of the common amino acid sequence shown in SEQ ID NO:60, such as in the peptide:HLA complexes disclosed herein.

In some embodiments, the Msln _530-538 specific binding protein comprises: (a) TCRVα and TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO:37 or 39, wherein TCRVβ optionally has at least about 85% identity with the amino acid sequence shown in SEQ ID NO:99 or 101; (b) TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO:34 or 36, and (b) TCRVα, wherein the TCRVα optionally has at least about 85% identity with the amino acid sequence set shown in SEQ ID NO:100 or 102; or (c) TCRVα comprising the CDR3 amino acid sequence as shown in SEQ ID NO:37 or 39 and TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO:38 or 40.

In some embodiments, the Msln _530-538 specific binding protein comprises the CDR3α amino acid sequence shown in SEQ ID NO:37 and the CDR3β amino acid sequence shown in SEQ ID NO:38. In some embodiments, the binding protein further comprises the CDR1α amino acid sequence shown in SEQ ID NO:89, the CDR2α amino acid sequence shown in SEQ ID NO:90, the CDR1β amino acid sequence shown in SEQ ID NO:83 or 87, and the CDR2β amino acid sequence shown in SEQ ID NO:88. In some embodiments, _Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:100, and/or _Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:99, wherein optionally CDR1α, CDR2α, CDR1β, and/or CDR2β remain unchanged.

In some embodiments, the Msln _530-538 specific binding protein comprises the CDR3α amino acid sequence as shown in SEQ ID NO:39 and the CDR3β amino acid sequence as shown in SEQ ID NO:40. In some embodiments, the binding protein further comprises the CDR1α amino acid sequence as shown in SEQ ID NO:93, the CDR2α amino acid sequence as shown in SEQ ID NO:94, the CDR1β amino acid sequence as shown in SEQ ID NO:83, 84, or 91, and the CDR2β amino acid sequence as shown in SEQ ID NO:92. In some embodiments, Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:102, and/or Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:101, wherein CDR1α, CDR2α, CDR1β, and/or CDR2β are optionally unchanged.

In some embodiments, the Msln _530-538 specific binding protein includes TCRVβ, which contains an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRBJ2-3*01 (e.g., an amino acid sequence TRBJ2-3*01 with a length of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 15 consecutive amino acids).

In some embodiments, mutagenesis of any one or more of residues 3, 5, 6, or 9 of SEQ ID NO:32 does not eliminate or substantially impair the binding of the Msln _530-538 -specific binding protein. In some embodiments, the Msln _530-538 -specific binding protein is capable of binding peptides comprising or consisting of the common amino acid sequence shown in SEQ ID NO:61, such as in the peptide:HLA complexes disclosed herein.

In some embodiments, mutagenesis of any one or more of residues 1, 5, or 9 of SEQ ID NO:32 does not eliminate or substantially impair the binding of the Msln _530-538 specific binding protein. In some embodiments, the Msln _530-538 specific binding protein is capable of binding peptides comprising or consisting of the common amino acid sequence shown in SEQ ID NO:62, such as in the peptide:HLA complexes disclosed herein.

In some embodiments, the Msln _530-538- specific binding protein of this disclosure does not bind to or binds nonspecifically to Msln _530-538 to the peptide:HLA complex, wherein the peptide comprises or is composed of the sequence amino acids shown in any one or more of SEQ ID NO:63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 and 77, and wherein the HLA optionally comprises HLA-A2, such as HLA-A:02*01.

In any of the currently disclosed embodiments, in the absence of or independently of CD8, Msln-specific binding proteins (i.e., Msln _20-28 -specific binding proteins, Msln530-538-specific binding proteins) are capable of binding the Msln peptide:HLA complex disclosed herein. In some embodiments, the binding proteins (e.g., when expressed on the cell surface of human T cells) have Msln peptide EC50 of about 9 μM, about 8 μM, about 7 μM, about 6 μM, about 5 μM, about 4 μM, about 3 μM, about 2 μM, about 1 μM, about 0.9 μM, about 0.8 μM, about 0.7 μM, about 0.6 μM, about 0.5 μM, about 0.4 μM, about 0.3 μM, about 0.2 μM, or less.

In the absence of the Msln peptide antigen, Msln-specific binding proteins do not exhibit isotropic reactivity across various human HLA types. In some embodiments, when the Msln peptide provided herein is absent, upon contact with cells expressing the following substances: (i) HLA-C6:02:01; (ii) HLA-B13:01:01 without HLA-B13:02:01; (iii) HLA-A3; (iv) HLA-A29; (v) HLA-B40; (vi) HLA-B44; (vii) HLA-C3; (viii) HLA-C16; (ix) HLA-A1; (x) HLA-24; (xi) HLA-B7; (xii) HLA-B57; (xiii) HLA-C7; (xiv) HLA-A11; (xv) HLA-B15; (xvi) HLA-C4; (xvii) HLA-C12; (xviii) HLA-B8; (xix) HLA-B 49; (xx)HLA-B51; (xxi)HLA-C15; (xxii)HLA-A30; (xxiii)HLA-A68; (xxiv)HLA-C2; (xxv)HLA-A32; (xxvi)HLA-A33; (xxvii)HLA-B55; (xxviii)HLA-C1; (xxvix)HLA-C5; (xxix)HLA-B8; (xxx)HLA-B35; or any combination of (xxxi)(i)-(xxx), where immune cells (e.g., T cells) expressing the Msln-specific binding protein of this disclosure do not produce IFN-γ and/or do not exhibit activation (e.g., CD8 expression, CD3 expression, Nur77 expression) and/or cytotoxic activity (e.g., specific killing, production, and release of perforin and/or granzymes).

Also provided are compositions encoding and/or expressing binding proteins and recombinant host cells (e.g., immune cells, such as T cells). In any of the currently disclosed embodiments, the binding protein is capable of being expressed on the cell surface by host T cells. In any of the currently disclosed embodiments, immune cells are activated by binding a Msln-specific binding protein expressed on the surface of immune cells (e.g., T cells) to a Msln peptide:HLA complex, wherein activation is optionally determined by the expression and/or activity of Nur77.

The currently disclosed binding proteins are highly sensitive to homologous Mlsn peptide antigens. In some embodiments, Nur77 expression increases when immune cells are present with approximately ^10⁻² μM peptide, approximately ^10⁻¹ μM peptide, approximately 1 μM peptide, or approximately ^10¹ μM peptide, wherein the peptide is optionally presented by an antigenic peptide, i.e., in a peptide:HLA complex.

On the other hand, polynucleotides encoding mesothelin-specific binding proteins as described herein are provided. In some embodiments, the polynucleotide encoding the binding protein comprises a polynucleotide having at least about 50% sequence identity with any of the polynucleotide sequences in SEQ ID NO: 1-5, 9-13, 17-21, 25-27, and 120 (i.e., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). Vectors comprising the polynucleotide are also provided.

The currently disclosed binding proteins, recombinant host cells, and related compositions can be used to treat subjects with diseases or conditions related to mesothelin expression and/or activity, such as cancer. In some embodiments, the cancer is a solid carcinoma. In some embodiments, solid carcinoma is or includes biliary carcinoma, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoidoma, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine cancer. In some embodiments, the compositions and recombinant host cells disclosed herein can be used to treat cancers in which the Msln _20-28 peptide is expressed on cancerous tumor cells, or cancers in which the Msln _530-538 peptide is expressed on tumor cells, such as mesothelioma, pancreatic cancer, ovarian cancer, or lung cancer.

This article also provides polynucleotides encoding binding proteins as described herein, vectors containing polynucleotides encoding binding proteins, and host cells containing the vectors.

Before elaborating on this disclosure in more detail, definitions of certain terms used herein may be provided to aid understanding. Unless otherwise expressly defined, the technical terms used herein have their normal meaning as understood in the art. Additional definitions are set forth throughout this disclosure.

In this specification, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, its fraction (e.g., one-tenth and one-hundredth of an integer), unless otherwise stated. Similarly, unless otherwise stated, any numerical range relating to any physical characteristic, such as polymer subunits, size, or thickness, as described herein shall be understood to include any integer within the range described. When referring to measurable values, ranges, or structures, the term "about" as used herein means a difference of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, unless otherwise stated.

It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the listed components. The use of alternatives (e.g., “or”) should be understood to refer to any, all, or any combination of the alternatives. As used herein, the terms “comprising,” “having,” and “including” are used synonymously, and these terms and their variations are intended to be interpreted as non-limiting.

"Optional" or "optional" means that the element, component, event, or condition described below may or may not occur, and the description includes both the cases in which the element, component, event, or condition occurs and the cases in which it does not occur.

Furthermore, it should be understood that this application discloses individual constructs or groups of constructs derived from various combinations of the structures and subunits described herein, to the same extent as each construct or group of constructs. Constructs are proposed individually. Therefore, the selection of a particular structure or a particular subunit is within the scope of this disclosure.

The term "substantially composed of" is not equivalent to "comprising," but rather refers to the material or step specified in the claim, or the material or step that does not substantially affect the essential characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, or linker) or a protein (which may have one or more domains, regions, or modules) is "substantially" composed of specific amino acids when the amino acid sequence of the domain, region, module, or protein includes a sequence consisting of extensions, deletions, mutations, or combinations thereof (e.g., amino acids at or between carboxyl terms or amino acids between domains) at most 20% of the length of the domain, region, module, or protein (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) and does not substantially affect (i.e., the reduced activity does not exceed 50%, e.g., not more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain, region, module, or protein (e.g., the target binding affinity of the binding protein).

As used herein, “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to that of naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that have been modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as natural amino acids, namely, the α-carbon bound to hydrogen, carboxyl, amino, and R groups, such as homoserine, oroleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., oroleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimics are compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to that of natural amino acids.

As used herein, “mutation” refers to a sequence change in a nucleic acid or polypeptide molecule compared to a reference or wild-type nucleic acid or polypeptide molecule, respectively. Mutations can result in several different types of sequence changes, including substitutions, insertions, or deletions of nucleotides or amino acids. In some embodiments, a mutation is a substitution of one or three codons or amino acids, a deletion of one to about five codons or amino acids, or a combination thereof.

"Conservative substitution" refers to amino acid substitutions that do not significantly affect or alter the binding properties of a specific protein. Generally, a conservative substitution is a substitution in which the substituted amino acid residue is replaced by an amino acid residue with a similar side chain. Conservative substitutions include substitutions found in one of the following groups: Group 1: Alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: Aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), glutamine (Gln or Q); Group 4: Arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: Isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); Group 6: Phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Alternatively, amino acids can be grouped into conserved substituents by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, aliphatic groups may include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other conserved substituents include: sulfur-containing: Met and cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar, or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and larger aromatic residues: Phe, Tyr, and Trp. Further information can be found in Creighton (1984) Protein, W.H. Freeman, and Company. In some embodiments, the variant proteins, peptides, polypeptides, and amino acid sequences of this disclosure may contain one or more conserved substitutions relative to a reference amino acid sequence.

As used herein, “protein” or “peptide” refers to a polymer of amino acid residues. The term “protein” applies to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids and non-naturally occurring amino acid polymers.

As used herein, a "fusion protein" refers to a protein having at least two distinct domains or motifs in a single strand, wherein said domains or motifs are not naturally present together (e.g., in a specified arrangement, order, or number, or not present at all). In some embodiments, the fusion protein comprises at least two distinct domains or motifs that are not naturally present together in a single peptide or polypeptide. The polynucleotide encoding the fusion protein can be constructed using PCR, recombinant engineering, etc., or such fusion proteins can be synthesized. The fusion protein may further comprise other components, such as tags, adapters, or transduction markers. In some embodiments, the fusion protein expressed or generated by a host cell (e.g., T cells) is located on the cell surface, wherein the fusion protein is anchored to the cell membrane (e.g., via a transmembrane domain) and comprises an extracellular or intracellular portion or component (e.g., comprising a binding domain, and in some embodiments, a adapter, a spacer, or both) and an intracellular portion or component.

"Linking amino acid" or "linking amino acid residue" refers to one or more (e.g., about 2-10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, such as adjacent constant domains between binding domains or between the TCR chain and an adjacent self-cleaving peptide. Linking amino acids can be generated by the design of the fusion protein construct (e.g., amino acid residues generated during the construction of the nucleic acid molecule encoding the fusion protein due to the use of restriction enzyme sites).

A "nucleic acid molecule" or "polynucleotide" is a polymeric compound comprising covalently linked nucleotides, which can be composed of native subunits (such as purine or pyrimidine bases) or non-native subunits (such as a morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, while pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polynucleotides (RNA), polydeoxyribonucleic acid (DNA), which can be single-stranded or double-stranded cDNA, genomic DNA, and synthetic DNA. If single-stranded, a nucleic acid molecule can be a coding strand or a non-coding strand (antisense strand). Nucleic acid molecules encoding amino acid sequences include all nucleotide sequences encoding the same amino acid sequence. Some forms of nucleotide sequences can also include introns, such that introns can be removed through co-transcriptional or post-transcriptional mechanisms. In other words, different nucleotide sequences can encode the same amino acid sequence due to redundancy or degeneracy of the genetic code or through splicing.

Variants of the nucleic acid molecules described in this disclosure are also considered. The variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the nucleic acid molecules of the polynucleotides defined or referenced herein, or hybridize under stringent hybridization conditions: 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68 °C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42 °C. The nucleic acid molecule variants retain the ability to encode fusion proteins or their binding domains having the functions described herein (e.g., specific binding to target molecules).

"Sequence identity percentage" refers to the relationship between two or more sequences determined by comparing sequences. Preferred methods for determining sequence identity are designed to yield the best match between the compared sequences. For example, sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment). Furthermore, non-homologous sequences may be ignored for comparison purposes. Unless otherwise stated, the sequence identity percentage referred to herein is calculated over the length of a reference sequence. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percentage identity calculations can be performed using BLAST programs (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithms used in BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. In the context of this disclosure, it will be understood that when sequence analysis software is used for analysis, the results of the analysis are based on the "default values" of the cited program. "Default value" refers to any set of values or parameters that are initially loaded with the software during initialization.

As understood in the art, the “similarity” between two peptides is determined by comparing the amino acid sequence and conserved amino acid substituents of one peptide with the sequence of the second peptide (e.g., using GNEWORKS™, Align, Clustal ^™ , BLAST algorithm, etc.). In some embodiments, the BLAST algorithm is preferred.

The term "isolated" refers to the removal of material from its original environment (e.g., its natural environment if it is naturally occurring). For example, instead of isolating naturally occurring nucleic acids or polypeptides present in a living animal, it means isolating the same nucleic acids or polypeptides that are isolated from some or all of the coexisting substances in the natural system. Such nucleic acids may be part of a vector and/or such nucleic acids or polypeptides may be part of a composition (e.g., cell lysate), and are still isolated because, for the nucleic acid or polypeptide, such a vector or composition is not part of the vector's natural environment. The term "gene" refers to a segment of DNA associated with the production of a polypeptide chain. It includes the regions before and after the coding region ("leader and tail regions") and the insertion sequences (introns) between the individual coding segments (exons).

In some cases, the term "variant" as used herein refers to at least one fragment of a full-length sequence, and more specifically, to one or more amino acid or nucleic acid sequences that are truncated at one or two ends by one or more amino acids relative to the full length. Such fragments comprise or encode peptides having at least 6, 7, 8, 10, 12, 15, 20, 25, 50, 75, 100, 150, or 200 consecutive amino acids having the original sequence or a variant thereof. The total length of the variant may be at least 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids.

In some embodiments, the term "variant" refers not only to at least one fragment, but also to a polypeptide or fragment thereof comprising at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the same amino acid sequence as a reference amino acid sequence or a fragment thereof, wherein amino acids other than those essential for biological activity or the folding or structure of the polypeptide are missing or substituted, one or more such essential amino acids are substituted in a conserved manner, and/or amino acids are added to such a polypeptide to maintain the polypeptide's biological activity. Prior art includes various methods that can be used to compare two given nucleic acid or amino acid sequences and calculate the degree of identity (see, for example, Arthur Lesk (2008), Introduction to Bioformatics, Oxford University Press, 2008, ^3rd edition). In some embodiments, the Clustal W software can be used with default settings (Larkin, MA, et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948).

In some embodiments, variants may additionally include chemical modifications, such as isotopic labeling or covalent modifications, such as glycosylation, phosphorylation, acetylation, decarboxylation, citrullination, hydroxylation, etc. Methods for modifying peptides are known and are generally employed to avoid eliminating or substantially reducing the desired activity of the peptide.

In one embodiment, the term "variant" of a nucleic acid molecule includes nucleic acids whose complementary strands hybridize, for example, with a reference or wild-type nucleic acid under stringent conditions. The stringency of the hybridization reaction can be readily determined by those skilled in the art and is typically determined by empirical calculations of probe length, washing temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured DNA to anneal with its complementary strand in an environment below its melting temperature: the higher the degree of homology required between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For further details and explanations regarding the stringency of the hybridization reaction, see Ausubel, F.M. (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Furthermore, those skilled in the art can refer to the manual (Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W., and Schleifer, K.H. (1991) International Journal of Systematic Bacteriology 41:255-260) for guidance on how to identify DNA sequences through hybridization. In one embodiment, stringent conditions apply to any hybridization, i.e., hybridization occurs only when the probe and target sequence have 70% or higher identity. Probes with low identity to the target sequence can hybridize, but such hybrids are unstable and will be removed in a washing step under stringent conditions (e.g., reducing the salt concentration to 2×SSC, or optionally and subsequently to 0.5×SSC, at temperatures, for example, approximately 50°C–68°C, approximately 52°C–68°C, approximately 54°C–68°C, approximately 56°C–68°C, approximately 58°C–68°C, approximately 60°C–68°C, approximately 62°C–68°C, approximately 64°C–68°C, or approximately 66°C–68°C). In one embodiment, the temperature is approximately 64°C–68°C or approximately 66°C–68°C. The salt concentration can be adjusted to 0.2×SSC or even 0.1×SSC. The sequence is separable from a reference or wild-type sequence by at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%. In one embodiment, as used herein, the term "variant of nucleic acid sequence" refers to any nucleic acid sequence that encodes the same amino acid sequence as the reference nucleic acid sequence and its variants, based on the degeneracy of the genetic code.

A “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to the parent or reference compound of this disclosure, but differs slightly in composition in certain circumstances (e.g., a base, atom, or functional group is different, added, or removed; or one or more amino acids are mutated, inserted, or deleted), such that the polypeptide or encoded polypeptide is capable of performing at least one functional efficiency of the encoded parent polypeptide at least 50%, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity level of the parent polypeptide. In other words, the polypeptide or functional variant of the encoded polypeptide of the present invention has “similar binding,” “similar affinity,” or “similar activity” when the functional variant exhibits a performance reduction of no more than 50% in a selected assay. Compared to the parent or reference peptide, it is used for assays to measure binding affinity (e.g., tetramer staining to measure association (Ka) or dissociation (KD) constants), affinity, or activation of host cells. As used herein, a “functional moiety” or “functional fragment” refers to a polypeptide or polynucleotide that contains only the domains, motifs, portions, or fragments of the parent or reference compound, and that the polypeptide or encoded polypeptide retains at least 50% of its activity. Preferably, at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity level of the parent peptide associated with the domains, portions, or fragments of the parent or reference compound, or that provides a biological benefit (e.g., effector function).

When the function of a "functional portion" or "functional fragment" of the polypeptide or encoded polypeptide of the present invention is reduced by no more than 50%, it has "similar binding" or "similar activity". Selectivity detection compared to a parent or reference polypeptide (preferably no more than 20% or 10% relative to affinity, or no more than a logarithmic difference compared to the parent or reference) is used, for example, to measure binding affinity or to measure effector function (e.g., cytokine release). In some embodiments, the functional portion refers to the "signal transduction portion" of an effector molecule, effector domain, co-stimulatory molecule, or co-stimulatory domain.

"Modified domain" or "modified protein" refers to a motif, region, domain, peptide, polypeptide, or protein that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%) different sequence identity from the wild-type motif, region, domain, peptide, polypeptide, or protein (e.g., wild-type TCRα chain, TCRβ chain, TCRα constant domain, or TCRβ constant domain).

As used herein, “heterologous,” “non-endogenous,” or “exogenous” means any gene, protein, compound, nucleic acid molecule, or activity that is not natural to the host cell or subject, or any gene, protein, compound, nucleic acid molecule, or the host cell or the subject whose natural activity has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered, resulting in a change in structure, activity, or both in the natural and altered form of the gene, protein, compound, or nucleic acid molecule. In some embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules (e.g., receptors, ligands, etc.) may not be endogenous to the host cell or subject, but rather the nucleic acid, protein, or nucleic acid molecule encoding such a gene may have been added to the host cell by means of conjugation, transformation, transfection, electroporation, etc., wherein the added nucleic acid molecule may be integrated into the host cell genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). It should be understood that in the case of a host cell containing a heterologous polynucleotide, the polynucleotide is “heterologous” to the host cell’s offspring, regardless of whether the offspring itself is manipulated (e.g., transduced) to contain the polynucleotide.

The term "homologous" or "homogeneous" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide can be homologous to a natural polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide can have altered structure, sequence, expression level, or any combination thereof. Non-endogenous polynucleotides or genes, and the polypeptides or activities they encode, can originate from the same species, different species, or combinations thereof.

As used herein, the terms “endogenous” or “natural” refer to polynucleotides, genes, proteins, compounds, molecules, or activities that are normally present in host cells or subjects.

As used herein, the term "expression" refers to the process of producing a polypeptide based on a coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The expressed nucleic acid molecule is typically operatively linked to an expression control sequence (e.g., a promoter).

The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment, such that the function of one is influenced by the function of the other. For example, when a promoter can affect the expression of a coding sequence (i.e., the coding sequence is under the transcriptional control of that promoter), the promoter is operably linked to that coding sequence. "Unrelated" means that the related genetic elements are not closely related to each other, and the function of one does not affect the function of the other.

In the context of inserting nucleic acid molecules into cells, the term “introduction” refers to “transfection,” “conversion,” or “transduction,” and includes references to the incorporation of nucleic acid molecules into eukaryotic or prokaryotic cells, where nucleic acid molecules can be incorporated into the cell’s genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA), converted into autonomous replicons, or transiently expressed (e.g., transfected mRNA). As used herein, the terms “engineered,” “recombinant,” “non-natural,” or “modified” refer to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or modification. Introduction of exogenous nucleic acid molecules, wherein such alteration or modification is introduced through genetic engineering (i.e., human intervention). Genetic alterations include, for example, the introduction of modifications to expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or other functional disruption of cellular genetic material by other nucleic acid molecules. Additional modifications include, for example, non-coding regulatory regions, where said modifications alter the expression of polynucleotides, genes, or operons; for example, the expression of endogenous nucleic acid molecules or genes is controlled, deregulated, or constitutively modified, wherein such alterations or modifications can be introduced through genetic engineering. Genetic alterations can include, for example, modifications to nucleic acid molecules encoding one or more proteins or enzymes (which may include expression control elements, such as promoters), or the addition, deletion, substitution, or other disruption or supplementation of the function of cellular genetic material. Exemplary modifications include modifications to the coding region or functional fragment of a heterologous or homologous polypeptide from a reference or parent molecule.

As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as a single nucleic acid molecule, as multiple separately controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a nucleic acid, as a fusion protein, or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it should be understood that the two or more heterologous nucleic acid molecules can enter the host chromosome as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into a single site or multiple sites, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities mentioned refers to the number of nucleic acid molecules encoding the nucleic acid molecule or the number of protein activities, not the number of separate nucleic acid molecules introduced into the host cell.

The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule. Constructs can be present in vectors (e.g., bacterial vectors, viral vectors) or can be integrated into the genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. Vectors can be, for example, plasmids, granules, viruses, RNA vectors, or linear or circular DNA or RNA molecules, which can include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. Vectors disclosed herein also include transposon systems (e.g., Sleeping Beauty, see, for example, Geurts et al., Mol. Ther. 8:108, 2003: Mátés et al., Nat. Genet. 41:753, 2009). Exemplary vectors are vectors capable of autonomous replication (attached vectors), vectors capable of delivering polynucleotides to the cellular genome (e.g., viral vectors), or vectors capable of expressing nucleic acid molecules linked to them.

As used herein, the term "host" refers to a cell (e.g., immune system cells as described herein) or microorganism that has been genetically modified with a heterologous nucleic acid molecule to produce a target polypeptide. In some embodiments, the host cell may optionally already possess or be modified to include other genetic modifications (e.g., including a detectable marker; a missing, altered, or truncated endogenous TCR; or increased expression of co-stimulatory factors) that confer desired properties related to or unrelated to the biosynthesis of, for example, heterologous proteins.

As used herein, a “binding domain” (also referred to as a “binding region” or “binding moiety”) refers to a molecule, such as a peptide, oligopeptide, polypeptide, or protein, that has the ability to specifically and nonvalently bind, conjugate, or bind to a target molecule (e.g., Msln _20-28 peptide (SEQ ID NO: 31) or Msln _530-538 peptide (SEQ ID NO: 32)), or an HLA molecule. A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinant binding partner against a biological molecule or other target. In some embodiments, the binding domain is an antigen-binding domain, such as an antibody or TCR, or a functional binding domain or antigen-binding fragment thereof. Exemplary binding domains include variable regions of single-chain antibodies (e.g., single-domain antibodies, sFv, scFv, and Fab), extracellular domains of receptors (e.g., TNF-α), ligands (e.g., cytokines and chemokines), antigen-binding regions of TCRs, such as single-chain TCRs (scTCRs), synthetic peptides selected based on their specific ability to bind biomolecules, aptamers, or single-domain antibodies (e.g., camelid or fish-derived single-domain antibodies; see Arbabi-Ghahroudi M (2017) Front. Immunol. 8:1589).

The term "variable region" or "variable domain" refers to a domain of the α-chain or β-chain (or γ-chain and δ-chain for γδTCR) of a TCR, or the heavy or light chain of an antibody, which participates in binding to an antigen (i.e., contains amino acids and/or other structures that contact the antigen and lead to binding). The variable domains of the α-chain and β-chain of a native TCR ( _Vα and _Vβ , respectively) typically have similar structures, each containing four generally conserved frame regions (FRs) and three core-derived domains (CDRs). The variable domains of the antibody heavy chain ( _VH ) and light chain ( _VL ) also typically contain four generally conserved frame regions (FRs) and three CDRs. In both TCRs and antibodies, the CDRs are frame-separated and located between the frame regions (i.e., in the primary structure).

The variable domains ( _Vα and _Vβ , respectively) of the α and β chains of natural TCRs typically have similar structures, with each domain containing four conserved frame regions (FRs) and three core regions (CDRs). The _Vα domain is encoded by two separate DNA segments, a variable gene segment and a linker gene segment (VJ). The _Vβ domain is encoded by three separate DNA segments of five (a variable gene segment, a diversity gene segment, and a linker gene segment (VDJ)). The V, D, and J alleles of human TCRs, including their nucleotides and encoded amino acid sequences, are known in the art. A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs binding to specific antigens can be isolated from antigen-binding TCRs using either the Vα or Vβ domain to screen libraries of complementary Vα or Vβ domains, respectively.

The terms “complementarity-determining region” and “CDR” are synonymous with “hypervariable region” or “HVR” and are known in the art to refer to the amino acid sequence in the TCR or antibody variable region that typically confers antigen specificity and/or binding affinity and is separated from each other by a framework sequence in the primary structure. In some cases, framework amino acids may also facilitate binding, for example, by also contacting the antigen or antigen-containing molecules. Typically, three CDRs are present in each variable region (i.e., three CDRs in each of the TCR α-chain and β-chain variable regions; and three CDRs in each of the antibody heavy chain and light chain variable regions). For the TCR, CDR3 is considered the major CDR responsible for recognizing and processing antigens. CDR1 and CDR2 interact primarily or, in some cases, only with the MHC. Variable domain sequences can be aligned with numbering schemes (e.g., Kabat, EU, International Immunogenetics Information System (IMGT) and Aho), and equivalent residue positions can be annotated and different molecules compared using the Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). In some embodiments herein, CDRs are numbered according to the IMGT numbering system.

As used herein, “specific binding” refers to the association or binding of a binding domain or a protein containing that binding domain with a target molecule by affinity or _Ka (i.e., the equilibrium association constant for a specific binding). It involves interacting with molecules of 1/M unit or greater than ^{10⁵ M⁻¹} without significantly associating or binding with any other molecules or components in the sample. Binding domains (or their fusion proteins) can be classified as “high-affinity” binding domains (or their fusion proteins) or “low-affinity” binding domains (or their fusion proteins). A “high-affinity” binding domain is defined as having at least ^{10⁷ M⁻¹} , at least ^{10⁸ M⁻¹} , at least ^{10⁹ M⁻¹} , at least ^{10¹⁰ M⁻¹} , at least ^{10¹¹ M⁻¹} , at least ^{10¹² M⁻¹} , or at least ^{10¹³ M⁻¹} . "Low affinity" binding domains refer to those binding domains with a _Ka value of up to ^{10⁷ M⁻¹} , up to ^{10⁶ M⁻¹} , or up to ^{10⁵ M⁻¹} . Alternatively, affinity can be defined as the equilibrium dissociation constant ( _Kd ) of a specific binding interaction with an M unit (e.g., ^10⁻⁵ M to ^10⁻¹³ M). In some embodiments, binding domains may have "enhanced affinity," which refers to a selective or engineered binding domain that binds more strongly to the target antigen compared to a wild-type (or parental) binding domain. For example, enhanced affinity might be due to a higher Ka _value (equilibrium association constant) for the target antigen than a wild-type binding domain, or a lower Kd _value for the target _antigen than a wild-type binding domain, or a lower dissociation rate ( _Koff ) for the target antigen than a wild-type binding domain. Various assays are known to be used to identify the binding domains of the present invention that specifically bind to a particular target, and to determine the affinity of the binding domains or fusion proteins, such as western blot, ELISA, and assays (see also, for example, Scatchard, et al., Ann. NYAcad. Sci. 57:660, 1949; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents).

Assays for assessing affinity, or apparent affinity, or relative affinity are known. In some instances, apparent affinity for the TCR is measured by evaluating binding to tetramers at various concentrations, for example, by flow cytometry using labeled tetramers. In some instances, the apparent K_{_d} of the TCR is measured using a 2-fold dilution of a labeled tetramer (i.e., peptide:MHC tetramer) within a certain concentration range, and the apparent K_{_d} is determined as the concentration of the ligand that produces the maximum binding half by determining the binding curve using nonlinear regression. In some embodiments, Msln _20-28- or Msln _530-538- specific binding proteins comprise, respectively, Msln _20-28- or Msln _530-538- specific immunoglobulin superfamily binding proteins or their binding moieties.

"MHC peptide tetramer staining" refers to an assay for detecting antigen-specific T cells, characterized by tetramers of MHC molecules, each molecule containing at least one epitope (e.g., Msln _20-28 or Msln _530-538 ) of the same peptide (e.g., identical or related) having a homologous amino acid sequence, wherein the complex is capable of binding to a TCR specific to the homologous epitope. Each MHC molecule can be labeled with a biotinylated molecule. The biotinylated MHC/peptide tetramer is formed by adding a fluorescently labeled streptavidin. The tetramer can be detected by flow cytometry via fluorescent labeling. In some embodiments, the MHC-peptide tetramer assay is used to detect or select the enhanced affinity TCR of this disclosure. Cytokine levels can be determined according to methods described herein and practiced in the art, including, for example, ELISA, ELISpot, intracellular cytokine staining, and flow cytometry, and combinations thereof (e.g., intracellular cytokine staining and flow cytometry). The proliferation and clonal expansion of immune cells induced or stimulated by antigen-specific immune responses can be determined by isolating lymphocytes, such as circulating lymphocytes in peripheral blood cell samples or circulating lymphocytes in lymph node cells, stimulating the cells with antigens, and measuring the antigens. Cytokine production, cell proliferation, and/or cell viability can be assessed, for example, by incorporating tritium-infused thymidine or by non-radioactive assays, such as the MTT assay. The role of the immunogens described herein in balancing the Th1 and Th2 immune responses can be examined, for example, by determining the levels of Th1 cytokines, such as IFN-γ, IL-12, IL-2, and TNF-β, and type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

As used herein, "antigen" or "Ag" refers to an immunogenic molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunologically active cells (e.g., T cells), or both. Antigens (immunogenic molecules) can be, for example, peptides, glycopeptides, polypeptides, glycopeptides, polynucleotides, polysaccharides, lipids, etc. It is evident that antigens can be synthesized, recombinantly generated, or derived from biological samples. Exemplary biological samples that may contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be chemically synthesized or produced by cells that have been modified or genetically engineered to express the antigen.

The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by associated binding molecules such as immunoglobulins, T-cell receptors (TCRs), chimeras, antigen receptors, or other binding molecules, domains, or proteins. Antigenic determinants typically contain chemically active surface groups of the molecule, such as amino acid or sugar side chains, and may have specific three-dimensional structural features as well as specific charge features.

As used herein, “Msln _20-28 ”, “Msln _20-28 peptide”, and “Msln20 peptide” refer to mesothelin amino acids 20-28 or peptides composed of them (human mesothelin, isotype 1) containing SEQ ID NO:50; namely, SLLFLLFSL (SEQ ID NO:31), which can associate with HLA-A*201.

As used herein, “Msln _530-538 ” and “Msln _530-538 peptide”, “Msln ₅₃₀ peptide” refers to mesothelin amino acids 530-538 containing SEQ ID NO:50 or peptides composed thereof; for example, VLPLTVAEV (SEQ ID NO:32), which can bind to HLA-A*201.

The term "Msln _20-28 specific binding protein" refers to a protein or polypeptide that specifically binds to and/or has specificity and/or high affinity for the Msln _20-28 peptide. In some embodiments, the protein or polypeptide binding to Msln _20-28 , such as the Msln _20-28 peptide complexed with MHC or HLA molecules on the cell surface, has or at least has about a specific affinity. The Msln _20-28 specific binding protein can bind to the Msln _20-28 peptide, its variants, or fragments thereof. For example, the Msln _20-28 specific binding protein can bind to the amino acid sequence of SEQ ID NO:31 (SLLFLLFSL), or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:31. In some embodiments, the Msln _20-28 specific binding protein binds the Msln _20-28 peptide:HLA complex (or Msln _20-28- derived peptide:MHC complex) with about the same affinity as, or greater than or equal to, the affinity exhibited by the exemplary Msln 20-28 specific binding proteins provided herein, such as any Msln _20-28 specific TCR provided herein, for example, as measured by the same assay. In some embodiments, the Msln _20-28 specific binding protein may bind the Msln _20-28 epitope as provided herein; for example, according to the concordant epitope sequence of SEQ ID NO:60. As disclosed herein, the Msln specific binding protein does not bind or substantially does not bind non-Msln human proteins or peptides that have high sequence homology or identity with SEQ ID NO:60.

The term "Msln _530-538- specific binding protein" refers to a protein or polypeptide that specifically binds to and/or has specificity and/or high affinity for the Msln _530-538 peptide. In some embodiments, a protein or polypeptide binding to Msln _530-538 , such as the Msln _530-538 peptide, complexes with MHC or HLA molecules, for example, on the cell surface with a specific affinity or at least approximately a specific affinity. The Msln _530-538- specific binding protein may bind to the Msln 530-538 peptide, its variants, or fragments thereof. For example, the Msln _530-538 -specific binding protein may bind to the amino acid sequence (VLPLTVAEV) of SEQ ID NO:32, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:32. In some embodiments, the Msln _530-538 -specific binding protein binds to the Msln _530-538 peptide:HLA complex (or Msln _530-538 -derived peptide:MHC complex) with an affinity of about the same as, or greater than or equal to, that exhibited by exemplary Msln _530-538- specific binding proteins provided herein (e.g., any Msln _530-538 -specific TCR provided herein), for example, as measured by the same assay. In some embodiments, the Msln _530-538- specific binding protein may bind to Msln _530-538 epitopes as provided herein; for example, according to the concordant epitope sequence of SEQ ID NO:61 or 62. As disclosed herein, the Msln-specific binding protein does not bind or substantially does not bind to non-Msln human proteins or peptides that are homologous to or identical to SEQ ID NO:61 or 62.

Target molecules specifically bound by the binding domains of this disclosure may be on or associated with target cells (“target cells”). Exemplary target cells include cancer cells, cells associated with autoimmune diseases or conditions or inflammatory diseases or conditions, and infectious organisms or cells (e.g., bacteria, viruses, or virus-infected cells). Cells of infectious organisms, such as mammalian parasites, are also considered target cells.

The term "functional affinity" refers to a biological measure or activation threshold of the response of immune cells (e.g., T cells, NK cells, NK-T cells) to a given concentration of ligand in vitro. This biological measure may include cytokine production (e.g., IFNγ production, IL-2 production, etc.), cytotoxic activity, and proliferation. For example, T cells that produce cytokines, exhibit cytotoxic or proliferative activity, and elicit a biological (immune) response to low doses of antigen in vitro are considered to have high functional affinity, while T cells with lower functional affinity require higher amounts of antigen prior to immunization to elicit a response similar to that of high-affinity T cells. It will be understood that functional affinity is different from affinity and kinetic affinity. Affinity refers to the strength of any given bond between a binding protein and its antigen/ligand. Some binding proteins are multivalent and can bind multiple antigens—in which case the strength of the overall bond is the affinity.

There are many correlations between functional affinity and the effectiveness of immune responses. Some in vitro studies have shown that different T cell functions (e.g., proliferation, cytokine production, etc.) can be triggered at different thresholds (see, for example, Bettset et al., J. Immunol. 172:6407, 2004; Langenkamp et al., Eur. J. Immunol. 32:2046, 2002). Factors affecting functional affinity include (a) the affinity of the TCR for the pMHC complex, i.e., the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 167:2577, 2001), (b) the expression levels of the TCR and CD4 or CD8 co receptors, and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273:104, 1996), as well as the expression levels of molecules that attenuate T cells, cell function, and TCR signaling.

The antigen concentration required to induce a half-maximal response between baseline and maximal response after a specified exposure time is referred to as the “half-maximal effective concentration” or “EC50”. EC50 values are typically expressed in moles (mol/L), but are usually converted to a logarithmic value as follows: log10(EC50). For example, if EC50 equals 1 μM ( ^10⁻⁶ M), then log10(EC50) is -6. Another value used is pEC50, defined as the negative logarithm of EC50 (-log10(EC50)). In the example above, pEC50 equals 1 μM of EC50 and has a value of 6. In some embodiments, the functional affinity of the binding protein of the present invention will be measured in terms of its ability to promote IFNγ production by T cells, and can be measured using the assays described herein. A “high functional affinity” TCR or its binding domain refers to those TCRs or their binding domains with an EC50 of at least ^10⁻⁴ M, at least about ^10⁻⁵ M, or at least about ^10⁻⁶ M.

In some embodiments, the mesothelin-specific binding protein or domain as described herein may be expressed by host T cells and may be functionally characterized according to any of the many art-recognized methods for analyzing T cell activity, including determining T cell binding, activation, or induction, and also determining antigen-specific T cell responses. In some embodiments, the binding protein is capable of promoting an antigen-specific T cell response against human mesothelin in a class I HLA-restricted manner. In further embodiments, the class I HLA-restricted response is associated with transport proteins and is independent of antigen processing (TAP). In some embodiments, the antigen-specific T cell response includes at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In related embodiments, the CTL response targets cells that overexpress mesothelin. Other examples of methods for assessing T cell activity include determining T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., by detecting ^51Cr release from preloaded target cells), changes in T cell phenotypic marker expression, and other measures of T cell function. For example, procedures for performing these and similar measurements can be found in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and the references cited therein.

As used herein, “immune system cells” refers to any cell in the immune system that originates from bone marrow hematopoietic stem cells, producing two main lineages: myeloid progenitor cells (which produce myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which produce lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8- double-negative T cells, γδ T cells, regulatory T cells, stem cell memory T cells, natural killer cells (such as NK cells or NK-T cells), B cells, and dendritic cells. Macrophages and dendritic cells may be referred to as “antigen-presenting cells” or “APCs”, and are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptor on the surface of APCs, which are complexed with peptides, interacts with the TCR on the surface of T cells.

T cells, or T lymphocytes, are immune system cells that mature in the thymus and produce TCRs. T cells can be naive (not exposed to antigens; compared to _TCM , the expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA is increased, while the expression of CD45RO is decreased), memory T cells ( _TMs ) (cells that have experienced antigens for a long time and survive), and effector cells (cells that have experienced the cytotoxicity of antigens). _TMs can be further divided into central memory T cells ( _TCMs , with increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA compared to naive T cells) and effector memory T cells ( _TMEs , with decreased expression of CD62L, CCR7, CD28, and CD45RA, and increased expression of CD127 compared to naive T cells or _TCMs ).

Effector T cells ( _TEs ) are antigen-experienced CD8+ cytotoxic T lymphocytes that, compared to TCM _cells , exhibit reduced expression of CD62L, CCR7, and CD28, and are positive for granzymes and perforin. Other exemplary T cells include regulatory T cells, such as CD4+CD25+ (Foxp3+) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8+CD28-, and Qa-1 restricted T cells.

Helper T cells ( _TH ) are CD4+ cells that influence the activity of other immune cells by releasing cytokines. CD4+ T cells can activate and suppress adaptive immune responses, and which of these two functions is induced depends on the presence of other cells and signals. T cells can be collected using known techniques, and various subsets or combinations thereof can be enriched or eliminated using known techniques such as affinity binding to antibodies, flow cytometry, or immunomagnetic selection.

"Cells of the T-cell lineage" refers to cells that exhibit at least one phenotypic characteristic of T cells, or precursor or progenitor cells that distinguish said cells from other lymphoid cells and cells of the erythroid or myeloid lineage. Such phenotypic characteristics may include the expression of one or more T-cell-specific proteins (e.g., CD3 ⁺ , CD4 ⁺ , CD8 ⁺ ) or T-cell-specific physiological, morphological, functional, or immunological characteristics. For example, cells of the T-cell lineage can be progenitor or precursor cells that are defined as T-cell lineages. These include CD25 ⁺ immature and inactivated T cells; cells that have undergone CD4 or CD8 linear commitment; CD4 ⁺ CD8 ⁺ double-positive thymocyte progenitor cells; single-positive CD4 ⁺ or CD8 ⁺ ; TCRαβ or TCRγδ; or mature, functional, or activated T cells.

"Hematopoietic progenitor cells" are cells derived from hematopoietic stem cells (HSCs) or fetal tissue that are capable of further differentiating into mature cell types (e.g., cells of the T-cell lineage). In some embodiments, ^{CD241L1 -} CD117 ⁺ hematopoietic progenitor cells are useful. As defined herein, hematopoietic progenitor cells may include embryonic stem cells capable of further differentiating into T-cell lineage cells. Hematopoietic progenitor cells can be derived from a variety of animals, including humans, mice, rats, or other mammals. "Thymocyte progenitor cells" or "thymocytes" are hematopoietic progenitor cells present in the thymus.

"Hematopoietic stem cells" or "HSCs" refer to undifferentiated hematopoietic cells that are capable of self-renewal in vivo, proliferate virtually without limit in vitro, and differentiate into other cell types, including cells of the T cell lineage. HSCs can be isolated, for example but not limited to, from fetal liver, bone marrow, and umbilical cord blood.

"Embryonic stem cells," "ES cells," or "ESCs" refer to undifferentiated embryonic stem cells with the ability to integrate into the germline of a developing embryo and become part of it. Embryonic stem cells can differentiate into hematopoietic progenitor cells and any tissue or organ. Embryonic stem cells used in this article include cells from the J1ES cell line, the 129JES cell line, the mouse stem cell line D3 (American Center for Type Culture Collection), the R1 or E14K cell lines derived from 129/Sv mice, cell lines derived from Balb/c and C57B1/6 mice, and human embryonic stem cells (e.g., from the Institute of [Institute Name]; or from the ES Cell International Organization in Melbourne, Australia).

The term "T-cell receptor" (TCR) refers to a member of the immunoglobulin superfamily (possessing a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, for example, Janeway, et al., Immunobiology: The Immune System in Health and Disease, ^3rd Ed., Current Biology Publications, p. 4: 33, 1997) that specifically binds to antigenic peptides that bind to MHC receptors. TCRs can be found on the cell surface or in soluble form and are typically composed of heterodimers with α and β chains (referred to as TCRα and TCRβ, respectively) or γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Like immunoglobulins, the extracellular portion of the TCR chain (e.g., the α and β chains) contains two immunoglobulin domains, a variable domain (e.g., the α chain variable domain or _Vα , the β chain variable domain or _Vβ ), typically an amino acid numbered from 1 to 116 at the N-terminus based on the terminal Kabat number (Kabat, et al., “Sequences of Proteins of Immunological Interest,” USDept. Health and Human Services, Public Health Service, National Institutes of Health, 1991, ^5th ed.), and a constant domain adjacent to the cell membrane (e.g., the α chain constant domain or _Cα , typically based on Kabat amino acids 117 to 259, the β chain constant domain or _Cβ , typically based on Kabat amino acids 117 to 295). Like immunoglobulins, the variable domain contains complementarity-determining regions (CDRs) separated by frame regions (FRs) (see, for example, Jores, et al., Proc. Nat'l Acad. Sci. USA 57:9138, 1990; Chothia, et al., EMBO J. 7:3745, 1988; see also Lefranc, et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, the TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. The TCRs used as in this disclosure can be derived from a variety of animal species, such as humans, mice, rats, cats, dogs, goats, horses, or other mammals.

"CD3" is a six-chain multiprotein complex (see Borst J, et al., J Biol Chem, 258(8):5135-41, 1983 and Janeway, et al., p.172 and 178, 1999, ibid.). In mammals, the complex comprises a homodimer of the CD3γ chain, the CD3δ chain, two CD3ε chains, and the CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are associated cell surface proteins of the immunoglobulin superfamily, each containing a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which is thought to be a feature that allows these chains to associate with positively charged regions of the TCR chain. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a conserved motif called an immune receptor tyrosine-based activation motif, or ITAM, while each CD3ζ chain has three. Unbound by any theory, it is believed that ITAM is important for the signal transduction capabilities of the TCR complex. As used in this disclosure, CD3 can be derived from various animal species, including humans, mice, rats, or other mammals.

As used herein, the "TCR complex" refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can consist of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of the CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex can consist of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of the CD3ζ chain, a TCRγ chain, and a TCRδ chain.

As used herein, “components of a TCR complex” refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed of two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex (TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

The major histocompatibility complex (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a transmembrane α-chain (containing three α-domains) and non-covalently bound β2-microglobulins. MHC class II molecules consist of two transmembrane glycoproteins, α and β, both of which are transmembrane. Each chain has two domains. MHC class I molecules deliver peptides originating from the cytosol to the cell surface, where CD8 ⁺ T cells recognize the peptide:MHC complex. MHC class II molecules deliver peptides originating from the vesicle system to the cell surface, where they are recognized by CD4 ⁺ T cells. Human MHC is known as human leukocyte antigen (HLA).

mesothelin-specific binding protein

In some aspects, this disclosure provides binding proteins (e.g., peptides comprising, or substantially comprising, the amino acid sequences shown in SEQ ID NO:31 or SEQ ID NO:32) capable of specifically binding to the mesothelin peptide antigen described herein. The binding proteins herein include TCRα chain variable domains (Vα) and TCRβ chain variable domains (Vβ). In any of the embodiments currently disclosed, the mesothelin-specific binding protein is capable of specifically binding to mesothelin peptide:HLA complexes, such as the mesothelin peptide:HLA-A*02:01 complex.

In some embodiments, a Msln _-530-538- specific binding protein is provided, comprising: (a) TCRVα and TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO:37 or 39, wherein TCRVβ optionally comprises an amino acid sequence having at least about 85% (i.e., at least about 86%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:99 or 101; (b) TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO:38 or 40; and (b) TCRVα, wherein TCRVα optionally comprises an amino acid sequence having at least about 85% (i.e., at least about 86%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:99 or 101; and (c) TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO:38 or 40; and (d) TCRVα, wherein TCRVα optionally comprises an amino acid sequence having at least about 85% (i.e., at least about 86%, 85%, 88%, 89%, 90%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity with the amino acid sequence shown in SEQ ID NO:99 or 101; The amino acid sequence shown in NO:100 or 102 is at least about 85% (i.e., at least about 86%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence shown in SEQ ID NO:37 or 39, wherein TCRVα optionally contains an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:100 or 102, and TCRVβ containing the CDR3 amino acid sequence shown in SEQ ID NO:38 or 40, wherein TCRVβ optionally contains an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:99 or 101.

In any of the embodiments described herein, the encoded polypeptides of this disclosure (e.g., TCR variable domains or TCR chains) may include a “signal peptide” (also referred to as a leader sequence, leader peptide, or transport peptide). The signal peptide targets the newly synthesized polypeptide to the appropriate intracellular or extracellular location. The signal peptide can be removed from the polypeptide during or after the completion of localization or secretion. A polypeptide having a signal peptide is referred to herein as a “preprotein,” while a polypeptide whose signal peptide has been removed is referred herein as a “mature” protein or polypeptide. Representative signal peptides include those amino acid sequences from position 1 to position 19 of any one of SEQ ID NO: 6, 14, 22, 28, or 29; from position 1 to position 17 of SEQ ID NO: 7; positions 1 to 22 of SEQ ID NO: 15; and positions 1 to 21 of SEQ ID NO: 23. Exemplary mature polypeptide sequences are provided in SEQ ID NO: 95-119.

In any of the currently disclosed embodiments, the Msln _530-538 specific binding protein may comprise TCRVα, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 100 or 102; and/or TCRVβ, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 99 or 101. In some embodiments, the binding protein comprises a variant domain sequence of a reference TCR variable, provided that at least three or four CDRs of the binding protein are not sequence-dependent according to the reference TCR variable domain sequence, wherein the CDRs with sequence variations have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or combinations thereof.

In some embodiments, the Msln _530-538 specific binding protein comprises the CDR3α amino acid sequence shown in SEQ ID NO:39 and the CDR3β amino acid sequence shown in SEQ ID NO:40. In some embodiments, the binding protein further comprises the CDR1α amino acid sequence shown in SEQ ID NO:93, the CDR2α amino acid sequence shown in SEQ ID NO:94, the CDR1β amino acid sequence shown in any one of SEQ ID NO:83, 84, or 91, and/or the CDR2β amino acid sequence shown in SEQ ID NO:92. In further embodiments, _Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:102, and/or _Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:101, wherein CDR1α, CDR2α, CDR1β, and/or CDR2β are optionally unchanged.

In some embodiments, the Msln _530-538 specific binding protein comprises TCRVβ, which contains an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRBJ2-3*01 (e.g., an amino acid sequence encoded by TRBJ2-3*01 of at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 15 consecutive amino acids) and/or an amino acid sequence encoded by TRAV21*01 or TRAV21*02 (e.g., an amino acid sequence encoded by TRAV21*01 or TRAV21*02 of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 107 consecutive amino acids). The amino acid sequence having at least 85% identity, and/or having an amino acid sequence having at least 85% identity with the amino acid sequence encoded by TRBV5-4*01 (e.g., an amino acid sequence encoded by TRBV5-4*01 with a length of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or 108 consecutive amino acids), and/or having an amino acid sequence encoded by TRAJ57*01 (e.g., an amino acid sequence encoded by TRAJ57*01 with a length of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive amino acids).

In some embodiments, the Msln _530-538 specific binding protein comprises TCRVα, which comprises or consists of the amino acid sequence shown in SEQ ID NO:102, and TCRVβ, which comprises or consists of the amino acid sequence shown in SEQ ID NO:101.

In other embodiments, the Msln _530-538 specific binding protein comprises the CDR3α amino acid sequence shown in SEQ ID NO:37 and the CDR3β amino acid sequence shown in SEQ ID NO:38. In some embodiments, the binding protein further comprises the CDR1α amino acid sequence shown in SEQ ID NO:89, the CDR2α amino acid sequence shown in SEQ ID NO:90, the CDR1β amino acid sequence shown in SEQ ID NO:83 or 87, and the CDR2β amino acid sequence shown in SEQ ID NO:88. In some embodiments, _Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:100, and/or _Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:99. Optionally, CDR1α, CDR2α, CDR1β, and/or CDR2β remain unchanged.

In some embodiments, the Msln _530-538 specific binding protein comprises an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRAV4-1*01 (e.g., an amino acid sequence encoded by a sequence of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 108 amino acids) and/or with TRAJ18*01 (e.g., an amino acid sequence encoded by TRAJ18*01 of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, ... An amino acid sequence that is at least about 85% identical to a sequence of 18, 19, 20 or 21 amino acids and/or an amino acid sequence that is at least about 85% identical to a sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids encoded by TRBJ1-1*01 and/or an amino acid sequence that is at least about 85% identical to a sequence of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 16 amino acids encoded by TRBJ2-3*01.

In some embodiments, the Msln _530-538 specific binding protein comprises TCRVα, which comprises or consists of the amino acid sequence shown in SEQ ID NO:100; and TCRVβ, which comprises or consists of the amino acid sequence shown in SEQ ID NO:99.

In some embodiments, mutagenesis of any one or more of residues 3, 5, 6, or 9 of SEQ ID NO:32 does not eliminate or substantially impair the binding of the Msln _530-538 -specific binding protein. In some embodiments, the Msln _530-538 -specific binding protein is capable of binding peptides comprising or consisting of the common amino acid sequence shown in SEQ ID NO:61, for example, in the peptide:HLA complexes disclosed herein.

In some embodiments, mutagenesis of any one or more alanine residues 1, 5, or 9 of SEQ ID NO:32 does not eliminate or substantially impair the binding of the Msln _530-538 specific binding protein. In some embodiments, the Msln _530-538 specific binding protein is capable of binding peptides comprising or consisting of the common amino acid sequence shown in SEQ ID NO:62, for example, in the peptide:HLA complexes disclosed herein.

Currently disclosed Msln-specific binding proteins advantageously exhibit a low to no alloreactivity risk to non-Msln targets; for example, they are expressed in healthy tissues. In short, this disclosure shows that Msln-specific binding proteins do not react or are substantially unreactive with human proteins having sequence homology with the Msln peptide antigens provided herein. Therefore, the binding proteins exhibit high specificity for the Msln peptide antigens.

For example, in some embodiments, the Msln _530-538- specific binding protein of the present invention does not bind to or binds nonspecifically to the peptide:HLA complex relative to the binding to Msln _530-538 , wherein the peptide comprises or consists of any one or more of the amino acid sequences shown in SEQ ID NO:63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 and 77, and wherein the HLA optionally comprises HLA-A2, such as HLA-A:02*01.

In some embodiments, a Msln _20-28 specific binding protein is provided, comprising: (a) TCRVα and TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO: 33 or 35, wherein TCRVβ optionally has at least about 85% (i.e., at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identity with the amino acid sequence shown in SEQ ID NO: 95 or 97; (b) TCRVβ comprising the CDR3 amino acid sequence as shown in SEQ ID NO: 34 or 36, and (b) TCRVα, wherein the TCRVα optionally has at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 96 or 98; or (c) TCRVα comprising the CDR3 amino acid sequence shown in SEQ ID NO: 33 or 35 and comprising SEQ ID NO: 34 or 35. TCRVβ of the CDR3 amino acid sequence shown in NO:34 or 36, wherein TCRVα optionally contains an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:95 or 97, and wherein TCRVβ optionally contains an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:96 or 98.

In any of the currently disclosed embodiments, the Msln _20-28 specific binding protein may comprise TCRVα, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:96 or SEQ ID NO:98, and/or TCRVβ, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:95 or 97. In some embodiments, the binding protein comprises a variant of a reference TCR variable domain sequence, provided that at least three or four CDRs of the binding protein are not sequence-dependent according to the reference TCR variable domain sequence, wherein the CDRs with sequence variations have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or combinations thereof.

In some embodiments, the Msln _20-28 specific binding protein comprises the CDR3α amino acid sequence shown in SEQ ID NO:33 and the CDR3β amino acid sequence shown in SEQ ID NO:34. In some embodiments, the binding protein further comprises the CDR1α amino acid sequence shown in SEQ ID NO:80, the CDR2α amino acid sequence shown in SEQ ID NO:81 or 118, the CDR1β amino acid sequence shown in any one of SEQ ID NO:78, 83 or 84, and the CDR2β amino acid sequence shown in SEQ ID NO:79. In some embodiments, _Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:96, and/or _Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:95, wherein optionally CDR1α, CDR2α, CDR1β and/or CDR2β are unchanged.

In some embodiments, the Msln _20-28 specific binding protein comprises (i) TCRVβ, which comprises (a) an amino acid sequence having at least about 85% (i.e., at least about 85%, 86%, 87%) of the amino acid sequence encoded by TRBV12-4*01 (e.g., an amino acid sequence encoded by TRBV12-4*01 with a length of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 108 consecutive amino acids). (a) an amino acid sequence with at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity; and/or (b) an amino acid sequence having at least about 85% identity with an amino acid sequence encoded by TRBJ2-7*01 (e.g., an amino acid sequence encoded by TRBJ2-7*01 with a length of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acids); and/or (ii) TCRVα, which contains (a) (a) an amino acid sequence having at least about 85% identity with an amino acid sequence encoded by TRAV1-1*01 (e.g., an amino acid sequence encoded by TRAV1-1*01 with a length of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 107 consecutive amino acids) and/or (b) an amino acid sequence having at least about 85% identity with an amino acid sequence encoded by TRAJ3*01 (e.g., an amino acid sequence encoded by TRAJ3*01 with a length of at least about 5, 6, ... The amino acid sequence encoded by an amino acid sequence of length 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids has at least about 85% identity with the amino acid sequence encoded by TRBJ2-3*01 (e.g., an amino acid sequence encoded by TRBJ2-3*01 of length at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids).

In some embodiments, the Msln _20-28 specific binding protein comprises TCRVα, which comprises or consists of the amino acid sequence shown in SEQ ID NO:96, and TCRVβ, which comprises or consists of the amino acid sequence shown in SEQ ID NO:95.

In some embodiments, Msln _20-28 specifically binds to proteins such as the CDR3α amino acid sequence shown in SEQ ID NO:35 and the CDR3β amino acid sequence shown in SEQ ID NO:36. In some embodiments, the binding protein further comprises the CDR1α amino acid sequence shown in SEQ ID NO:85, the CDR2α amino acid sequence shown in SEQ ID NO:86 or 119, the CDR1β amino acid sequence shown in any one of SEQ ID NO:82, 83 or 84, and the CDR2β amino acid sequence shown in SEQ ID NO:79. In some embodiments, _Vα comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:98, and/or _Vβ comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:97, wherein CDR1α, CDR2α, CDR1β and/or CDR2β optionally remain unchanged.

In some embodiments, the Msln _20-28 specific binding protein comprises TCRVα, which comprises (a) an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRAV12-3*01 (e.g., with the amino acid sequence encoded by TRAV12-3*01 of a length of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 108 consecutive amino acids), and/or (b) an amino acid sequence having at least about 85% identity with the amino acid sequence encoded by TRAJ29*01 (e.g., with the amino acid sequence encoded by TRAJ29). *01 encodes an amino acid sequence having at least about 85% identity with an amino acid sequence having a length of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 amino acids) and/or (c) and/or an amino acid sequence having at least about 85% identity with an amino acid sequence encoded by TRBJ2-3*01 (e.g., an amino acid sequence having a length of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids) encoded by TRBJ2-3*01.

In some embodiments, the Msln _20-28 specific binding protein comprises TCRVα, which comprises or consists of the amino acid sequence shown in SEQ ID NO:98; and TCRVβ, which comprises or consists of the amino acid sequence shown in SEQ ID NO:97.

In some embodiments, mutagenesis of any one or more of residues 1, 2, 7, 8, or 9 of SEQ ID NO:31 does not eliminate or substantially impair the binding of the Msln _20-28 specific binding protein. In some embodiments, the Msln _20-28 specific binding protein is capable of binding peptides comprising or consisting of the common amino acid sequence shown in SEQ ID NO:60, for example, in the peptide:HLA complex disclosed herein.

In any of the currently disclosed embodiments, the Msln-specific binding protein is capable of binding the Msln peptide:HLA complex, wherein the Msln peptide comprises the amino acid sequence shown in SEQ ID NO:31 or 32, and wherein HLA is or comprises HLA-A2, such as HLA-A*02:01.

In any of the currently disclosed embodiments, immune cells (e.g., T cells) expressing the Msln-specific binding protein of this disclosure do not produce IFN-γ and/or do not exhibit activation (e.g., CD8 expression, CD3 expression, Nur77 expression) and/or cytotoxic activity (e.g., specific killing, production, and release of perforin and/or granzymes) when contacted with cells expressing the following: (i) HLA-C6:02:01; (ii) HLA-B13:01:01 without HLA-B13:02:01; (iii) HLA-A3; (iv) HLA-A29; (v) HLA-B40; (vi) HLA-B44; (vii) HLA-C3; (viii) HLA-C16; (ix) HLA-A1; (x) HLA-24; (xi) HLA-B7; (xii) HLA -B57; (xiii)HLA-C7; (xiv)HLA-A11; (xv)HLA-B15; (xvi)HLA-C4; (xvii)HLA-C12; (xviii)HLA-B8; (xix)HLA-B49; (xx)HLA-B51; (xxi)HLA-C15; (xxii)HLA-A30; (xxiii)HLA-A68; (xxiv)HLA-C2; (xxv)HLA-A32; (xxvi)HLA-A33; (xxvii)HLA-B55; (xxviii)HLA-C1; (xxvix)HLA-C5; (xxix)HLA-B8; (xxx)HLA-B35; or any combination of (xxxi)(i)-(xxx), when the Msln peptide provided herein is not present.

In any of the currently disclosed embodiments, when expressed on the surface of a host cell, the Msln-specific binding protein is able to bind to the Msln peptide:HLA complex disclosed herein in the absence of or independently of CD8.

In some embodiments, the binding protein according to the invention has a Msln peptide EC50 of about 9 μM, about 8 μM, about 7 μM, about 6 μM, about 5 μM, about 4 μM, about 3 μM, 2 μM, about 1 μM, about 0.9 μM, about 0.8 μM, about 0.7 μM, about 0.6 μM, about 0.5 μM, about 0.4 μM, about 0.3 μM, about 0.2 μM or less.

In any of the currently disclosed embodiments, when the Msln-specific binding protein is expressed in host T cells or NK-T cells, the Msln-specific binding protein is able to bind more effectively to CD3 protein and/or has increased expression on the cell surface relative to endogenous TCR.

In some embodiments, the binding protein is a TCR, a single-chain TCR (scTCR), or a CAR.

In some embodiments, the binding protein is a TCR. In some embodiments, the binding protein comprises TCR Vβ, TCR Cβ, TCR Vα, and TCR Cα, wherein Vβ and Cβ together comprise a TCRβ chain (TCRβ), and wherein Vα and Cα together comprise a TCRα chain (TCRα), and wherein TCRβ and TCRα are capable of associating to form a dimer. In a further embodiment, TCRCβ contains a cysteine amino acid at amino acid position 57 (e.g., GV(S→C)TD) instead of native serine, while TCRCα contains a cysteine amino acid at amino acid position 48 (e.g., DK(T→C)VL; see, for example, Cohen et al., Cancer Res. 67(8):3898-3903(2007)) instead of native threonine.

In other embodiments, the Msln-specific binding protein is a TCR comprising TCRβ and TCRα, wherein TCRβ and TCRα comprise amino acid sequences having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequences shown in SEQ ID NO: (i) 103 or 6 (TCRβ) and 104 or 7 (TCRα); (ii) 105 or 14 (TCRβ) and 106 or 15 (TCRα); (iii) 107 or 22 (TCRβ) and 108 or 23 (TCRα); or (iv) 109 or 28 (TCRβ) and 110 or 29 (TCRα).

In some embodiments, the binding protein is a soluble TCR, which optionally includes or is coupled to cytotoxic and/or detectable elements or reagents. (See, for example, Walseng et al., PLoS One doi:10.1371/journal.pone.0119559 (2015)). For instance, a method for isolating and purifying recombinant-derived soluble TCRs may include obtaining a supernatant from a suitable host cell/carrier system that secretes the recombinant soluble TCR into a culture medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse-phase HPLC steps may be used to further purify the recombinant peptide. These purification methods can also be used when isolating immunogens from the natural environment. Large-scale production of one or more isolated/recombinant soluble TCRs described herein involves batch cell culture, monitoring and controlling it to maintain suitable culture conditions. Purification of soluble TCRs can be performed according to the methods described herein and those known in the art, and in accordance with the laws and guidelines of domestic and international regulatory authorities.

In some embodiments, two or more distinct polypeptide domains or sequences are linked by a linker (e.g., TCRVα and TCRVβ in the case of scTCR or CAR). A “linker” is an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and can provide a spacer function compatible with the interaction of the two sub-binding domains, thereby allowing the resulting polypeptide to maintain specific binding affinity to the target molecule (e.g., scTCR) or retain signal transduction activity (e.g., TCR complex). In some embodiments, the linker comprises about 2 to about 35 amino acids, about 4 to about 20 amino acids, about 8 to about 15 amino acids, about 15 to about 25 amino acids, or other suitable numbers of amino acids. Typically, the linker is preferably chemically inert, flexible, non-immunogenic, or minimally immunogenic. The linker sequence can be repeated to achieve a desired length, for example, to facilitate desired protein interactions through or between the linker domains. Exemplary linkers (including glycine-serine linkers) and the properties of linkers are discussed, for example, in Chen et al., Adv. Drug Deliv Rev., 65(10): 1357-1369 (2013), and in van Rosmalen et al., Biochemistry 56(60): 6565-6574 (2017), whose linker amino acid sequences and design characteristics are incorporated herein by reference.

In a particular embodiment, the Msln-specific binding protein is or contains scTCR (e.g., a single-chain αβTCR protein, such as Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vα, or Vα-L-Vβ-Cβ, wherein Vα and Vβ are the TCR α and β variable domains, respectively, Cα and Cβ are the TCR α and β constant domains, respectively, and L is a linker).

In some embodiments, the Msln-specific binding protein is or comprises a CAR. A “chimeric antigen receptor” (CAR) is a fusion protein of the present invention engineered to comprise two or more naturally occurring (or engineered) amino acid sequences linked together in a manner unnaturally occurring or unnaturally occurring in the host. When present on the cell surface, the fusion protein can function as a receptor. The CAR of this disclosure comprises an extracellular portion comprising an antigen-binding domain (e.g., derived from or derived from immunoglobulins or immunoglobulin-like molecules, such as scFv or scTCR derived from antibodies or TCRs specific to cancer antigens, or an antigen-binding domain derived from a cytotoxic immune receptor, from NK cells, or from another protein (natural, recombinant, or synthetic) having or engineered to have the ability to specifically bind to antigens), which is coupled with a transmembrane linking domain and one or more intracellular signaling domains (optionally including a co-stimulatory domain) (see, for example, Sadelainet al., Cancer Discov., 3(4):388(2)). 013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016); Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014)). In some embodiments, the binding protein comprises a CAR containing an antigen-specific TCR binding domain (see, for example, Walseng et al., Scientific Reports 7:10713, 2017; TCR CAR constructs and methods are incorporated herein by reference in their entirety).

Polynucleotides, vectors, and host cells

This document also provides polynucleotides encoding Msln-specific binding proteins or portions thereof (e.g., TCR variable domains) of the present invention. Those skilled in the art will understand that, due to the degeneracy of the genetic code, as described herein, there are many nucleotide sequences encoding binding proteins or portions thereof. Some of these polynucleotides have limited or minimal sequence identity with the nucleotide sequences of natural, original, or identified polynucleotide sequences. However, this disclosure explicitly considers polynucleotides that vary due to differences in codon usage.

In some embodiments, codon-optimized sequences for expression in host cells, such as mammalian cells, are particularly considered. Codon optimization can be performed using known techniques and tools, for example, using the OptimumGene ^™ tool. Codon-optimized sequences include at least partially codon-optimized sequences (i.e., one or more codons are optimized for expression in host cells) and fully codon-optimized sequences. Codon optimization for expression in certain immune host cells is described, for example, in Scholten et al., Clin. Immunol. 119:135, 2006.

Exemplary polynucleotide sequences encoding the TCR chain of the present invention are provided in SEQ ID NO: 1-4, 9-12, 17-20, 25 and 26. Therefore, in some embodiments, the polynucleotide binding protein encoding Msln-specific polynucleotides comprises a polynucleotide having at least about 50% (i.e., at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identity with the polynucleotide sequence shown in any of SEQ ID NO: 1-4, 9-12, 17-20, 25 and 26.

In some embodiments, polynucleotides encoding the TCRα chain and the TCRβ chain are provided, each having at least about 50% identity with the polynucleotide sequences shown below: SEQ ID NO: (i) 1 and 3, respectively; (ii) 2 and 4, respectively; (iii) 9 and 11, respectively; (iv) 10 and 12, respectively; (v) 17 and 19, respectively; (vi) 18 and 20, respectively; or (vii) 25 and 26, respectively.

In some embodiments, two or more components or portions of a binding protein or TCR encoding the present disclosure are contained within two or more coding sequences operably bound in a single open reading frame. Such an arrangement can advantageously allow for the co-expression of desired gene products, e.g., the simultaneous expression of the α and β chains of a TCR, such that they are produced in an approximately 1:1 ratio. In some embodiments, two or more substituent gene products of the binding protein of the present disclosure, such as a TCR (e.g., α and β chains), are expressed as separate molecules and bound post-translationally. In further embodiments, two or more substituent gene products of the binding protein of the present disclosure are expressed as a single peptide, portions of which are separated by cleavable or removable fragments.

For example, self-cleaving peptides (also known as "ribosomal jumping elements") can be used to express separable polypeptides encoded by a single polynucleotide or vector, as is known in the art, including, for example, the P2A peptide, which is a peptide encoded by a polynucleotide having the nucleotide sequence shown in any one of SEQ ID NO:41-46; the East Asian virus 2A (T2A) peptide, which is a peptide encoded by a polynucleotide having the nucleotide sequence shown in SEQ ID NO:47; and the vest. Equine rhinitis virus (ERAV) 2A (E2A) peptide, for example, a peptide encoded by a polynucleotide having the nucleotide sequence shown in SEQ ID NO:48, and foot-and-mouth disease virus 2A (F2A) peptide, for example, a peptide encoded by a polynucleotide having the nucleotide sequence shown in SEQ ID NO:49. Exemplary amino acid sequences of self-cleaving peptides are provided in SEQ ID NO:113-117.

Exemplary polynucleotides encoding the Msln-specific TCR of the present invention, wherein a polynucleotide encoding a self-cleaving peptide is positioned between a polynucleotide encoding the TCRβ chain and a polynucleotide encoding the TCRα chain, including those polynucleotides encoding amino acid sequences as shown in any one of SEQ ID NO: 8, 16, 24, and 30. Exemplary such polynucleotides have polynucleotide sequences as shown in any one of SEQ ID NO: 5, 13, 21, 27, and 120; in some embodiments, a polynucleotide having at least about 50% identity with the polynucleotide sequence shown in any one of SEQ ID NO: 5, 13, 21, 27, and 120 is provided.

In further embodiments, the binding protein is expressed as part of the encoded transgenic construct, and/or the host immune cell containing the polynucleotide encoding the binding protein may further encode: one or more additional accessory proteins, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor β chain; a CD8 co-receptor α chain or both; or any combination thereof. Polynucleotides and transgenic constructs, including their nucleotide and amino acid sequences, which can be used to encode and express binding proteins and accessory components (e.g., safety switch proteins, selection markers, CD8 co-receptor β chains or CD8 co-receptor α chains), are described in published PCT application no. WO 2018/058002 and are incorporated herein by reference. It should be understood that any or all of the binding proteins, safety switch proteins, tags, selection markers, CD8 co-receptor β chains or CD8 co-receptor α chains of this disclosure may be encoded by individual nucleic acid molecules or may be encoded by polynucleotide sequences present or contained in separate polynucleotide sequences.

For example, exemplary safety switch proteins include truncated EGF receptor peptides (huEGFRt) that lack an extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity, but retain their native amino acid sequence, have type I transmembrane cell surface localization, and possess a conformationally intact binding epitope to the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011); caspase peptides (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; Di Stasi et al., N. Engl. J. Med. 365:1 673-1683, 2011; Zhou and Brenner, Exp. Hematol. pii:S0301-472X(16)30513-6. doi:10.1016/j.exphem.2016.07.011), RQR8 (Philip et al., Blood 124:1277-1287, 2014); 10-amino acid tag derived from human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Sci. USA 105:623-628, 2008); and tag/safety switch peptides, such as RQR (CD20+CD34; Philip et al., 2014).

Other auxiliary components that can be used with the recombinant host cells disclosed herein include tags or selection markers that allow for the identification, sorting, separation, enrichment, or tracking of cells. For example, labeled cells with desired properties (e.g., antigen-specific TCRs and safety switch proteins) can be sorted from unlabeled cells in a sample and more efficiently activated and expanded for inclusion in a product of desired purity.

As used herein, the term "selection marker" includes a nucleic acid construct (and encoded gene product) that confers cellular recognition of changes, thereby allowing detection and positive selection of immune cells transduced with a polynucleotide containing the selection marker. RQR is a selection marker comprising a major CD20 extracellular loop and two minimal CD34 binding sites. In some embodiments, the polynucleotide encoding RQR comprises a polynucleotide encoding a 16-amino acid CD34 minimal epitope. In some embodiments, the CD34 minimal epitope is incorporated into the N-terminal position of the CD8 co-receptor stem domain (Q8). In a further embodiment, the CD34 minimal binding site sequence may bind to a target epitope of CD20 to form a dense marker/suicide gene for T cells (RQR8) (Philip et al., 2014, incorporated herein by reference). This construct allows for the selective deletion of transgenic engineered T cells expressing the construct, such as CD34-specific antibodies that bind to magnetic beads (Miltenyi), and the use of clinically accepted drug antibody rituximab (Philip et al., 2014).

Other exemplary selection markers include several truncated type I transmembrane proteins that are not typically expressed on T cells: truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (see, for example, Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011; Mavilio et al., Blood 83:1988-1997, 1994; Fehseet et al., Mol. Ther. 1:448-456, 2000; each incorporated herein by reference). A useful feature of CD19 and CD34 is that they can be used with the readily available Miltenyi CliniMACs™ selection system for clinical-grade sorting. However, CD19 and CD34 are relatively large surface proteins, which may increase vector packaging capacity and transcriptional efficiency of integrated vectors. Surface markers containing extracellular non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR) may also be used. Any selection marker can be employed and should be acceptable for good manufacturing practices. In some embodiments, the selection marker is expressed together with a polynucleotide encoding a target gene product (e.g., a binding protein of the present disclosure, such as a TCR or CAR). Other examples of selection markers include, for example, reporter molecules such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In some embodiments, the selection marker, for example, CD34, is expressed by cells, and CD34 can be used to selectively enrich or isolate (e.g., by immunomagnetic selection) target cells of interest for use in the methods described herein. As used herein, the CD34 marker is distinguished from anti-CD34 antibodies or, for example, scFv, TCR, or other antigen recognition moieties that bind to CD34.

In some embodiments, the selected markers include RQR peptides, truncated low-affinity nerve growth factor (tNGFR), truncated CD19 (tCD19), truncated CD34 (tCD34), or any combination thereof.

In practicing the various embodiments of this disclosure, standard techniques can be used for the synthesis of recombinant DNA, peptides, and oligonucleotides; immunoassays; tissue culture; and transformations (e.g., electroporation and lipid transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly performed in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods known in the art, and various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology techniques are incorporated in or disclosed throughout this specification. (See, e.g., Sambrook, et al, Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: APractical Approach, vol.I&II (IRL Press, Oxford Univ. Press USA, 1985); CurrentProtocols in Immunology (Edited by: John E.Coligan, Ada M.Kruisbeek, DavidH.Margulies, Ethan M.Shevach, Warren Strober 2001John Wiley&Sons, NY, NY); Real-Time PCR:Current Technology and Applications,Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic AcidHybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning(1984);Next-Generation GenomeSequencing (Janitz, 2008Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (Park, Ed., ^3rd Edition, 2010Humana Press); Immobilized Cells And Enzymes (IRLPress, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., NY); GeneTransfer Vectors For Mammalian Cells (JHMiller and MPCalos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In CellAnd Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV(DMWeir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem CellProtocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001); and Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008)).

Vectors containing the polynucleotides according to the invention are also provided. Any suitable expression vector can be used, including the exemplary expression vectors disclosed herein. Furthermore, the expression vector can be configured or enabled to deliver the polynucleotides to a host cell.

Typical vectors can include nucleic acid molecules capable of transporting another nucleic acid linked to them or capable of replicating in a host organism. As described herein, some examples of vectors include plasmids, viral vectors, granules, and others.

Some vectors may be able to replicate autonomously in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free-type mammalian vectors), while others may integrate into the host cell's genome after introduction and replicate along with the host genome. Additionally, some vectors are capable of directing the expression of genes operatively linked to them (these vectors may be referred to as "expression vectors"). According to relevant embodiments, it should also be understood that if one or more reagents (e.g., polynucleotides encoding Msln-specific binding proteins or variants thereof, as described herein) are co-administered to a subject, each reagent may reside in separate or identical vectors, and multiple vectors (each containing a different reagent or the same reagent) may be introduced into cells or cell populations or administered to a subject.

As used herein, an "expression vector" refers to a DNA construct containing a nucleic acid molecule operatively linked to a suitable control sequence that influences the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter that enables transcription, an optional operon sequence that controls such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences that control the termination of transcription and translation. A vector can be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself. In this specification, "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.

In some embodiments, the viral vector is used to introduce a non-endogenous nucleic acid sequence encoding a target-specific polypeptide. The viral vector may be a retroviral vector or a lentiviral vector. The viral vector may also include a nucleic acid sequence encoding a transduction marker.

Viral vectors suitable for use with the compositions of the present invention include those identified for human gene therapy applications (see Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectors include RNA virus-based vectors, such as vectors derived from retroviruses, such as vectors derived from Moloney murine leukemia virus (MLV), and more complex vectors derived from retroviruses, such as vectors derived from lentiviruses. HIV-1-derived vectors belong to this class.

Viral vectors include retroviruses, adenoviruses, parvoviruses (such as adeno-associated virus), coronaviruses, negative-strand RNA viruses (such as orthomyxoviruses, such as influenza virus), rhabdoviruses (such as rabies and vesicular stomatitis virus), paramyxoviruses (such as measles and Sendai virus), positive-strand RNA viruses (such as piconemaviruses and A viruses), and double-stranded DNA viruses, including adenoviruses, herpesviruses (such as herpes simplex virus types 1 and 2 and Epstein-Barr virus and cytomegalovirus), and poxviruses (such as cowpox, fowlpox, and canarypox). Other viruses include, but are not limited to, norovirus, cloacal virus, flavivirus, reovirus, papillomavirus, and hepatitis virus. And hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma, mammalian C, B, and D viruses, HTLV-BLV groups, lentiviruses, and foam viruses (Coffin, J.M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B.N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

Retroviruses are viruses with an RNA genome that are reverse transcribed into DNA using reverse transcriptase, and then incorporated into the host cell's genome. Gamma retroviruses refer to the genus *Retroviridae*. Examples of gamma retroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

As used herein, “lentiviral vector” refers to an HIV-based lentiviral vector used for gene delivery. It can be integrated or non-integrated, has a relatively large packaging capacity, and can transduce a variety of different cell types. Lentiviral vectors are typically generated after transient transfection of three or more plasmids (packaging, envelope, and transfer) into production cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with cell surface receptors. Upon entry, the viral RNA undergoes reverse transcription, mediated by the viral reverse transcriptase complex. The reverse transcription product is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of the infected cell. “Lentivirus” refers to the genus of retroviruses capable of infecting both dividing and non-dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV types 1 and 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Vesna virus (sheep lentivirus).

Methods for transducing mammalian host cells with viral particles containing chimeric antigen receptor transgenes using retroviral and lentiviral vectors and packaging cells are known in the art and have been previously described, for example, in U.S. Patent No. 8,119,772; Walchli, et al., PLoS One 6:327930, 2011; Zhao, et al., J. Immunol. 174:4415, 2005; Engels, et al., Hum. Gene Ther. 14:1155, 2003; Frecha, et al., Mol. Ther. 75:1748, 2010; and Verhoeyen, et al., Methods Mol. Biol. 506:91, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

In some embodiments, the viral vector may be a gamma retrovirus, such as a vector derived from Moloney murine leukemia virus (MLV). In other embodiments, the viral vector may be a more complex retroviral vector, such as a lentiviral vector. HIV-1-derived vectors belong to this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Vesna virus (sheep lentivirus). Methods for transducing mammalian host cells with viral particles containing TCR or CAR transgenes using retroviral and lentiviral vectors and packaging cells are known in the art and have been previously described, for example, in U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors can also be used for polynucleotide delivery, including DNA viral vectors, such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

Other vectors developed for gene therapy purposes may also be used with the compositions and methods disclosed herein. Such vectors include those derived from baculoviruses and alpha viruses (Jolly, DJ. 1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (e.g. Sleeping Beauty or other transposon vectors).

When a viral vector genome contains multiple polynucleotides that will be expressed as individual transcripts in the host cell, the viral vector may also include additional sequences between two (or more) transcripts that allow bicistronic or polycistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

In some embodiments, polynucleotides encoding Msln-specific binding proteins can be operatively linked to one or more specific elements of the vector. For example, polynucleotide sequences that influence the expression and processing of the coding sequences to which they are linked can be efficiently linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancement sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences enhancing translation efficiency (i.e., Kozak concordant sequences); sequences enhancing protein stability; and sequences that may enhance protein secretion. If the expression control sequence is adjacent to the target gene and the expression control sequence, the expression control sequence can be efficiently linked, with the expression control sequence controlling the target gene in a trans or long-range manner. In some embodiments, the viral or plasmid vector also includes transconduction markers (e.g., green fluorescent protein, tEGFR, tCD19, tNGFR, etc.).

In some embodiments, the vector is capable of delivering a polynucleotide construct to a host cell (e.g., hematopoietic progenitor cells or human immune system cells). In specific embodiments, the vector is capable of delivering the construct to human immune system cells, such as CD4+ T cells, CD8+ T cells, CD4-CD8- double-negative T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof. In other embodiments, the vector is capable of delivering the construct to naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof. In some embodiments, the vector encoding the construct of this disclosure may further comprise a polynucleotide encoding a nuclease that can be used for chromosome knockout in a host cell (e.g., a CRISPR-Cas endonuclease or another endonuclease disclosed herein), or for delivering a therapeutic transgene or a portion thereof into a host cell in gene therapy replacement or gene repair therapies. Alternatively, a nuclease for chromosome knockout or gene replacement or gene repair therapies may be delivered to a host cell independently of the vector encoding the construct of this disclosure.

The construction of expression vectors for recombinantly generating Msln-specific binding proteins can be accomplished using any suitable molecular biology engineering techniques known in the art, including digestion with restriction endonucleases, ligation, transformation, plasmid purification, and DNA sequencing, as described, for example, in Sambrook, et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel, et al. (Current Protocols in Molecular Biology (2003)). For efficient transcription and translation, each recombinant expression construct includes at least one suitable expression control sequence (also known as a regulatory sequence), such as a leader sequence, and in particular a promoter operatively (i.e., operablely) linked to the nucleotide sequence encoding the target protein or peptide.

In some embodiments, nucleic acid molecules encoding binding proteins specific to Msln _20-28 or Msln _530-538 peptides are used to transfect/transduct host cells (e.g., T cells) for adoptive transfer therapy. Advances in TCR sequencing have been described (e.g., Robins, et al., 2009 Blood 114:4099; Robins, et al., 2010 Sci. Translat. Med. 2:47ra64, PMID:20811043; Robins, et al., 2011 (Sept. 10) J. lmm. Meth. Epub ahead of print, PMID:21945395; and Warren, et al., 2011 Genome Res. 21:790) and can be used in the practice of embodiments according to this disclosure.

Similarly, methods for transfecting/transducing T cells with desired nucleic acids have been described (e.g., US2004/0087025), as well as adoptive transfer procedures using T cells with desired antigen specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 77:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:112, 2007; Kuball et al., Blood 109:2331, 2007; US 2011/0243972; US 2011/0189141; and Leen et al.). (al., Ann. Rev. Immunol. 25:243, 2007), to adapt these methodologies to the teachings herein, the “binding” of embodiments of this disclosure is contemplated, including proteins that specifically bind to Msln _20-28 (SEQ ID NO:31) or Msln _530-538 (SEQ ID NO:32) peptides that are complexed with HLA receptors.

Recombinant expression vectors may include, for example, tissue-specific transcriptional regulatory elements (TREs), such as B lymphocyte, T lymphocyte, or dendritic cell-specific TREs. Tissue-specific TREs are known in the art (see, for example, Thompson, et al., Mol. Cell. Biol. 72:1043, (1992); Todd et al., J. Exp. Med. 177:1663, (1993); and Penix, et al., J. Exp. Med. 775:1483, (1993)).

Also provided are recombinant (e.g., modified) host cells encoding (e.g., containing encoded heteropolynucleotides) and/or expressing Msln-specific binding proteins as disclosed herein. In some embodiments, the host cell may be a hematopoietic progenitor cell or an immune system cell as disclosed herein, such as a human immune system cell. In any of the currently disclosed embodiments, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double-negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a dendritic cell, or any combination thereof. Additionally, the T cell may be a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof. In some embodiments, the host cell is modified to contain or contain heteropolynucleotides using vectors disclosed herein.

The recombinant host cell can be allogeneic, syngeneic, or autologous (e.g., for a subject receiving treatment with this host cell). In some embodiments where the host cell encodes an endogenous TCR, the heterologous binding protein or TCR expressed by the T cell is able to associate with the CD3 protein more effectively than the endogenous TCR. In some embodiments, the Msln-specific binding protein expressed by the host T cell is able to associate with the CD3 complex and exhibit functional surface expression and immune activity, such as cytokine production and/or killing of target cells expressing antigens. In some embodiments, the Msln-specific binding protein may have higher cell surface expression than the endogenous TCR.

In any of the currently disclosed embodiments, the host cell, such as a host immune cell, may contain a chromosomal gene knockout of an endogenous immune cell protein, such as PD-1, TIM3, LAG3, CTLA4, TIGIT, HLA components, or TCR components, or any combination thereof. As used herein, the term "chromosomal gene knockout" refers to a genetic alteration or the introduction of an inhibitor in the host cell that prevents (e.g., reduces, delays, inhibits, or eliminates) the production of a functionally active endogenous polypeptide product by the host cell. Alterations leading to chromosomal gene knockout may include, for example, the introduction of nonsense mutations (including premature termination codon formation), missense mutations, gene deletions and strand breaks, and the heterologous expression of repressive nucleic acid molecules that inhibit the expression of endogenous genes in the host cell.

After using knockout procedures or reagents, chromosomal gene knockout can be directly confirmed by DNA sequencing of host immune cells. Chromosomal gene knockout can also be inferred from the absence of gene expression after knockout (e.g., the absence of mRNA or polypeptide products encoded by the gene).

In some embodiments, chromosomal gene knockout or knock-in is performed via chromosome editing of the host cell. Chromosomal editing can be performed using, for example, endonucleases. As used herein, "endonuclease" refers to an enzyme capable of catalyzing the cleavage of phosphodiester bonds within a polynucleotide chain. In some embodiments, endonucleases are capable of cleaving a target gene, thereby inactivating or "knocking out" the target gene. Endonucleases can be naturally occurring, recombinant, genetically modified, or fusion endonucleases. Nucleic acid chain breaks induced by endonucleases are typically repaired through the unique mechanism of homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, donor nucleic acid molecules can be used for donor gene "knock-in," for target gene "knock-out," and optionally to inactivate the target gene via a donor gene knock-in or target gene knock-out event. NHEJ is an error-prone repair process that typically results in a change in the DNA sequence at the cleavage site, such as the substitution, deletion, or addition of at least one nucleotide. NHEJ can be used to "knock out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE nucleases, CRISPR-Cas nucleases, a wide range of nucleases, and megaTAL.

As used herein, a “zinc finger nuclease” (ZFN) is a fusion protein containing a zinc finger DNA-binding domain fused to a nonspecific DNA cleavage domain, such as the Fok1 endonuclease. Each zinc finger motif, approximately 30 amino acids long, binds to approximately 3 base pairs of DNA, and amino acids at certain residues can be altered to change the specificity of the triplet sequence (e.g., see Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285:1917-1934, 1999). Multiple zinc finger motifs can be tandemly linked to produce binding specificity to desired DNA sequences, such as regions ranging in length from approximately 9 to approximately 18 base pairs. As background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome and facilitate targeted integration of transgenes by including flanking sequences homologous to the DSB location. Alternatively, DSBs generated by ZFNs can lead to the knockout of target genes via non-homologous end joining (NHEJ) repair, an error-prone cellular repair pathway that results in nucleotide insertions or deletions at the cleavage site. In some embodiments, gene knockout comprises insertions, deletions, mutations, or combinations thereof using ZFN molecules.

As used herein, “transcription activator-like effector nuclease” (TALEN) refers to a fusion protein containing a TALE DNA-binding domain and a DNA-cutting domain, such as the FokI endonuclease. The “TALE DNA-binding domain” or “TALE” consists of one or more TALE repeat domains/units, each typically having a highly conserved 33-35 amino acid sequence and distinct 12th and 13th amino acids. The TALE repeat domain is involved in the binding of the TALE to the target DNA sequence. The distinct amino acid residues, called repeatable variable residues (RVDs), are associated with specific nucleotide recognition. Natural (canonical) codes for DNA recognition of these TALEs have been determined such that the HD (histidine-aspartate) sequence at positions 12 and 13 of the TALE results in the TALE binding to cytosine (C), NG (asparagine-glycine) to T nucleotides, NI (asparagine-isoleucine) to A, NN (asparagine-asparagine) to G or A nucleotides, and NG (asparagine-glycine) to T nucleotides. Non-classical (atypical) RVDs are also known (see, for example, U.S. Patent Publication No. 2011/0301073, atypical RVDs are incorporated herein by reference in their entirety). TALENs can be used to guide site-specific double-strand breaks (DSBs) in the T cell genome. Non-homologous end joining (NHEJ) joins DNA from both sides of a double-strand break where there is little or no sequence overlap for annealing, thus introducing an error that knocks out gene expression. Alternatively, if homologous flanking sequences are present in the transgene, direct homology repair can introduce the transgene into the DSB site. In some embodiments, gene knockout comprises insertions, deletions, mutations, or combinations thereof, and is prepared using TALEN molecules.

As used in this article, the “clustered regularly spaced short palindromic repeats/Cas” (CRISPR/Cas) nuclease system refers to a system in which a CRISPR RNA (crRNA)-guided Cas nuclease recognizes a target site within the genome (called the protospacer region) through base pairing complementarity, and then cleaves the 3’ of the DNA sequence upon the subsequent short, conserved protospacer-associated motif (PAM). Based on the sequence and structure of the Cas nuclease, the CRISPR/Cas system is classified into three types (i.e., type I, type II, and type III). Type I and type III crRNA-guided surveillance complexes require multiple Cas subunits. The most studied type II system contains at least three components: an RNA-guided Cas9 nuclease, crRNA, and trans-crRNA (tracrRNA). The tracrRNA contains a double-strand-forming region. crRNA and tracrRNA form a double-stranded RNA that interacts with the Cas9 nuclease and guides the Cas9/crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base pairing between the crRNA spacer region and the protospacer region upstream of the PAM. The Cas9 nuclease cleaves the double-stranded RNA within the region defined by the crRNA spacer region. NHEJ repair results in insertions and/or deletions, thereby disrupting the expression of the target locus. Alternatively, homology-guided repair can introduce transgenes with homologous flanking sequences into the DSB site. crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012). Furthermore, regions of the guide RNA complementary to the target site can be altered or programmed to target the desired sequence (Xie et al., PLOS One 9:e100448, 2014; US Patent Publication No. 2014/0068797, US Patent Publication No. 2014/0186843; US Patent Publication No. 8,697,359, and PCT Publication No. WO 2015/071474; each of which is incorporated herein by reference). In some embodiments, gene knockout includes insertion, deletion, mutation, or a combination thereof, and is prepared using a CRISPR/Cas nuclease system.

Exemplary gRNA sequences and methods for knocking out endogenous genes encoding immune cell proteins using gRNA sequences include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266(2017), gRNA, CAS9 DNA, vectors, and gene knockout techniques, which are incorporated herein by reference in their entirety.

As used in this article, "large-scale nucleases," also known as "homing endonucleases," refer to endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of approximately 12 to 40 base pairs). Based on sequence and structural motifs, they are classified into five families: LAGLIDADG (SEQ ID NO: 121), GIY-YIG (SEQ ID NO: 122), HNH, His-Cys box, and PD-(D/E)XK (SEQ ID NO: 123). Exemplary wide-ranging nucleases include I-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII, and I-TevIII, whose recognition sequences are known (see, for example, U.S. Patents Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res. 25:3379-338). 8, 1997; Dujon et al., Gene 82:115-118, 1989; Perler et al., Nucleic Acids Res. 22:1125-1127, 1994; Jasin, TrendsG enet. 12:224-228, 1996; Gimble et al., J. Mol. Biol. 263: 163-180, 1996; Argast et al., J. Mol. Biol. 280: 345-353, 1998).

In some embodiments, a wide range of naturally occurring nucleases can be used to promote site-specific genomic modifications targeting genes selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, HLA-encoding genes, or TCR component-encoding genes.

In other embodiments, engineered broad-spectrum nucleases with novel binding specificity to target genes are used for site-specific genome modification (see, for example, Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952-62, 2003; Chevalier et al.). Molec. Cell 10:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49-66, 2007; US Patent Publication Nos. US 2007/0117128; US 2006/0206949; US 2006/0153826; US 2006/0078552; and US2004/0002092). In a further embodiment, a homing endonuclease is used to generate a chromosomal gene knockout, said homing endonuclease having been modified with the modular DNA-binding domain of TALEN to prepare a fusion protein called megaTAL. When used in conjunction with an exogenous donor template encoding a target polypeptide, MegaTALs can be used not only to knock out one or more target genes, but also to introduce (knock out) heterologous or exogenous polynucleotides.

In some embodiments, chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) containing a heteropolynucleotide encoding an antigen-specific receptor that specifically binds to a tumor-associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor, and wherein the encoded target-specific inhibitor inhibits the expression of an endogenous gene (i.e., PD-1, TIM3, LAG3, CTLA4, TIGIT, an HLA component, or a TCR component, or any combination thereof) in the host immune cell.

In some embodiments, the target binding protein can be knocked into a host cell; or it can be bound to a host cell. For example, any currently disclosed technique or reagent that can be used to knock the target polynucleotide into a host cell can be used. In some embodiments, the polynucleotide encoding the binding protein is knocked into the host cell without integration into an endogenous chromosome, such as in the cell nucleus. In some embodiments, the polynucleotide encoding the binding protein is knocked into the host cell at an endogenous locus, optionally disrupting the coding sequence of the endogenous locus. In some embodiments, the polynucleotide encoding the binding protein is knocked into an endogenous TCR locus, thereby knocking out the endogenous TCR and knocking in the target protein. See, for example, Eyquem et al., Nature 543(7643):113–117 (2017).

In some embodiments, a polynucleotide encoding a mesothelin-specific binding protein (e.g., a polypeptide comprising, constituting, or substantially composed of one or more amino acid sequences from SEQ ID NO: 6-8, 14-16, 22-24, 28-40, 78-110, 118, 119) is knocked into a host cell. The binding protein described herein includes a TCRα chain variable domain (Vα) and a TCRβ chain variable domain (Vβ). In any of the currently disclosed embodiments, the mesothelin-specific binding protein is capable of specifically binding to a mesothelin peptide:HLA complex, such as the mesothelin peptide:HLA-A*02:01 complex.

In some embodiments, gene knock-in can be used to introduce a polynucleotide encoding a binding protein capable of specifically binding to the mesothelin peptide antigen described herein (e.g., comprising, constituting, or substantially composed of an amino acid sequence having at least about 85% (i.e., at least about 86%, 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) identity with any of the following amino acid sequences: SEQ ID NO: 6-8, 14-16, 22-24, 28-40, 78-110, 118 or 119).

In some embodiments, a polynucleotide encoding a mesothelin-specific binding protein (e.g., TCR) is knocked into a host cell, and the host cell further contains polynucleotides encoding different binding proteins. In some embodiments, the different binding proteins are heterologous to the host cell. In other embodiments, the different binding proteins are endogenous to the host cell. In some embodiments, a polynucleotide encoding a different binding protein is knocked into the host cell. In some embodiments, the different binding proteins are for different antigens (e.g., different Msln antigens, or antigens from different proteins or targets, such as BCMA, CA19-9, BRAF, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAG). E-A4), KRAS, HER2, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor- or pathogen-associated peptides bound to HLA, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, LTβR, LIFRβ, LRP5, MUC1, OSMRβ, TCRα, TCRβ, CD19, CD20 Specific binding proteins for proteins such as CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, HA1-H, Robo1, alpha-fetoprotein (AFP), cyclophosphamide, OX40, PRAME, and SSX-2. For example, host cells may contain knock-in polynucleotides encoding binding proteins that specifically bind to the Msln antigen:HLA complex and (e.g., knock-in) polynucleotides encoding binding proteins (e.g., TCR or CAR). Proteins that specifically bind to the CA19-9 antigen.

In some embodiments, host immune cells encoding and/or expressing the Msln-specific binding protein of this disclosure are able to preferentially migrate or localize in vivo to target tissues expressing homologous Msln antigens, such as tumors, but are present in statistically significantly reduced amounts in non-adjacent tissues of the same type. By way of illustration, host immune cells may be present in lung tumors (e.g., as determined by deep sequencing of the TCR V region encoding the binding protein), but at low levels or not at all in the same lung tissue not adjacent to the tumor. In some embodiments, non-adjacent tissue includes or refers to tissue removed at least 3 cm from diseased or malignant tissue.

In some embodiments, host cells are enriched with the cell composition, for example, which may be administered to a subject. As used herein, "enrichment" or "depletion" relative to the amount of cell type in a mixture means that, in a cell mixture produced during one or more enrichment or depletion processes or steps, the number of "enriched" cells increases, the number of "depleted" cells decreases, or both. Thus, depending on the source of the original cell population undergoing the enrichment process, the mixture or composition may contain 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more (number or quantity) of "enriched" cells. Cells undergoing a depletion process can result in a mixture or composition containing 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less (in quantity or count) of “depleted” cells. In some embodiments, the mixture will be enriched in quantity of one cell type and depleted in quantity of another cell type, for example, enriching CD4+ cells while depleting CD8+ cells, or enriching CD62L+ cells while depleting CD62L- cells, or a combination thereof.

use

On the other hand, this disclosure provides a method for treating a subject (i.e., someone suffering from or suspected of suffering from a disease or condition related to mesothelin antigen) by administering an effective amount of the composition described herein (e.g., a binding protein, recombinant host cell, polynucleotide, carrier, or related composition) to the subject. Such compositions are also provided for treating such diseases or for preparing medicaments for treating such diseases. These diseases include various forms of proliferative or hyperproliferative diseases, such as solid tumors and hematologic malignancies.

"Treatment," "cure," or "improvement" refers to the medical management of a disease, symptom, or condition in an object (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). Typically, a suitable dose or treatment regimen comprising a host cell expressing the binding protein of this disclosure and optionally an adjuvant is administered in an amount sufficient to cause a therapeutic or preventive benefit. Therapeutic or preventive/preventive benefits include improved clinical outcomes; reduction or alleviation of disease-related symptoms; reduction in the occurrence of symptoms; improved quality of life; a longer disease-free state; a reduction in disease severity; a stable disease state; delayed disease progression; remission; survival; prolonged survival; or any combination thereof.

As used herein, the terms “adoptive immunotherapy” or “adoptive cell therapy” refer to the administration of naturally occurring or genetically engineered disease-antigen-specific immune cells (e.g., T cells). Adoptive cell immunotherapy can be autologous (immune cells are derived from the recipient), allogeneic (immune cells are derived from a donor of the same species), or syngeneic (immune cells are derived from a donor with the same genes as the recipient).

The term "therapeutic effective amount" or "effective amount" for binding proteins or host cells in this disclosure refers to the amount of binding proteins or host cells sufficient to produce a therapeutic effect, including improved clinical outcomes; reduction or alleviation of disease-related symptoms; reduction in the occurrence of symptoms; improved quality of life; longer disease-free status; reduction in disease severity; stabilization of disease status; delayed disease progression; remission; survival or statistically significant extension of survival. When referring to a single active ingredient administered alone or cells expressing a single active ingredient, the therapeutic effective amount refers to the effect of that ingredient or cells expressing that ingredient alone. When referring to a combination, the therapeutic effective amount refers to the combined amount of an active ingredient or a co-active ingredient in a combination with cells expressing the therapeutic effect, whether administered sequentially or concurrently. A combination may also consist of cells expressing more than one active ingredient, such as two different binding proteins that specifically bind to an antigen, or the fusion proteins of this disclosure.

As used in this article, “statistical significance” means that when calculated using the Student’s t-test, the p-value is equal to or less than 0.050, and it indicates that the specific event or measurement result is unlikely to occur by chance.

As used in this article, "hyperproliferative disorders" refer to cells that grow or proliferate excessively compared to normal or disease-free cells. Typical hyperproliferative disorders include tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, malignant pre-cells, and non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenomas, fibromas, lipomas, leiomyomas, hemangiomas, fibrosis, restenosis, and autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, etc.). In contrast to hyperproliferative disorders, certain diseases involving abnormal or excessive growth that develop more slowly than slow-proliferative disorders can be termed "proliferative disorders," including certain tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, malignant pre-cells, and non-neoplastic or non-malignant diseases.

In addition, “cancer” can refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, hematologic or lymphomas or other malignant tumors; malignant tumors of connective tissue; metastatic diseases; minimal residual disease after organ or stem cell transplantation; multidrug-resistant cancers; primary or secondary malignant tumors; angiogenesis or other forms of cancer associated with malignant tumors.

The currently disclosed binding proteins, host cells, polynucleotides, carriers, and compositions can be used to treat or prepare drugs for treating cancer, wherein the Msln _20-28 peptide is expressed on cancer tumor cells, and/or wherein the Msln _530-538 peptide is expressed on cancer tumor cells; exemplary cancers for treatment include mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer.

In some embodiments, the currently disclosed binding proteins, host cells, polynucleotides, carriers, and compositions can be used to treat and/or prepare medicaments for treating cancer, such as solid tumors or hematologic malignancies. In some embodiments, the solid tumors are selected from or include biliary tract cancer, bladder cancer, bone and soft tissue cancer, brain tumors, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoidoma, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumors, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine cancer.

In some embodiments, cancers treatable according to the currently disclosed methods and uses include carcinoma, sarcoma, glioma, lymphoma, leukemia, myeloma, or any combination thereof. In some embodiments, cancers include head or neck cancer, melanoma, pancreatic cancer, bile duct cancer, hepatocellular carcinoma, breast cancer including triple-negative breast cancer (TNBC), gastric cancer, non-small cell lung cancer, prostate cancer, esophageal cancer, mesothelioma, small cell lung cancer, colorectal cancer, glioblastoma, or any combination thereof. In some embodiments, cancers include Askin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's sarcoma, PNET, malignant angioendothelioma, malignant schwannoma, osteosarcoma, alveolar soft tissue sarcoma, angiosarcoma, cystic sarcoma, dermatofibrosarcoma protuberans (DFSP), desmoid tumor, fibroproliferative small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), perivascular tumor, etc. Cytosarcoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymph node sarcoma, undifferentiated pleomorphic sarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, plastic dermatitis, vasoactive intestinal peptide tumor, bile duct carcinoma, hepatocellular carcinoma, adenoid cystic carcinoma, renal cell carcinoma, Grawitz's tumor, ependymoma, astrocytoma Glioma, brainstem glioma, optic glioma, mixed glioma, Hodgkin's lymphoma, B-cell lymphoma, non-Hodgkin's lymphoma (NHL), Burkitt lymphoma, small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunogenic large cell lymphoma, precursor B-cell lymphoblastic lymphoma and mantle cell lymphoma, macroglobulinemia, CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma Marginal zone B-cell lymphoma of extranodal mucosa-associated (MALT) lymphoid tissue, nodular marginal zone B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, adult T-cell lymphoma, extranodal NK/T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, hepatosplenic T-cell lymphoma, primitive NK-cell lymphoma, Sezary syndrome, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, or any combination thereof.

In some embodiments, cancer includes solid tumors. In some embodiments, a solid tumor is a sarcoma or carcinoma. In some embodiments, a solid tumor is selected from: chondrosarcoma; and fibrosarcoma (fibroblastic sarcoma); dermatosarcoma protuberans (DFSP); osteosarcoma, rhabdomyosarcoma, Ewing's sarcoma; gastrointestinal stromal tumor; leiomyosarcoma; angiosarcoma (angiosarcoma); Kaposi's sarcoma; liposarcoma, pleomorphic sarcoma, or synovial sarcoma.

In some embodiments, the solid tumor is selected from lung cancer (e.g., adenocarcinoma, squamous cell carcinoma (squamous cell carcinoma); squamous cell carcinoma; adenocarcinoma; adenosquamous carcinoma; anaplastic carcinoma; large cell carcinoma; small cell carcinoma); and breast cancer (e.g., ductal carcinoma in situ (non-invasive), lobular carcinoma in situ (non-invasive), invasive ductal carcinoma, invasive lobular carcinoma, non-invasive carcinoma); liver cancer (e.g., hepatocellular carcinoma, cholangiocarcinoma, or bile duct cancer); large cell undifferentiated carcinoma, bronchoalveolar carcinoma); ovarian cancer (e.g., surface epithelial-stromal tumor (adenocarcinoma)) or ovarian epithelial carcinoma (including serous tumors, endometrioid tumors, and mucinous tumors). Solid tumors include: cystadenocarcinoma, epidermoid (squamous cell carcinoma), embryonal carcinoma, and choriocarcinoma (germ cell tumors); renal cancer (e.g., renal adenocarcinoma, adrenal carcinoma, transitional cell carcinoma (renal pelvis), squamous cell carcinoma, Bellini ductal carcinoma, clear cell adenocarcinoma, transitional cell carcinoma, renal pelvis carcinoid); adrenal cancer (e.g., adrenocortical carcinoma); testicular cancer (e.g., germ cell carcinoma (seminoma, choriocarcinoma, embryonal carcinoma, teratoma), serous carcinoma); gastric cancer (e.g., adenocarcinoma); intestinal cancer (e.g., duodenal adenocarcinoma); colorectal cancer; or skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma). In some embodiments, solid tumors are ovarian cancer, epithelial ovarian cancer, cervical adenocarcinoma or small cell carcinoma, pancreatic cancer, colorectal cancer (e.g., adenocarcinoma or squamous cell carcinoma), lung cancer, breast ductal carcinoma, or prostate adenocarcinoma.

In any of the currently disclosed embodiments, the host cells are allogeneic cells, syngeneic cells, or autologous cells. Subjects treatable by the invention are typically human and other primate subjects, such as monkeys and apes for veterinary purposes. In any of the above embodiments, the subject can be a human subject. The subject can be male or female and can be of any suitable age, including infants, adolescents, teenagers, adults, and elderly subjects. As determined by those skilled in the art, the cells according to the invention can be administered in a manner suitable for the disease, condition, or disorder to be treated. In any of the above embodiments, cells containing the fusion protein described herein are administered intravenously, intraperitoneally, intratumorally, or into the bone marrow, lymph nodes, or cerebrospinal fluid to encounter the labeled cells to be ablated. The appropriate dosage, appropriate duration, and frequency of administration of the composition will be determined by factors such as the patient's condition; these factors include: the size, type, and severity of the disease, condition, or disorder; the undesirable type or level or activity of the labeled cells; the specific form of the active ingredient; and the method of administration.

Typically, appropriate dosages and treatment regimens deliver the active molecules or cells in amounts sufficient to provide benefit. This response can be monitored in treated subjects by establishing improved clinical outcomes (e.g., more frequent remissions, complete or partial or longer disease-free survival) compared to untreated subjects. Increased pre-existing immune responses to tumor proteins are often associated with improved clinical outcomes. Such immune responses can usually be assessed using standard proliferative, cytotoxic, or cytokine assays.

For prophylactic use, the dosage should be sufficient to prevent, delay, or reduce the onset or severity of the disease or condition. The prophylactic benefit of the immunogenic composition administered according to the methods described herein can be determined by conducting preclinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained through appropriate statistical, biological, and clinical methods and techniques, all of which can be readily practiced by those skilled in the art.

In the case of adoptive cell therapy, an effective dose is the amount of host cells encoding or expressing Msln-specific binding proteins used in the adoptive transfer that is capable of producing clinically desired results in the treated human or non-human mammal (i.e., a specific T-cell immune response against cells expressing Msln-specific antigens, such as cytotoxic T-cell responses, in a statistically significant manner). In a particular embodiment, the T cells are naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof.

Pharmaceutical compositions (compositions) comprising the Msln-specific binding protein disclosed herein, host (i.e., modified) immune cells, polynucleotides or carriers, and pharmaceutically acceptable carriers, diluents, or excipients are also considered. The terms “pharmaceutically acceptable excipient or carrier” or “physiologically acceptable excipient or carrier” refer to biocompatible carriers suitable for human or other non-human mammals, such as physiological saline, described in more detail herein, and generally considered safe or unlikely to cause serious adverse events in subjects. Suitable excipients include water, saline, dextran, glycerol, and combinations thereof. In the examples, compositions comprising the fusion protein or host cells disclosed herein also comprise a suitable infusion medium. A suitable infusion medium can be any isotonic preparation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), a 5% aqueous glucose solution, or Ringer's lactate may be used. The infusion medium may be supplemented with human serum albumin or other human serum components.

Pharmaceutical compositions may be administered in a manner appropriate for the disease or condition to be treated (or prevented) as determined by a person skilled in the medical field. The appropriate dosage and the appropriate duration and frequency of administration of the composition will depend on factors such as the patient's health status, the patient's body type (i.e., weight, mass, or body surface area), the type and severity of the patient's condition, the specific form of the active ingredient, and the method of administration. Generally, appropriate dosage and treatment regimens provide the composition in an amount sufficient to provide therapeutic and/or preventive benefits (as described herein, including improved clinical outcomes such as more frequent complete or partial remissions, longer disease-free and/or overall survival, or reduction in symptom severity).

This article also provides unit doses of either an effective amount of modified immune cells or a composition containing modified immune cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of modified CD4+ T cells, combined with (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of modified CD8+ T cells, in a ratio of about 1:1, wherein the unit dose comprises a reduced amount or substantially no naive T cells (i.e., the percentage of naive T cells present in the unit dose is less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% compared to a patient sample having a comparable number of PBMCs).

In some embodiments, a unit dose comprises (i) a composition comprising at least about 50% modified CD4+ T cells in a ratio of about 1:1, and (ii) a composition comprising at least about 50% modified CD8+ T cells, wherein the number of T cells contained in the unit dose is reduced or substantially absent. In a further embodiment, a unit dose comprises (i) a composition comprising at least about 60% modified CD4+ T cells in a ratio of about 1:1, and (ii) a composition comprising at least about 60% modified CD8+ T cells, wherein the unit dose comprises a reduced amount or substantially no naive T cells. In another embodiment, a unit dose comprises (i) a composition comprising at least about 70% engineered CD4+ T cells, and (ii) a composition comprising at least about 70% engineered CD8+ T cells in a ratio of about 1:1, wherein the number of T cells contained in the unit dose is reduced or substantially absent. In some embodiments, a unit dose comprises (i) a composition comprising at least about 80% modified CD4+ T cells and (ii) a composition comprising at least about 80% modified CD8+ T cells in a ratio of about 1:1, wherein the unit dose comprises a reduced amount or substantially no naïve T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 85% modified CD4+ T cells and (ii) a composition comprising at least about 85% modified CD8+ T cells in a ratio of about 1:1, wherein the unit dose comprises a reduced amount or substantially no naïve T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 90% modified CD4+ T cells and (ii) a composition comprising at least about 90% modified CD8+ T cells in a ratio of about 1:1, wherein the unit dose comprises a reduced amount or substantially no naïve T cells.

The number of cells in the composition or unit dose is at least one cell (e.g., a recombinant CD8+ T cell subset (e.g., optionally comprising memory and/or naive CD8+ T cells); a recombinant CD4+ T cell subset (e.g., optionally comprising memory and/or naive CD4+ T cells)), or generally greater than ^10² cells, for example, up to ^10⁴ , up to ^10⁵ , up to ^10⁶ , up to ^10⁷ , up to ^10⁸ , up to ^10⁹ , or more than ^10¹⁰ cells. In some embodiments, cells are applied in the range of about ^10⁴ to about ^10¹⁰ cells/ ^m² , preferably in the range of about ^10⁵ to about ^10⁹ cells/ ^m² . In some embodiments, the application dose includes up to about 3.3 × ^10⁵ cells/kg. In some embodiments, the application dose includes up to about 1 × ^10⁶ cells/kg. In some embodiments, the application dose includes up to about 3.3 × 10⁵ cells/kg. ⁶ cells/kg. In some embodiments, the administration dose includes up to about 1 × ^10⁷ cells/kg. In some embodiments, the administration dose of recombinant host cells to the subject includes up to about 5 × ^10⁴ cells/kg, ⁵ × 10⁵ cells/kg, 5 × ^10⁶ cells/kg, or up to about 5 × ^10⁷ cells/kg. In some embodiments, the administration dose of recombinant host cells to the subject includes at least about 5 × ^10⁴ cells/kg, 5 × ^10⁵ cells/kg, 5 × ^10⁶ cells/kg, or up to about 5 × 10⁷ cells/kg. ⁷ cells/kg. The number of cells will depend on the intended end use of the composition and the type of cells included therein. For example, cells modified to express or encode binding proteins will comprise at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the purposes described herein, cell volumes are typically 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In examples, the desired cell density is typically greater than ^10⁴ cells/ml and typically greater than ^10⁷ cells/ml, typically ^10⁸ cells/ml or higher. Cells can be administered over a period of time in the form of a single infusion or multiple infusions. In some embodiments, a clinically relevant number of cells may be allocated as multiple infusions, the cumulative number of which equals or exceeds ^10⁶ , ^10⁷ , ^10⁸ , ^10⁹ , ^10¹⁰ , or 10¹⁰. ^Eleven cells. In some embodiments, the unit dose of cells may be co-administered with hematopoietic stem cells from an allogeneic donor (e.g., simultaneously or concurrently). In some embodiments, one or more cells included in the unit dose are autologous to the subject.

It should be understood that unit doses, compositions, or treatment regimens of this disclosure may comprise Msln-specific binding proteins or recombinant host cells as described herein, and may also comprise (e.g., modified) immune cells expressing binding proteins specific to different antigens, such as: different Msln antigens or antigens derived from different proteins or targets, such as BCMA, CA19-9, BRAF, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, E rbB3, ErbB4, EphA2, IGF1R, GD2, O-acetylGD2, O-acetylGD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CE A, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4), KRAS, HER2, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor- or pathogen-associated peptides bound to HLA, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, LTβR, LIFRβ, LRP5, MUC1, OSMR β, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, HA1-H, Robo1, alpha-fetoprotein (AFP), fuzzy protein, OX40, PRAME, and SSX-2, etc. For example, a unit dose or treatment regimen may contain modified CD4+ T cells expressing a binding protein that specifically binds to the Msln antigen:HLA complex and modified CD4+ T cells (and/or modified CD8+ T cells) expressing a binding protein that specifically binds to the CA19-9 antigen (e.g., TCR or CAR).

In any of the embodiments described herein, the unit dose comprises an equal or approximately equal number of engineered CD45RA-CD3+CD8+ and modified CD45RA-CD3+CD4+TM cells.

The pharmaceutical compositions described herein may be contained in single-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to maintain the stability of the formulation until it is infused into a patient.

As used herein, administration of the composition means delivery to a subject, regardless of the route or manner of delivery, such as intravenous, oral, vaginal, rectal, subcutaneous, etc. Administration may be continuous or intermittent and parenteral. Administration may be for the treatment of a subject who has been shown to have a recognized symptom, disease, or disease state, or for the treatment of a subject who is susceptible to or in the process of developing such a symptom, disease, or disease state. Co-administration with adjuvant therapy may include the simultaneous and/or sequential delivery of multiple drugs in any order and according to any dosing regimen (e.g., recombinant host cells with one or more cytokines; immunosuppressive therapies such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolate mofetil, or any combination thereof).

If the compositions of the present invention are administered parenterally, the compositions may further comprise sterile aqueous or oily solutions or suspensions. Suitable non-toxic, parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol mixed with water. Aqueous solutions or suspensions may further comprise one or more buffers, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, any materials used to prepare any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts used. Additionally, the active compound may be incorporated into sustained-release formulations and preparations. As used herein, dosage unit form refers to physically discrete units suitable as a unit dose to the target of treatment; each unit may contain a predetermined amount of recombinant cells or active compound, which is calculated to bind with a suitable drug carrier to produce the desired therapeutic effect.

In some embodiments, multiple doses of the composition described herein (e.g., recombinant host cells) may be administered to a subject at intervals between approximately two weeks and approximately four weeks.

The treatment or prevention methods disclosed herein can be administered to a subject as part of a treatment procedure or regimen, which may include additional treatment before or after administration of the immediately disclosed unit dose, cell, or composition. For example, in some embodiments, a subject receiving a unit dose of HCT (e.g., recombinant host cells) is undergoing or has previously undergone hematopoietic cell transplantation (HCT; including myeloablative and non-myeloablative HCT). Transplantation of any suitable donor cells is known in the art and may include, for example, cells from umbilical cord blood, bone marrow, or peripheral blood, hematopoietic stem cells, mobilized stem cells, or amniotic fluid cells. In some embodiments, the recombinant host cells of the present invention may be administered in a modified HCT therapy together with hematopoietic stem cells or shortly after hematopoietic stem cells. In some embodiments, the HCT comprises donor hematopoietic cells comprising a chromosomal knockout of a gene encoding an HLA component, a chromosomal knockout of a gene encoding a TCR component, or both.

The level of CTL immune response can be determined by any of the various immunological methods described herein and those commonly practiced in the art. The level of CTL immune response can be determined before and after administration of any of the Msln-specific binding proteins (or host cells encoding and/or expressing the same protein) or immunogenic compositions described herein. Cytotoxicity assays for determining CTL activity can be performed using any of the several techniques and methods commonly practiced in the art (see, for example, Henkart, et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127–50, and references cited therein).

Antigen-specific T cell responses are typically determined by comparing observed T cell responses to any of the T cell functional parameters described herein (e.g., proliferation, cytokine release, CTL activity, altered cell surface marker phenotypes, etc.). Antigen specificity is indicated by the difference between T cells exposed to a homologous antigen (e.g., an antigen used to initiate or activate T cells when presented by immunocompatible antigen-presenting cells) and T cells from the same source population exposed to a structurally different or unrelated control antigen. A statistically significant greater response to a homologous antigen than to a control antigen indicates antigen specificity.

Biological samples may be obtained from the subject to determine the presence and level of an immune response to the Msln peptide as described herein. As used herein, “biological samples” may be blood samples from which serum or plasma may be prepared, biopsy samples, body fluids (e.g., bronchoalveolar lavage fluid, ascites, mucosal washings, synovial fluid, etc.), bone marrow, lymph nodes, tissue explants, organ cultures, or any other tissue or cell preparation from the subject or biological sources. Biological samples may also be obtained from the subject prior to receiving any immunogenic composition and may be used as a control to establish baseline (i.e., pre-immunization) data.

In some embodiments, the subject receiving the subject composition has previously received chemotherapy, such as lymph node phagocytosis chemotherapy. In further embodiments, the lymphoblastic chemotherapy comprises cyclophosphamide, fludarabine, anti-thymocyte globulin, oxaliplatin, or combinations thereof. In some embodiments, the subject composition has previously received radiation therapy, immunotherapy comprising cytokines, antibodies, antibody-drug conjugates, or Fc fusion proteins, antisense nucleotide therapy, gene therapy, vaccines, or surgery, or any combination thereof.

The methods and uses according to this disclosure may further include administering one or more other agents in combination therapy to treat a disease or condition. For example, in some embodiments, combination therapy includes administering (simultaneously, concurrently, or sequentially) a composition of an immune checkpoint inhibitor (e.g., any one or more binding proteins encoding and/or expressing the same polynucleotide, a carrier-modified host cell). In some embodiments, combination therapy includes administering the composition of the present invention together with an agonist of a stimulating immune checkpoint agent. In further embodiments, combination therapy includes administering the composition of the present invention together with a secondary therapy, such as a chemotherapy agent, radiation therapy, surgery, antibodies, antibody-drug conjugates, cytokines, antisense therapy, gene therapy, a vaccine, or any combination thereof.

As used herein, the term "immunosuppressant" or "immunosuppressant" refers to one or more cells, proteins, molecules, compounds, or complexes that provide inhibitory signals to help control or suppress an immune response. For example, immunosuppressants include molecules that partially or completely block immune stimulation; for example, reducing, preventing, or delaying immune activation; or increasing, activating, or upregulating immunosuppression. Exemplary targeted immunosuppressants (e.g., those with immune checkpoint inhibitors) include PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

Immunosuppressants (also known as immune checkpoint inhibitors) can be compounds, antibodies, antibody fragments or fusion peptides (e.g., Fc fusion proteins, such as CTLA4-Fc or LAG3-Fc), antisense molecules, ribozymes or RNAi molecules or low molecular weight organic molecules. In any of the embodiments disclosed herein, the method may comprise, alone or in any combination, a composition of the present disclosure having one or more inhibitors having any of the following immunosuppressive components.

Therefore, in some embodiments, the treatment method according to the present invention may further include administering a PD-1 inhibitor to the subject. PD-1 inhibitors may include nivolumab, pembrolizumab, ipilimumab + nivolumab, cimipril; IBI-308; nivolumab + relatlimab; BCD-100; kamizumab; JS-001; spartazumab; tislelizumab; AGEN-2034; BGBA-333 + tislelizumab; CBT-501; dostarlimab; durvalumab + MEDI-0680; JNJ-3283 Pazopanib hydrochloride + pembrolizumab; pidilizumab; REGN-1979 + cimiprizumab; ABBV-181; ADUS-100 + spartazumab; AK-104; AK-105; AMP-224; BAT-1306; BI-754091; CC-90006; cimiprizumab + REGN-3767; CS-1003; GLS-010; LZM-009; MEDI-5752; MGD-013; P F-06801591; Sym-021; Tislelizumab + Pamipanib; XmAb-20717; AK-112; ALPN-202; AM-0001; Antibody against PD-1 in Alzheimer's disease; BH-2922; BH-2941; BH-2950; BH-2954; Biologic agents against CTLA-4 and PD-1 in solid tumors; Bispecific monoclonal antibodies targeting PD-1 and LAG-3 in tumors; BLSM-101; CB -201; CB-213; CBT-103; CBT-107; Cellular immunotherapy + PD-1 inhibitor; CX-188; HAB-21; HEISCOIII-003; IKT-202; JTX-4014; MCLA-134; MD-402; mDX-400; MGD-019; Monoclonal antibody antagonizing PD-CD1 tumors; Monoclonal antibody antagonizing PD-1 tumors; Oncolytic virus inhibiting PD-1 tumors; OT-2; PD-1 antagonist Agents + rope interferon α-2b; PEGMP-7; PRS-332; RXI-762; STIA-1110; TSR-075; vaccines targeting HER2 and PD-1 tumors; PD-1 vaccines for tumors and autoimmune diseases; XmAb-23104; antisense oligonucleotides inhibiting PD-1 for oncology; AT-16201; bispecific monoclonal antibody inhibiting PD-1 in oncology; IMM-1802; antagonists of PD-1 and CTLA-4 Monoclonal antibodies for solid tumors and hematologic malignancies; nivolumab biosimilars; recombinant proteins for antagonizing CD278 and CD28 and PD-1 in oncology; recombinant proteins for activating PD-1 in autoimmune and inflammatory diseases; SNA-01; SSI-361; YBL-006; AK-103; JY-034; AUR-012; BGB-108; drugs for solid tumors that inhibit PD-1, Gal-9, and TIM-3; ENUM-244C8; ENU M-388D4; MEDI-0680; Monoclonal antibody antagonizing PD-1 in metastatic melanoma and metastatic lung cancer; Monoclonal antibody inhibiting PD-1 in oncology; Monoclonal antibody targeting CTLA-4 and PD-1 against tumors; Monoclonal antibody antagonizing PD-1 in NSCLC; Monoclonal antibody inhibiting PD-1 and TIM-3 in tumors; Monoclonal antibody inhibiting PD-1 in oncology; Recombinant protein inhibiting PD-1 and VEGF-A for hematologic malignancies and solid tumors; For Small molecules against PD-1 tumors; Sym-016; inebilizumab + MEDI-0680; vaccines against PDL-1 and IDO for metastatic melanoma; anti-PD-1 monoclonal antibodies + cell immunotherapy for glioblastoma; antibodies antagonizing PD-1 for oncology; monoclonal antibodies inhibiting PD-1/PD-L1 in hematologic malignancies and bacterial infections; monoclonal antibodies inhibiting PD-1-induced HIV infection; and/or small molecules inhibiting PD-1 in solid tumors.

In some embodiments, the compositions disclosed herein are used in combination with LAG3 inhibitors such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

In some embodiments, the compositions of the present invention are used in combination with an inhibitor of CTLA4. In particular embodiments, the compositions are used in combination with a CTLA4-specific antibody or a binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (e.g., abatacept, belatacept), or any combination thereof.

In some embodiments, the compositions of the present invention are used in combination with a B7-H3 specific antibody or a binding fragment thereof, such as enoxazumab (MGA271), 376.96, or both. The B7-H4 antibody binding fragment may be scFv or a fusion protein thereof, as described, for example, in Dangaj et al., Cancer Res. 73:4820, 2013, and U.S. Patent No. 9,574,000 and PCT Patent Publications Nos. WO/201640724A1 and WO 2013/025779A1.

In some embodiments, the compositions of this disclosure are used in combination with an inhibitor of CD244.

In some embodiments, the compositions of the present invention are used in combination with inhibitors of BLTA, HVEM, CD160, or any of their inhibitors. Anti-CD-160 antibodies are described, for example, in PCT Publication No. WO 2010/084158.

In some embodiments, the compositions of the present invention are used in combination with inhibitors of TIM3.

In some embodiments, the compositions of the present invention are used in combination with inhibitors of Gal9.

In some embodiments, the compositions of this disclosure are used in combination with an adenosine signaling inhibitor, such as a decoy adenosine receptor.

In some embodiments, the compositions of the present invention are used in combination with A2aR inhibitors.

In some embodiments, the compositions of this disclosure are used in combination with KIR inhibitors such as rilimab (BMS-986015).

In some embodiments, the compositions of the present invention are used in combination with inhibitory cytokines (typically cytokines other than TGFβ) or inhibitors of Treg development or activity.

In some embodiments, the compositions of this disclosure are used in combination with an IDO inhibitor, such as levo-1-methyltryptophan, epakalstat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem.49:591-600, 2010), indosimod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyltryptophan (1-MT)-tira-pazamine, or any combination thereof.

In some embodiments, the compositions of this disclosure are used in combination with arginase inhibitors, such as N(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-L-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronococcal acid (ABH), S-(2-boronethyl)-L-cysteine (BEC), or any combination thereof.

In some embodiments, the compositions disclosed herein are used in combination with VISTA inhibitors such as CA-170 (Curis, Lexington, Mass.).

In some embodiments, the compositions of this disclosure are used in combination with a TIGIT inhibitor such as COM902 (Compugen, Toronto, Ontario, Canada), a CD155 inhibitor such as COM701 (Compugen), or both.

In some embodiments, the compositions of the present invention are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO 2016/134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO 2017/021526.

In some embodiments, the compositions of the present invention are used in combination with LAIR1 inhibitors.

In some embodiments, the composition n of the present invention is used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

In some embodiments, the compositions of this disclosure are used in combination with agents that increase the activity of stimulating immune checkpoint molecules (i.e., are agonists). For example, the compositions may be used with CD137 (4-1BB) agonists (e.g., urelumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, CD27 agonists (e.g., CDX-1127), CD28 agonists (e.g., TGN1412, CD80, or CD86), and CD40 agonists (e.g., CP-870, 8...). The composition may be used in combination with agonists of the following: 93, rhuCD40L or SGN-40), CD122 agonists (e.g., IL-2), GITR agonists (e.g., humanized monoclonal antibodies as described in PCT Patent Publication No. WO2016/054638), ICOS (CD278) (e.g., GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof). In any of the embodiments disclosed herein, a method may include administering the composition of the disclosed composition together with one or more agonists of stimulating immune checkpoint molecules, including any of the above, alone or in any combination.

In some embodiments, the combination therapy comprises the compositions of this disclosure and a secondary therapy comprising one or more of the following: an antibody specific to a cancer antigen expressed by a non-inflammatory solid tumor or an antigen-binding fragment thereof, radiation therapy, surgery, chemotherapy agents, cytokines, RNAi, or any combination thereof.

In some embodiments, the combined treatment method includes administering the composition of the present invention followed by further radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma-ray therapy, and radiopharmaceutical therapy. Surgical and surgical techniques suitable for treating a given cancer in a subject are well known to those skilled in the art.

Cytokines that can be used to promote immune anticancer or antitumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, alone or in any combination with the compositions of the present invention. In a further embodiment, the cytokines are administered sequentially if the subject has been given the Msln-specific composition at least three or four times prior to administration of the cytokines. In some embodiments, the cytokines are administered subcutaneously. In some embodiments, the subject may have received or be receiving immunosuppressive therapy, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolate mofetil, or any combination thereof. In another embodiment, the treated subject has received a non-myeloablative or hematopoietic cell transplant, wherein the treatment may be administered at least 2 to at least 3 months after the non-myeloablative hematopoietic cell transplant.

In some embodiments, the combined treatment method includes administration of the composition of the invention according to the present invention and further administration of a chemotherapeutic agent. Chemotherapy agents include, but are not limited to, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (e.g., intercalating agents), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following group: antimetabolites/anticancer agents, such as pyrimidine analogs (5-fluorouracil, fluorouracil, capecitabine, gemcitabine, and cytarabine) and purine analogs, folic acid antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents (including natural products, such as vinca alkaloids (vincrine, vincristine, and vinorelbine)); microtubule inhibitors; and other chemotherapeutic agents. Interfering agents (e.g., taxanes (paclitaxel, docetaxel)), vincristine, vinblastine, nocodazole, epothilone and nabiveb, epipodophyllotoxin (etoposide, teniposide), DNA damaging agents (actinomycin, sulfanilic acid, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cyclophosphamide, actinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, chlorpyrifos, chlorpyrifos Pyrimidines, glyphosate, glyphosate, glyphosate, glyphosate, glyphosate, glyphosate, glyphosate, glyphosate, glyphosate, paclitaxel, texotetra, temozolomide, teniposide, triethylene thiophosphoramide and etoposide (VP16); antibiotics (such as actinomycin (actinomycin D)), daunorubicin, doxorubicin (doxorubicin), idarubicin, anthracyclines, mitoxantrone, bleomycin, procainoxam (nemomycin) and mitomycin; enzymes (L-asparaginase, total... Systemic metabolism of L-asparagine (depriving cells unable to synthesize their own asparagine); antiplatelet drugs; antiproliferative/antimitotic alkylating agents (e.g., nitrogen mustard gas (methyl ethylamine, cyclophosphamide and its analogues, melphalan, chlorambucil)), acetylinium imine and methyl melamine (hexamethyl melamine and thiotepa), alkyl sulfonates-cyclobutyran, nitrosoureas (carmustine (BCNU) and its analogues, streptozotocin), fluorene-dacarbazine DTICs; antiproliferative/antimitotic antimetabolites (e.g., folic acid analogues (methotrexate)); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutamine; hormones, hormone analogues (estrogens, tamoxifen, goserelin, bicalutamide, nilumethoxazole) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (e.g., DTICs); antiproliferative/antimitotic antimetabolites ...pro Examples of anti-migration agents include tissue plasminogen activators, streptokinase, and urokinase; aspirin, dipyridamole, ticlopidine, clopidogrel, and abciximab; anti-migration agents; anti-secretion agents (breveldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, and mycophenolate mofetil); anti-angiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors and fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab and rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (retinoic acid); mTOR inhibitors; and topoisomerase inhibitors (doxorubicin (adriamycin), acridine, camptothecin, daunorubicin, actinomycin, eniposide, epirubicin, etoposide, and idarubicin). Star, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpannisolone, prednisone and prednisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers, cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin and caspase activators; and chromatin interfering substances.

In some embodiments, the treatment further includes administration of a usable T-cell-based vaccine (see, for example, PCT Publication No. WO 2017/192924, wherein the T-cell vaccine, immunogenic enhancer, transposon expression construct and related methods are incorporated herein by reference in their entirety). In some embodiments, the vaccine composition comprises a liposomal RNA preparation (see, for example, Kreiter, et al, Nature 520:692, 2015, the preparation and methods thereof are incorporated herein by reference in their entirety). In some embodiments, the vaccine composition is used to prepare dendritic cells or other antigen-presenting cells for peptide pulses, which may be performed ex vivo, in vitro or in vivo.

This disclosure also provides a method for preparing antigen-pulsing antigen-presenting cells. In some embodiments, the method includes in vitro contact with (i) an antigen-presenting cell population immunocompatible with a subject, and (ii) polynucleotides, peptides, immunogenic compositions, and/or expression vectors as described herein, under conditions and time sufficient for the antigen-presenting cells to process and present antigens, thereby obtaining antigen-pulsing antigen-presenting cells capable of eliciting an antigen-specific T cell response to the Msln peptide as described herein. The method may further include contacting the antigen-stimulated antigen-presenting cells with one or more immunocompatible T cells under certain conditions and for a period of time sufficient to generate Msln-specific T cells.

In some embodiments, the method further includes transfecting or transducing a population of immune cells in vitro or in vitro with a polynucleotide containing a nucleic acid sequence encoding a binding protein so defined that an amount of engineered Msln-specific immune cells may be adopted or conferred an antigen-specific T-cell response against the Msln antigen when the cells are administered to a subject.

In some embodiments, immune cell lines can be prepared as described by Ho et al. (see 2006 J Immunol Methods 310(1-2):40-52). For example, dendritic cells (DCs) can be obtained from the plastic-adhesive portion of PBMCs (GELLGENIX ^™ , Freiburg, Germany) by culturing them for two days (days -2 to 0) in DC medium supplemented with GM-CSF (800 U/ml) and IL-4 (1000 U/ml). On day -1, the maturation cytokines TNFα (1100 U/ml), IL-1β (2000 U/ml), IL-6 (1000 U/ml), and PGE2 (1 μg/ml) can be added. On day 0, DCs can be harvested in serum-free DC medium over 2 to 4 hours, washed, and pulsed with peptides (10 μg/ml of a single peptide or 2 μg/ml of a peptide library). CD8 T cells can be isolated from PBMCs using anti-CD8 microbeads (MILTENYI BIOTEC ^™ , Auburn, Calif) and stimulated with DCs at an effector-target (E:T) ratio of 1:5 to 1:10 in the presence of IL-21 (30 ng/ml). On day 3, IL-2 (12.5 U/ml), IL-7 (5 ng/ml), and IL-15 (5 ng/ml) can be added. Two hours after IL-21 peptide pulses, cells can be restimulated as antigen-presenting cells (APCs) using the plastic-adhesive portion of irradiated autologous PBMCs from day 10 to day 14. Following restimulation, cells can be supplemented with IL-2 (25 U/ml), IL-7 (5 ng/ml), and IL-15 (5 ng/ml) starting from day 1. T-cell clones can be generated by plated cells at a limited dilution and expanded using TM-LCL coated with OKT3 (ORTHOBIOTECH ^TM , Bridgewater, NJ) and allogeneic PBMCs as described as feeder cells (REP protocol) (see Ho et al., 2006 J Immunol Methods 310(1-2):40-52).

In addition to other embodiments, the present disclosure provides the following embodiments.

In one embodiment, a binding protein is present comprising a T-cell receptor (TCR) α-chain variable domain ( _Vα ) and a TCR _β- chain variable domain ( _Vβ ), wherein: (a) _Vα comprises a CDR3 amino acid sequence as shown in SEQ ID NO:39 or 37, and _Vβ optionally comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:101 or 99; (b) _Vβ comprises a CDR3 amino acid sequence as shown in SEQ ID NO:40 or SEQ ID NO:38, and _Vα optionally comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:102 or 100; and/or (c) _Vα comprises a CDR3 amino acid sequence as shown in SEQ ID NO:39 or 37, and _Vβ comprises a CDR3 amino acid sequence as shown in SEQ ID NO:40 or 38, wherein the binding protein is capable of specifically binding to the mesothelin (Msln) peptide:HLA complex. In one embodiment, (i) the _Vα of (a), (b) and/or (c) of the binding protein disclosed herein comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 102 or 100, provided that at least three or four CDRs have no sequence changes, wherein the CDRs having sequence changes have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or combinations thereof; and/or (ii) the _Vβ of (a), (b) and/or (c) of the binding protein disclosed herein comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 101 or 99, provided that at least three or four CDRs have no sequence changes, wherein the CDRs having sequence changes have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or combinations thereof. Furthermore, the binding protein of one embodiment may comprise: (a) the CDR1α amino acid sequence shown in SEQ ID NO:93; (b) the CDR2α amino acid sequence shown in SEQ ID NO:94; (c) the CDR3α amino acid sequence shown in SEQ ID NO:39; (d) the CDR1β amino acid sequence shown in SEQ ID NO:83, optionally as shown in SEQ ID NO:84, and further optionally as shown in SEQ ID NO:91; (e) the CDR2β amino acid sequence shown in SEQ ID NO:92; and (f) the CDR3β amino acid sequence shown in SEQ ID NO:40. Even further, the binding protein of one embodiment may comprise an amino acid sequence having at least 85% identity with the amino acid sequence encoded by: (a) TRBJ2-3*01; and (b) TRAV21*01 or TRAV21*02; (c) TRBV5-4*01; (d) TRAJ57*01; and/or (e) TRBD1*01 or TRBD2*02. Furthermore, the binding protein of one embodiment may comprise Vα, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:102, and Vβ, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:101. In some cases, the embodiment may comprise a binding protein wherein Vα comprises or is composed of the amino acid sequence shown in SEQ ID NO:102, and wherein Vβ comprises or is composed of the amino acid sequence shown in SEQ ID NO:101. Furthermore, the binding protein of one embodiment may comprise a TCRα chain (TCRα) and a TCRβ chain (TCRβ), wherein TCRα comprises an amino acid sequence or an amino acid sequence composed of having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:110 or 29, and/or wherein TCRβ comprises an amino acid sequence or an amino acid sequence composed of having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:109 or 28. Some embodiments may include a binding protein, wherein TCRα comprises or consists of the amino acid sequence shown in SEQ ID NO: 110 or 29, and wherein TCRβ comprises or consists of the amino acid sequence shown in SEQ ID NO: 109 or 28. In further embodiments, the binding protein comprises: (a) the CDR1α amino acid sequence shown in SEQ ID NO: 89; and (b) the CDR2α amino acid sequence shown in SEQ ID NO: 90; (c) the CDR3α amino acid sequence shown in SEQ ID NO: 37; (d) the CDR1β amino acid sequence shown in SEQ ID NO: 83, optionally as shown in SEQ ID NO: 87; (e) the CDR2β amino acid sequence shown in SEQ ID NO: 88; and (f) the CDR3β amino acid sequence shown in SEQ ID NO: 38. In one embodiment, the binding protein may comprise an amino acid sequence having at least 85% identity with the amino acid sequence encoded by: (a) TRBJ1-1*01 or TRBJ2-3*01; (b) TRAV4-1*01; (c) TRAJ18*01; and/or (d) TRBD1*01 or TRBD2*02. In one embodiment, the binding protein may comprise Vα, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:100, and Vβ, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:99. In one embodiment, the binding protein may comprise Vα, which comprises or consists of the amino acid sequence shown in SEQ ID NO:100, and wherein Vβ comprises or consists of the amino acid sequence shown in SEQ ID NO:99. In some embodiments, the binding protein comprises a TCRα chain (TCRα) and a TCRβ chain (TCRβ), wherein TCRα comprises or is composed of an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 108 or 23, and/or wherein TCRβ comprises or is composed of an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 107 or 22. In some embodiments, the binding protein comprises or is composed of the amino acid sequence shown in SEQ ID NO: 108 or 23, and wherein TCRβ comprises or is composed of the amino acid sequence shown in SEQ ID NO: 107 or 22. In some embodiments, the binding protein comprises a binding protein capable of specifically binding to the SEQ ID NO: 32: human leukocyte antigen (HLA) complex, and in some such cases, the HLA comprises HLA-A*201. In some embodiments, mutagenesis of any one or more of residues 3, 5, 6, or 9 of SEQ ID NO: 32 does not eliminate or substantially does not weaken the binding of the binding protein to the Msln peptide:HLA complex. In some embodiments, the binding protein is capable of binding the peptide:HLA complex, wherein the peptide comprises or is composed of the common amino acid sequence shown in SEQ ID NO:61. In some embodiments, mutagenesis of any one or more alanine residues 1, 5, or 9 of SEQ ID NO:32 does not eliminate or substantially weaken the binding of the binding protein to the Msln peptide:HLA complex. In some embodiments, the binding protein is capable of binding the peptide:HLA complex, wherein the peptide comprises or is composed of the common amino acid sequence shown in SEQ ID NO:62. In some embodiments, the binding protein does not bind or nonspecifically binds the peptide:HLA complex, wherein the peptide comprises or is composed of any one or more amino acid sequences shown in SEQ ID NO:63-77, and wherein HLA is optionally HLA-A:02*01.

In one embodiment, the binding protein comprises a T-cell receptor (TCR) α-chain variable domain ( _Vα ) and a TCR β-chain variable domain ( _Vβ ), wherein: (a) _Vα comprises the CDR3 amino acid sequence shown in SEQ ID NO: 33 or 35, and _Vβ optionally comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 95 or 97; (b) _Vβ comprises the CDR3 amino acid sequence shown in SEQ ID NO: 34 or 36, and _Vα optionally comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 96 or 98; and/or (c) _Vα comprises the CDR3 amino acid sequence shown in SEQ ID NO: 33 or 35, and _Vβ comprises the CDR3 amino acid sequence shown in SEQ ID NO: 40 or 38, wherein the binding protein is capable of specifically binding the mesothelin (Msln) peptide:HLA complex. In one embodiment, Vα of (i)(a), (b), and/or (c) comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 96 or 98, provided that at least three or four CDRs have no sequence changes, wherein the CDRs with sequence changes have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or combinations thereof; and/or _Vβ of (ii)(a), (b), and/or (c) comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 95 or 97, provided that at least three or four CDRs have no sequence changes, wherein the CDRs with sequence changes have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or combinations thereof. In one embodiment, the binding protein comprises: (a) the CDR1α amino acid sequence shown in SEQ ID NO:80; (b) the CDR2α amino acid sequence shown in SEQ ID NO:81 or 118; (c) the CDR3α amino acid sequence shown in SEQ ID NO:33; (d) the CDR1β amino acid sequence shown in SEQ ID NO:83, optionally as shown in SEQ ID NO:84, and further optionally as shown in SEQ ID NO:78; (e) the CDR2β amino acid sequence shown in SEQ ID NO:79; and (f) the CDR3β amino acid sequence shown in SEQ ID NO:34. In one embodiment, the binding protein comprises an amino acid sequence having at least 85% identity with the amino acid sequence encoded by: (a) TRBJ2-7*01 or TRBJ2-3*01; (b) TRAV1-1*01; (c) TRBV12-4*01; (d) TRAJ3*01; and/or (e) TRBD1*01 or TRBD2*02. In one embodiment, the binding protein comprises: Vα, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:96; and Vβ, which comprises an amino acid sequence having at least about 35% identity with the amino acid sequence shown in SEQ ID NO:95. In one embodiment, the binding protein comprises Vα, which comprises or is composed of the amino acid sequence shown in SEQ ID NO:96, and Vβ, which comprises or is composed of the amino acid sequence shown in SEQ ID NO:95. In one embodiment, the binding protein comprises a TCRα chain (TCRα) and a TCRβ chain (TCRβ), wherein TCRα comprises or is composed of an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:104 or 7, and/or wherein TCRβ comprises or is composed of an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:103 or 6. In one embodiment, the binding protein comprises TCRα, which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 104 or 7, and wherein TCRβ comprises or is composed of the amino acid sequence shown in SEQ ID NO: 103 or 106. In one embodiment, the binding protein comprises: (a) the CDR1α amino acid sequence shown in SEQ ID NO: 85; and (b) the CDR2α amino acid sequence shown in SEQ ID NO: 86 or 119; (c) the CDR3α amino acid sequence shown in SEQ ID NO: 35; (d) the CDR1β amino acid sequence shown in SEQ ID NO: 83, optionally as shown in SEQ ID NO: 84, and further optionally as shown in SEQ ID NO: 82; (e) the CDR2β amino acid sequence shown in SEQ ID NO: 79; and (f) the CDR3β amino acid sequence shown in SEQ ID NO: 36. In one embodiment, the binding protein comprises an amino acid sequence having at least 85% identity with the amino acid sequence encoded by: (a) TRBJ2-3*01; (b) TRAV12-3*01; (c) TRBV12-3*01; (d) TRAJ29*01; and/or (e) TRBD1*01 or TRBD2*02. In one embodiment, the binding protein comprises: Vα, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:98; and Vβ, which comprises an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO:97. In one embodiment, the binding protein comprises Vα, which is composed of or constitutes the amino acid sequence of SEQ ID NO:98, and Vβ, which comprises or constitutes the amino acid sequence of SEQ ID NO:97. In one embodiment, the binding protein comprises a TCRα chain (TCRα) and a TCRβ chain (TCRβ), wherein TCRα comprises or consists of an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 106 or 15, and/or wherein TCRβ comprises or consists of an amino acid sequence having at least about 85% identity with the amino acid sequence shown in SEQ ID NO: 105 or 14. In one embodiment, the binding protein comprises TCRα, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 106 or 15; and TCRβ, which comprises or consists of the amino acid sequence shown in SEQ ID NO: 105 or 14. In one embodiment, the binding protein is capable of specifically binding to the SEQ ID NO: 31: human leukocyte antigen (HLA) complex, and wherein the HLA is optionally HLA-A*201. In one embodiment, the binding protein is or comprises a TCR, wherein the TCR is optionally a soluble antigen-binding fragment of a TCR, scTCR, or CAR. In one embodiment, the binding protein is human, humanized, or chimeric. In one embodiment, the binding protein is able to bind the mesothelin:HLA complex in the absence of or independently of CD8.

In one embodiment, the binding protein has a Msln peptide EC50 of about 9 μM, about 8 μM, about 7 μM, about 6 μM, about 5 μM, about 4 μM, about 3 μM, about 2 μM, about 1 μM, about 0.9 μM, about 0.8 μM, about 0.7 μM, about 0.6 μM, about 0.5 μM, about 0.4 μM, about 0.3 μM, about 0.2 μM or smaller.

In one embodiment, a composition is provided comprising the binding protein described herein and a pharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the polynucleotide encodes the binding protein described herein. In some embodiments, the polynucleotide is codon-optimized for expression in a host cell, wherein the host cell is optionally a human immune system cell, preferably a T cell. Similarly, in some embodiments, the polynucleotide has at least about 50% identity with the polynucleotide sequence shown in any one of SEQ ID NO: 1-4, 9-12, 17-20, 25, and 26. The polynucleotide comprises a polynucleotide encoding a TCRα chain and a polynucleotide encoding a TCRβ chain, respectively having at least about 50% identity with the polynucleotide sequences shown below: SEQ ID NO: (i) 1 and 3, respectively; (ii) 2 and 4, respectively; (iii) 9 and 11, respectively; (iv) 10 and 12, respectively; (v) 17 and 19, respectively; (vi) 18 and 20, respectively; or (vii) 25 and 26, respectively. In some embodiments, the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide located between the TCRβ chain-encoding polynucleotide and the TCRα chain-encoding polynucleotide. In some embodiments, the encoded polypeptide comprises the amino acid sequence as described in any one of SEQ ID NO: 8, 16, 24, and 30. In some embodiments, the polynucleotide encoding the binding protein has at least about 50% identity with the polynucleotide sequence described in any one of SEQ ID NO: 5, 13, 21, 27, and 120. In a particular embodiment, the polynucleotide encoding the binding protein comprises or consists of the polynucleotide sequence shown in SEQ ID NO: 120.

In some embodiments, an expression vector is provided comprising a polynucleotide operatively linked to an expression control sequence as described herein. In some embodiments, the expression vector is capable of delivering the polynucleotide to a host cell. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In some embodiments, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8-double-negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a dendritic cell, or any combination thereof. In some embodiments, the immune system cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector or a γ-retroviral vector.

In one embodiment, the recombinant host cell comprises a heteropolynucleotide encoding a binding protein as described herein and/or an expression vector as described herein, wherein the recombinant host cell is capable of expressing the encoded binding protein on its cell surface. In some embodiments, the recombinant host cell is a hematopoietic progenitor cell or a human immune system cell. In some embodiments, the recombinant host cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double-negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a dendritic cell, or any combination thereof. In some embodiments, the recombinant host cell is a T cell. In some embodiments, the recombinant host cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof. In some embodiments, the recombinant host cell is a T cell encoding an endogenous TCR or an NK-T cell, and wherein the mesothelin-specific binding protein encoded by the heteropolynucleotide is more effectively associated with the CD3 protein than the CD3 protein compared to the endogenous TCR. In some embodiments, Nur77 expression is increased in recombinant host cells in the presence of the Msln peptide, which is encoded by a binding protein, at a concentration of about ^10⁻² μM peptide, about ^10⁻¹ μM peptide, about 1 μM peptide, or about ^10¹ μM peptide, wherein the peptide is optionally presented to the host cells via antigen-presenting cells. In some embodiments, the recombinant host cell is a recombinant host cell that, when contacted with cells expressing the following, does not produce IFN-γ and/or does not exhibit activating and/or cytotoxic activity: (i) HLA-C6:02:01; (ii) HLA-B13:01:01 without HLA-B13:02:01; (iii) HLA-A3; (iv) HLA-A29; (v) HLA-B40; (vi) HLA-B44; (vii) HLA-C3; (viii) HLA-C16; (ix) HLA-A1; (x) HLA-24; (xi) HLA-B7; (xii) HLA-B57; (xiii) HLA-C7; (xiv) HLA-A11; (x v)HLA-B15; (xvi)HLA-C4; (xvii)HLA-C12; (xviii)HLA-B8; (xix)HLA-B49; (xx)HLA-B51; (xxi)HLA-C15; (xxii)HLA-A30; (xxiii)HLA-A68; (xxiv)HLA-C2; (xxv)HLA-A32; (xxvi)HLA-A33; (xxvii)HLA-B55; (xxviii)HLA-C1; (xxvix)HLA-C5; (xxix)HLA-B8; (xxx)HLA-B35; or any combination of (xxxi)(i)-(xxx), provided that there is no mesothelin peptide encoded by the binding protein. In some embodiments, the recombinant host cell comprises T cells or NK-T cells encoding an endogenous TCR, wherein the binding protein encoded by a heterologous polynucleotide has higher cell surface expression compared to the endogenous TCR.

In one embodiment, a cell composition is provided comprising the recombinant host cells described herein and a pharmaceutically acceptable carrier, excipient, or diluent.

In one embodiment, a unit dose comprising an effective amount of the recombinant host cell or cell composition described herein is provided.

In one embodiment, a method is provided for treating a disease or condition associated with mesothelin expression and/or activity in a subject, wherein the method comprises: administering to the subject an effective amount of the binding protein described herein, a recombinant host cell described herein, a composition described herein, or a unit dose described herein. In one embodiment, the method includes treating a disease or condition associated with mesothelin expression and/or activity in a subject, wherein the disease or condition is a hyperplastic disease or proliferative disorder. In one embodiment, the method includes treating a disease or condition associated with mesothelin expression and/or activity in a subject, wherein the disease or condition is cancer, and optionally, the cancer is a solid tumor or a hematologic malignancy. In one embodiment, the method includes treating a subject with a disease or condition associated with mesothelin expression and/or activity, wherein the disease or condition is biliary tract cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoidoma, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine cancer. In one embodiment, the method includes treating a subject with a disease or condition associated with mesothelin expression and/or activity, wherein the disease or condition is pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colorectal cancer, mesothelioma, or lung cancer. In one embodiment, the binding protein, host cells, composition, or unit dose is administered parenterally or intravenously. In one embodiment, the method includes administering multiple doses of the binding protein, host cells, composition, or unit dose to the subject, and optionally, administering multiple doses at intervals of approximately two weeks to approximately four weeks. In one embodiment, the method further includes administering cytokines to the subject. In one embodiment, the method includes administering IL-2, IL-15, IL-21, or any combination thereof. In one embodiment, the method includes further receiving or having received an immune checkpoint inhibitor, an agonist of a stimulating immune checkpoint agent, radiotherapy, an antibody, an antibody-drug conjugate, an Fc fusion protein, antisense nucleotide therapy, gene therapy, a vaccine, surgery, chemotherapy, or any combination thereof.

In one embodiment, the binding protein described herein, the composition described herein, the polynucleotide described herein, the expression vector described herein, the recombinant host cell described herein, the cell composition described herein, or the unit dose described herein is for the treatment of a disease or condition characterized by mesothelin expression and/or activity.

In one embodiment, the binding protein, composition, polynucleotide, expression vector, recombinant host cell, cellular composition, or unit dose described herein are used in adoptive immunotherapy for a disease or condition characterized by mesothelin expression and/or activity.

In one embodiment, the binding protein described herein, the composition described herein, the polynucleotide described herein, the expression vector described herein, the recombinant host cell described herein, the cell composition described herein, or the unit dose described herein is for the manufacture of a medicament for treating a disease or condition characterized by mesothelin expression and/or activity.

In one embodiment, the binding protein, composition, polynucleotide, expression vector, recombinant host cell, cell composition, or unit dose described herein is used for diseases or conditions characterized by mesothelin expression and/or activity, wherein the disease or condition is mesothelioma, pancreatic cancer, ovarian cancer, lung cancer, cancer in which Msln _20-28 peptide is expressed on the tumor cells of the cancer, or cancer in which Msln _530-538 peptide is expressed on the tumor cells of the cancer.

In one embodiment, the binding protein, composition, polynucleotide, expression vector, recombinant host cell, cell composition, or unit dose described herein is used in the context of a disease or condition characterized by mesothelin expression and/or activity, wherein the disease or condition is pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, colorectal cancer, mesothelioma, or lung cancer.

In one embodiment, an isolated polynucleotide is provided encoding a binding protein capable of specifically binding to the SEQ ID NO:32:HLA-A:02*01 complex, wherein the polynucleotide comprises or is composed of the polynucleotide sequence of SEQ ID NO:120. In one embodiment, an expression vector is provided comprising a polynucleotide encoding a binding protein capable of specifically binding to the SEQ ID NO:32:HLA-A:02*01 complex, wherein the polynucleotide comprises or is composed of a polynucleotide sequence operatively linked to the sequence shown in SEQ ID NO:120 of the expression control sequence. In such embodiments, the expression vector is capable of delivering the polynucleotide to a host cell. In such embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In such embodiments, the immune system cell of the expression vector is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double-negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a dendritic cell, or any combination thereof. In such embodiments, the immune system cells expressed via the expression vector are naive T cells, central memory T cells, stem cell memory T cells, effector memory T cells, or any combination thereof. The expression vector according to any one of claims 86-90 is a viral vector. In such embodiments, the expression vector is a viral vector, specifically a lentiviral vector or a gamma-retroviral vector.

In one embodiment, a recombinant host cell is provided, comprising the polynucleotides described herein and/or the expression vectors described herein, wherein the recombinant host cell is capable of expressing a binding protein encoded on its cell surface. In such embodiments, the recombinant host cell is a hematopoietic progenitor cell or a human immune system cell. In such embodiments, the recombinant host cell is an immune system cell, specifically a CD4+ T cell, CD8+ T cell, CD4-CD8- double-negative T cell, γδ T cell, natural killer cell, natural killer T cell, macrophage, dendritic cell, or any combination thereof. In such embodiments, the recombinant host cell is a T cell. In such embodiments, the recombinant host cell is a naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof.

Example

Example 1

TCR identification and selection for MSLN ₂₀ or MSLN ₅₃₀ .

Exemplary TCR clones specific to Msln ₂₀ or Msln ₅₃₀ are shown in Figures 1 through 4. Referring to Figures 1A and 1B, the enrichment score of each TCR clonal type in the proposed next-generation sequencing (NGS)-based TCR isolation method is compared to T cell frequency. The enrichment score on the y-axis is correlated with the degree of binding to the peptide:HLA tetramer. All TCRs were synthesized and their function was tested when expressed in reporter T cells and primary CD8 ⁺ PBMCs. TCRs with the highest functional affinity were circled when transferred to recipient CD8 ⁺ T cells. These data suggest that the TCRs identified as having the highest functional activity did not have the highest tetramer binding and were rare in peptide-amplified T cell populations. Unbound from theoretical constraints, this may be partly attributed to reduced TCR surface expression in these highly active clonal types. More than 100 TCR constructs were synthesized and evaluated (Msln ₂₀ = 60 TCRs synthesized; Msln ₅₃₀ = 42 TCRs synthesized). Mesothelin-specific T cell lines were generated from 18 donors. Cells were stained with titrated concentrations of tetramer, sorted, and analyzed by single-cell TCR sequencing (approximately 8 sorting experiments in total).

Example 2

Tetramer binding via MSLN ₅₃₀ TCR

Msln ₅₃₀ -specific TCR-encoding constructs were transduced virally into CD8-Jurkat T cells lacking endogenous TCRα/β chains (Jurkat76 – dark gray) or Jurkat76 cells were transduced to express CD8αβ (Jurkat76-CD8αβ – light gray) (see Figure 2). In the absence of TCRα/β chains, CD3 cannot be expressed on the cell surface. Therefore, CD3 expression is a substitute for TCR surface expression in these cells, allowing for the assessment of tetramer binding relative to TCR surface expression. In Figure 2, TCRs appear in the order of tetramer binding relative to CD3 expression, and TCRs above the indicator line are considered CD8-independent and characterized by high affinity.

Example 3

Assessment of antigen-specific T cell responses

Using the reporter Jurkat T cell line with the tdTomato transgene knock-in Nur77 locus, the antigen-specific function of four TCRs exhibiting the highest levels of tetramer binding was evaluated. T2 target cells were pulsed with titrated concentrations of peptide, and tdTomato expression in T cells expressing the TCRs was assessed, as shown in Figure 3A2. The percentage of tdTomato-positive cells detected at each peptide concentration was plotted and fitted to a dose-response curve using nonlinear regression (Figure 3B). The calculated EC50 for each TCR was plotted, and TCR B11 (also referred to as 11B in this paper) was identified as the most average TCR (see arrows in Figures 3B and 3C). The two TCRs with the highest tetramer binding levels (A16 and A3, also referred to as 16A and 3A, respectively) were found to have lower antigen sensitivity, also indicated by arrows in Figure 3B.

Example 4

Functional evaluation of MSLN ₅₃₀ dedicated TCR

Since the function is independent of tetramer binding to the first four tetramer binders, the expression of tdTomato by all selected Msln ₅₃₀ -specific TCRs was evaluated in response to a lower concentration of peptide (0.1 μm) as an alternative for antigen sensitivity (see Figure 4). It was found that two TCRs (B9 and A11) that bound the tetramer at lower levels mediated high levels of tdTomato expression in response to the antigen. Data from these and other TCRs are shown in the boxes in Figure 4; these have been included in the TCR set for further investigation. Despite the lower tetramer binding rates, several TCRs, including B9 and A11, still exhibited high antigen-specific activity. In Figure 4, the TCRs are presented in order of rank of tetramer binding.

Example 5

Functional evaluation of the selected TCR

Referring to Figures 5A-5C, CD8+ T cells were purified from donor PBMCs and transduced with a TCR lentivirus specific to Msln ₅₃₀ or Msln ₂₀ (selected using a method similar to that described in Examples 3 and 4). After 8 days, tetramer cells were sorted and further expanded for 8-10 days. Transduced T cells were stained with tetramer and CD8 to confirm purity and uniform high levels of CD8 expression.

As shown in Figures 6A-6C, effector CD8 ⁺ T cells transduced with Msln ₂₀ (A) or Msln ₅₃₀ (B) specific TCRs were incubated with target T2 cells from peptide pulses, and dose-dependent IFN-γ production was assessed by flow cytometry analysis of intracellular IFN-γ (effector cells = TCR-transduced primary CD8 ⁺ T cells (sorted); target = peptide pulsed T2 cells). The percentage of IFN-γ-positive cells detected at each peptide concentration was plotted and fitted to a dose-response curve using nonlinear regression. The calculated EC50 for each TCR was plotted. In Figure 6C, arrows indicate the most affinity TCRs for each epitope (“20-B3” and “530-B11”, respectively).

Example 6

CD8 ⁺ T cells transformed by TCR kill tumor cells.

Two Msln-expressing tumor cell lines, MDA-MB-231 and MDA-MB-468, were used to measure specific tumor cell lysis using a chromium release assay, based on the titration ratio of Msln-specific TCR-transduced CD8 ⁺ T cells (see Figures 7A-7C). The results for TCR Msln ₅₃₀ -B11, i.e., the highest affinity TCR identified, are indicated by arrows.

Example 7

Phenotypic analysis of MSLN ₂₀ and MSLN ₅₃₀ peptides by scanning with alanine.

Figure 8 illustrates the epitope analysis assay, in which each consecutive amino acid of the Msln target peptide sequence was replaced with alanine, and TCR-transduced T cells were incubated with HLA-A2 ⁺ target cells pulsed with the variant peptide. Representative data for IFN-γ produced by Msln ₂₀ -specific TCRs in response to each variant peptide are shown at the bottom of the figure.

The results of the alanine scan assay show the percentage of IFN-γ+ T cells in response to each alanine-substituted peptide for each of the four tested TCRs, as shown in Figures 9A-9D. Essential residues are identified by a one-letter amino acid code, and non-essential residues are indicated by X.

Example 8

Epitope homology analysis of MSLN ₅₃₀ in the proteome

Using the ScanProsite tool, human peptides expected to have potential cross-reactivity with Msln-specific TCRs were identified by searching for the indicated common epitope motifs of each indicated Msln ₅₃₀ -specific TCR (A11 and B11) in the human proteome, as shown in Figure 10. HLA-A2 binding of the identified peptides was analyzed using three different prediction algorithms: SITHPATHI, PanMHCnet, and IEDB. The suggested threshold values for each method are listed in parentheses next to the algorithm name. Potentially cross-reactive peptides are shown in the figure and were synthesized for further analysis.

Example 9

Analysis of synthetic peptides with potential homology to MSLN ₅₃₀ in the proteome

T2 target cells were pulsed with peptides that have the potential to cross-react with Msln ₅₃₀ -A11 and -B11 TCRs and incubated with T cells transduced to express these TCRs and sorted for purity. (See Figures 11A-11B; effector T cells = CD8+ T cells transduced with tetramer-sorted TCRs; target cells = T2 cells pulsed with 10 μM peptide). A high dose of peptide (10 μM) was used to assess potential reactivity. The percentage of T cells transduced with IFN-γ-positive TCRs of Msln _530-11A and Msln _530-11B is shown in Figure 11A. The right side of the figure shows the response to 10 μM wild-type Msln530 peptide and the maximum response obtained with a mixture of nonspecific T cell activation peptides. Only one peptide (#10) elicited a low-level (<20%) response in T cells transduced with TCR Msln530-11B at 10 μM peptide. Figure 11B shows dose-response curves of Msln _530-11B -transduced T cells to the Msln ₅₃₀ peptide and several potential cross-reactive peptides (including peptide #10) to determine responsiveness at physiological levels. Percentage IFN-γ ⁺ data were fitted to the dose-response curves using nonlinear regression, and EC50 values were calculated and shown below the graph. These data indicate that the Msln _530-11B EC50 for peptide #10 is more than 3000 times higher than that for Msln _530. Therefore, Msln _530-11B is more specific to Msln ₅₃₀ than to peptide #10.

Example 10

Assimilation reactivity analysis was performed using LCLs from different donor sources.

To determine the potential allogeneic reactivity of T cells expressing Msln _20-3B or Msln _530-11A or -11B, TCR-transduced T cells were co-cultured with allogeneic LCLs naturally expressing different HLA alleles, including many of the more common alleles. LCL lines and corresponding HLA allele expression are listed in the table in Figure 12A. For each cell line (Figures 12B-12I), the percentage of IFN-γ expression (presented by transduced T cells when the LCL cell line lacks HLA-A2 expression) is shown when T cells and LCL cells are co-cultured with or without the added Msln530 peptide.

Further analysis of the targeting of different LCL cell lines to T cells is shown in Figures 13A-13H. To determine the potential allogeneic reactivity of T cells expressing Msln _20-3B or Msln _530-11A or -11B, TCR-transduced T cells were co-cultured with allogeneic LCLs that naturally express multiple HLA alleles, including many of the more common alleles. LCL lines and corresponding HLA allele expression are listed in the table in Figure 13A. For each cell line, the percentage of IFN- _γ expression (presented by transduced T cells when the LCL cell line lacks HLA-A2 expression) is shown after co-culturing target and effector cells with or without the added Msln ₅₃₀ peptide for each cell line. The second group of LCLs contained several lines expressing HLA-C6 and HLA-B13, which exhibited linkage disequilibrium and were often found together. Several of these LCLs elicited responses in Msln 530-11B-transduced T cells. These data indicate that HLA-B13:02:01 is an allelic reactive allele, because only cells expressing HLA-B13:02:01 elicit a response, while cells expressing HLA-C6:02:01 or HLA-B13:01:01 do not elicit a response if HLA-B13:02:01 is not present.

Table 1 shows the frequencies of HLA-B13:02:01 and HLA-A2:01:01 co-expression in different populations. Some HLA-B13:02:01-specific alloantigen reactions were detected. However, given the small frequency of haplotypes in the populations, cross-reactive alleles in patients are rare events.

Table 1: HLA A2:01/B13:02 Haplotype Frequencies

Europeans 0.845% (B13:02) (29.6% A2:01) African Americans 0.177% (B13:02) (12.5% A2:01) Asians and Pacific Islanders 0.110% (B13:02) (9.5% A2:01) Hispanic Americans 0.129% (B13:02) (19.4% A2:01)

The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications mentioned in this specification and/or shown in the application data sheet, including U.S. Provisional Patent Application No. 62/758,397, filed November 9, 2018, are incorporated herein by reference in their entirety. If it is necessary to employ the concepts of various patents, applications and publications to provide other embodiments, aspects of the embodiments may be modified.

These and other changes can be made to the embodiments based on the detailed description above. Generally, the terminology used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but should be interpreted to include all possible embodiments and the full scope of the claims and their equivalents. Therefore, the claims are not limited to the disclosure.

Sequence List

<110> Fred Hutchinson Cancer Center

Juno Therapeutics Co., Ltd.

T.M. Schmitt

A.G. Sapio

P.D. Greenberg

<120> Mesothelin-specific T-cell receptor and its application in immunotherapy

<130> 360056.475WO

<140> PCT

<141> 2019-11-08

<150> US 62/758,397

<151> 2018-11-09

<160> 123

<170> FastSEQ for Windows Version 4.0

<210> 1

<211> 939

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B WT-TCR β

<400> 1

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gctggagtta tccagtcacc ccggcacgag gtgacagaga tgggacaaga agtgactctg 120

agatgtaaac caatttcagg acacgactac cttttctggt agagacagac catgatgcgg 180

ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240

gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300

tcagaaccca gggactcagc tgtgtacttc tgtgccagca gttttactag cggggagctac 360

gagcagtact tcgggccggg caccaggctc acggtcacag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc agagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc accgatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggctag 939

<210> 2

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<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B codon optimization - TCR

β

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aacacacaga tgctggagtg attcagagcc ctagacacga ggtgacagag atgggacagg 120

aagtgacact gagatgtaag cccattagcg gacacgacta cctgttctgg tacaggcaga 180

ccatgatgag aggactggaa ctgctgatct acttcaacaa caacgtgccc atcgacgata 240

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gcgagaacga cgagtggacc caggaccggg ccaagcccgt gacccagatc gtgtctgctg 780

aggcctgggg cagagccgat tgcggcttca ccagcgagag ctaccagcag ggcgtgctga 840

gcgccaccat cctgtacgag atcctgctgg gcaaggccac cctgtacgcc gtgctggtgt 900

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<210> 3

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<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B WT-TCR α

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taccagacat ctgggtttta tgggctgtcc tggtaccagc aacatgatgg cggagcaccc 180

acatttcttt cttacaatgc tctggatggt ttggaggaga caggtcgttt ttcttcattc 240

cttagtcgct ctgatagtta tggttacctc cttctacagg agctccagat gaaagactct 300

gcctcttact tctgcgctgt gaatgatgct tccaagataa tctttggatc agggaccaga 360

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gatgtcaagc tggtcgagaa aagctttgaa acagatacga acctaaactt tcaaaacctg 720

tcagtgattg ggttccgaat cctcctcctg aaagtggccg ggtttaatct gctcatgacg 780

ctgcggctgt ggtccagctg a                           801

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<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B codon optimization - TCR

α

<400> 4

atgtggggcg cttttcttct gtatgtgagc atgaagatgg gcggaacagc tggacagtct 60

ctggaacagc ctagcgaggt tacagctgtt gaaggagcta ttgtgcagat caactgcacc 120

taccagacaa gcggcttcta cggcctgagc tggtatcaac agcacgatgg aggagctcct 180

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ctgagcatca ggcccaatat ccagaatcca gatcctgctg tgtaccagct gcggggacagc 420

aagagcagcg acaagagcgt gtgcctgttc accgacttcg acagccagac caacgtgtcc 480

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gacttcaaga gcaacagcgc cgtggcctgg tccaacaaga gcgacttcgc ctgcgccaac 600

gccttcaaca acagcattat ccccgaggac acattcttcc caagccccga gagcagctgc 660

gacgtgaagc tggtggaaaa gagcttcgag acagacacca acctgaactt ccagaacctc 720

agcgtgatcg gcttccggat cctgctgctg aaggtggccg gcttcaacct gctgatgacc 780

ctgcggctgt ggtccagctg a                           801

<210> 5

<211> 1820

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B CO-TCR β-P2A-TCR

α

<400> 5

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aacacacaga tgctggagtg attcagagcc ctagacacga ggtgacagag atgggacagg 120

aagtgacact gagatgtaag cccattagcg gacacgacta cctgttctgg tacaggcaga 180

ccatgatgag aggactggaa ctgctgatct acttcaacaa caacgtgccc atcgacgata 240

gcggcatgcc tgaggacaga tttagcgcca agatgcctaa tgccagcttt tctaccctga 300

agatccagcc ctctgagccc agagattctg ccgtgtactt ttgtgccagc agctttacat 360

ctggctctta tgagcagtac ttcggcccag gcacaaggct gacagtgaca gaggacctga 420

agaacgtgtt ccccccagag gtggccgtgt tcgagcctag cgaggccgag atcagccaca 480

cccagaaagc caccctcgtg tgcctggcca ccggctttta ccccgaccac gtggaactgt 540

cttggtgggt caacggcaaa gaggtgcaca gcggcgtctg caccgacccc cagcccctga 600

aagagcagcc cgccctgaac gacagccggt actgtctgag cagcagactg agagtgtccg 660

ccaccttctg gcagaacccc cggaaccact tcagatgcca ggtgcagttc tacggcctga 720

gcgagaacga cgagtggacc caggaccggg ccaagcccgt gacccagatc gtgtctgctg 780

aggcctgggg cagagccgat tgcggcttca ccagcgagag ctaccagcag ggcgtgctga 840

gcgccaccat cctgtacgag atcctgctgg gcaaggccac cctgtacgcc gtgctggtgt 900

ccgccctggt gctgatggcc atggtcaagc ggaaggacag ccggggcggt tccggagcca 960

cgaacttctc tctgttaaag caagcaggag acgtggaaga aaaccccggt cccatgtggg 1020

gcgcttttct tctgtatgtg agcatgaaga tgggcggaac agctggacag tctctggaac 1080

agcctagcga ggttacagct gttgaaggag ctattgtgca gatcaactgc acctaccaga 1140

caagcggctt ctacggcctg agctggtatc aacagcacga tggaggagct cctacatttc 1200

tgagctataa tgccctggat ggcctggagg agacaggcag atttagcagc ttcctgagca 1260

gatctgactc ttacggatat ctgctgctgc aggagctgca gatgaaggat agcgccagct 1320

acttttgtgc cgtgaatgat gcctctaaga tcatcttcgg cagcggcacc agactgagca 1380

tcaggcccaa tatccagaat ccagatcctg ctgtgtacca gctgcgggac agcaagagca 1440

gcgacaagag cgtgtgcctg ttcaccgact tcgacagcca gaccaacgtg tcccagagca 1500

aggacagcga cgtgtacatc accgataagt gcgtgctgga catgcggagc atggacttca 1560

agagcaacag cgccgtggcc tggtccaaca agagcgactt cgcctgcgcc aacgccttca 1620

acaacagcat tatccccgag gacacattct tcccaagccc cgagagcagc tgcgacgtga 1680

agctggtgga aaagagcttc gagacagaca ccaacctgaa cttccagaac ctcagcgtga 1740

tcggcttccg gatcctgctg ctgaaggtgg ccggcttcaa cctgctgatg accctgcggc 1800

tgtggtccag ctgagtcgac 1820

<210> 6

<211> 312

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B protein TCRβ -

Contains signal peptide

<400> 6

Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Phe Thr Ser Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 7

<211> 266

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B protein TCRα -

Contains signal peptide

<400> 7

Met Trp Gly Ala Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr

1 5 10 15

Ala Gly Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly

20 25 30

Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly

35 40 45

Leu Ser Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser

50 55 60

Tyr Asn Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe

65 70 75 80

Leu Ser Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln

85 90 95

Met Lys Asp Ser Ala Ser Tyr Phe Cys Ala Val Asn Asp Ala Ser Lys

100 105 110

Ile Ile Phe Gly Ser Gly Thr Arg Leu Ser Ile Arg Pro Asn Ile Gln

115 120 125

Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp

130 135 140

Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser

145 150 155 160

Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp

165 170 175

Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn

180 185 190

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro

195 200 205

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu

210 215 220

Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu

225 230 235 240

Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn

245 250 255

Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265

<210> 8

<211> 600

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B protein CO-TCR

β-P2A-TCR α

<400> 8

Met Gly Ser Trp Thr Leu Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asp Tyr Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Phe Thr Ser Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser

305 310 315 320

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Trp

325 330 335

Gly Ala Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr Ala Gly

340 345 350

Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly Ala Ile

355 360 365

Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly Leu Ser

370 375 380

Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser Tyr Asn

385 390 395 400

Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe Leu Ser

405 410 415

Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln Met Lys

420 425 430

Asp Ser Ala Ser Tyr Phe Cys Ala Val Asn Asp Ala Ser Lys Ile Ile

435 440 445

Phe Gly Ser Gly Thr Arg Leu Ser Ile Arg Pro Asn Ile Gln Asn Pro

450 455 460

Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser

465 470 475 480

Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser

485 490 495

Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg

500 505 510

Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser

515 520 525

Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp

530 535 540

Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu

545 550 555 560

Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val

565 570 575

Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

580 585 590

Met Thr Leu Arg Leu Trp Ser Ser

595 600

<210> 9

<211> 939

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B - WT-TCR β

<400> 9

atggactcct ggaccttctg ctgtgtgtcc ctttgcatcc tggtagcgaa gcatacagat 60

gctggagtta tccagtcacc ccgccatgag gtgacagaga tgggacaaga agtgactctg 120

agatgtaaac caatttcagg ccacaactcc cttttctggt agacacagac catgatgcgg 180

ggactggagt tgctcattta ctttaacaac aacgttccga tagatgattc agggatgccc 240

gaggatcgat tctcagctaa gatgcctaat gcatcattct ccactctgaa gatccagccc 300

tcagaaccca gggactcagc tgtgtacttc tgtgccagca gttcgcctga caggctgggc 360

gagcagtact tcgggccggg caccaggctc acggtcacag aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc agagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc accgatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggctag 939

<210> 10

<211> 939

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B-CO-TCRβ

<400> 10

atggatagct ggaccttctg ttgcgtgagc ctgtgtatcc tggtggccaa acacacagat 60

gctggagtga ttcagagccc tagacatgag gtgaccgaaa tgggacagga ggtgacactg 120

agatgtaagc ccatttctgg ccacaacagc ctgttctggt agacacagac catgatgagg 180

ggactggaac tgctgatcta cttcaacaac aacgtgccca tcgacgatag cggcatgcct 240

gaggacagat ttagcgccaa gatgcctaat gccagctttt ctaccctgaa gatccagccc 300

tctgagccca gagattctgc cgtgtacttt tgtgccagct cttctcctga tagactggga 360

gagcagtact ttggccctgg cacaagactg acagtgacag aggacctgaa gaacgtgttc 420

cccccagagg tggccgtgtt cgagcctagc gaggccgaga tcagccacac ccagaaagcc 480

accctcgtgt gcctggccac cggcttttac cccgaccacg tggaactgtc ttggtgggtc 540

aacggcaaag aggtgcacag cggcgtctgc accgaccccc agcccctgaa agagcagccc 600

gccctgaacg acagccggta ctgtctgagc agcagactga gagtgtccgc caccttctgg 660

cagaaccccc ggaaccactt cagatgccag gtgcagttct acggcctgag cgagaacgac 720

gagtggaccc aggacgggc caagcccgtg accgatcg tgtctgctga ggcctggggc 780

agagccgatt gcggcttcac cagcgagagc taccagcagg gcgtgctgag cgccaccatc 840

ctgtacgaga tcctgctggg caaggccacc ctgtacgccg tgctggtgtc cgccctggtg 900

ctgatggcca tggtcaagcg gaaggacagc cggggctga 939

<210> 11

<211> 819

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B - WT-TCR α

<400> 11

atgatgaaat ccttgagagt tttactggtg atcctgtggc ttcagttaag ctgggtttgg 60

agccaacaga aggaggtgga gcaggatcct ggaccactca gtgttccaga gggagccatt 120

gtttctctca actgcactta cagcaacagt gcttttcaat acttcatgtg gtacagacag 180

tattccagaa aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat 240

ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt catcagagac 300

tcacagccca gtgattcagc cacctacctc tgtgcaggag ggggaaacac acctcttgtc 360

tttggaaagg gcacaagact ttctgtgatt gcaaatatcc agaaccctga ccctgccgtg 420

taccagctga gagactctaa atccagtgac aagtctgtct gcctattcac cgattttgat 480

tctcaaacaa atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg 540

ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag caacaaatct 600

gactttgcat gtgcaaacgc cttcaacaac agcattattc cagaagacac cttcttcccc 660

agcccagaaa gttcctgtga tgtcaagctg gtcgagaaaa gctttgaaac agatacgaac 720

ctaaactttc aaaacctgtc agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780

tttaatctgc tcatgacgct gcggctgtgg tccagctga 819

<210> 12

<211> 819

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B-CO-TCR α

<400> 12

atgatgaaga gcctgcgagt cctcctggtg attctgtggt tacagctgtc ttgggtgtgg 60

tctcagcaga aagaagtgga gcaggatcct ggacctctgt ctgtgcctga aggagctatt 120

gtgagcctga attgcaccta cagcaatagc gccttccagt acttcatgtg gtaccggcag 180

tacagcagaa aggggccctga actgctgatg tacacctact ctagcggcaa taaggaagat 240

ggccggttta cagctcaggt ggacaagagc agcaagtaca tcagcctgtt catcagggat 300

tctcagccta gcgattctgc cacctacctg tgtgctggag gcggaaatac acctctggtt 360

tttggaaaag gcaccagact gtctgtgatc gccaacatcc agaaccccga ccctgctgtg 420

taccagctgc gggacagcaa gagcagcgac aagagcgtgt gcctgttcac cgacttcgac 480

agccagacca acgtgtccca gagcaaggac agcgacgtgt acatcaccga taagtgcgtg 540

ctggacatgc ggagcatgga cttcaagagc aacagcgccg tggcctggtc caacaagagc 600

gacttcgcct gcgccaacgc cttcaacaac agcattatcc ccgaggacac attcttccca 660

agccccgaga gcagctgcga cgtgaagctg gtggaaaaga gcttcgagac agacaccaac 720

ctgaacttcc agaacctcag cgtgatcggc ttccggatcc tgctgctgaa ggtggccggc 780

ttcaacctgc tgatgacct gcggctgtgg tccagctga 819

<210> 13

<211> 1838

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthesis of Meso20-16B-CO-TCR β-P2A-

TCR α

<400> 13

ggcgcgccac catggatagc tggaccttct gttgcgtgag cctgtgtatc ctggtggcca 60

aacacacaga tgctggagtg attcagagcc ctagacatga ggtgaccgaa atgggacagg 120

aggtgacact gagatgtaag cccatttctg gccacaacag cctgttctgg tacagacaga 180

ccatgatgag gggactggaa ctgctgatct acttcaacaa caacgtgccc atcgacgata 240

gcggcatgcc tgaggacaga tttagcgcca agatgcctaa tgccagcttt tctaccctga 300

agatccagcc ctctgagccc agagattctg ccgtgtactt ttgtgccagc tcttctcctg 360

atagactggg agagcagtac tttggccctg gcacaagact gacagtgaca gaggacctga 420

agaacgtgtt ccccccagag gtggccgtgt tcgagcctag cgaggccgag atcagccaca 480

cccagaaagc caccctcgtg tgcctggcca ccggctttta ccccgaccac gtggaactgt 540

cttggtgggt caacggcaaa gaggtgcaca gcggcgtctg caccgacccc cagcccctga 600

aagagcagcc cgccctgaac gacagccggt actgtctgag cagcagactg agagtgtccg 660

ccaccttctg gcagaacccc cggaaccact tcagatgcca ggtgcagttc tacggcctga 720

gcgagaacga cgagtggacc caggaccggg ccaagcccgt gacccagatc gtgtctgctg 780

aggcctgggg cagagccgat tgcggcttca ccagcgagag ctaccagcag ggcgtgctga 840

gcgccaccat cctgtacgag atcctgctgg gcaaggccac cctgtacgcc gtgctggtgt 900

ccgccctggt gctgatggcc atggtcaagc ggaaggacag ccggggcggt tccggagcca 960

cgaacttctc tctgttaaag caagcaggag acgtggaaga aaaccccggt cccatgatga 1020

agagcctgcg agtcctcctg gtgattctgt ggttacagct gtcttgggtg tggtctcagc 1080

agaaagaagt ggagcaggat cctggacctc tgtctgtgcc tgaaggagct attgtgagcc 1140

tgaattgcac ctacagcaat agcgccttcc agtacttcat gtggtaccgg cagtacagca 1200

gaaagggccc tgaactgctg atgtacacct actctagcgg caataaggaa gatggccggt 1260

ttacagctca ggtggacaag agcagcaagt acatcagcct gttcatcagg gattctcagc 1320

ctagcgattc tgccacctac ctgtgtgctg gaggcggaaa tacacctctg gtttttggaa 1380

aaggcaccag actgtctgtg atcgccaaca tccagaaccc cgaccctgct gtgtaccagc 1440

tgcggggacag caagagcagc gacaagagcg tgtgcctgtt caccgacttc gacagccaga 1500

ccaacgtgtc ccagagcaag gacagcgacg tgtacatcac cgataagtgc gtgctggaca 1560

tgcggagcat ggacttcaag agcaacagcg ccgtggcctg gtccaacaag agcgacttcg 1620

cctgcgccaa cgccttcaac aacagcatta tccccgagga cacattcttc ccaagccccg 1680

agagcagctg cgacgtgaag ctggtggaaa agagcttcga gacagacacc aacctgaact 1740

tccagaacct cagcgtgatc ggcttccgga tcctgctgct gaaggtggcc ggcttcaacc 1800

tgctgatgac cctgcggctg tggtccagct gagtcgac 1838

<210> 14

<211> 312

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B - Protein TCR β -

Contains signal peptide

<400> 14

Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asn Ser Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Ser Pro Asp Arg Leu Gly Glu Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 15

<211> 272

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B - protein TCR α

- Contains signal peptide

<400> 15

Met Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu

1 5 10 15

Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro

20 25 30

Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser

35 40 45

Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys

50 55 60

Gly Pro Glu Leu Leu Met Tyr Thr Tyr Tyr Ser Ser Gly Asn Lys Glu Asp

65 70 75 80

Gly Arg Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu

85 90 95

Phe Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala

100 105 110

Gly Gly Gly Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser

115 120 125

Val Ile Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg

130 135 140

Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp

145 150 155 160

Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr

165 170 175

Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser

180 185 190

Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe

195 200 205

Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser

210 215 220

Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn

225 230 235 240

Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu

245 250 255

Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

260 265 270

<210> 16

<211> 606

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B - protein TCR

β-P2A-TCR α

<400> 16

Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asn Ser Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Ser Pro Asp Arg Leu Gly Glu Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser

305 310 315 320

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Met

325 330 335

Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser Trp

340 345 350

Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser

355 360 365

Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser

370 375 380

Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro

385 390 395 400

Glu Leu Leu Met Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg

405 410 415

Phe Thr Ala Gln Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile

420 425 430

Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Gly Gly

435 440 445

Gly Asn Thr Pro Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile

450 455 460

Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser

465 470 475 480

Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln

485 490 495

Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys

500 505 510

Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val

515 520 525

Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn

530 535 540

Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys

545 550 555 560

Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn

565 570 575

Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val

580 585 590

Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600 605

<210> 17

<211> 930

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A WT-TCR β

<400> 17

atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tggggagcagt tcccatagac 60

actgaagtta cccagacacc aaaacacctg gtcatgggaa tgacaaataa gaagtctttg 120

aaatgtgaac aacatatggg gcacagggct atgtattggt acaagcagaa agctaagaag 180

ccaccggagc tcatgtttgt ctacagctat gagaaactct ctataaatga aagtgtgcca 240

agtcgcttct cacctgaatg ccccaacagc tctctcttaa accttcacct acacgccctg 300

cagccagaag actcagccct gtatctctgc gccagcagcc acgggtccct gaacactgaa 360

gctttctttg gacaaggcac cagactcaca gttgtagagg acctgaacaa ggtgttccca 420

cccgaggtcg ctgtgtttga gccatcagaa gcagagatct cccacaccca aaaggccaca 480

ctggtgtgcc tggccacagg cttcttcccc gaccacgtgg agctgagctg gtgggtgaat 540

gggaaggagg tgcacagtgg ggtcagcacg gacccgcagc ccctcaagga gcagcccgcc 600

ctcaatgact ccagatactg cctgagcagc cgcctgaggg tctcggccac cttctggcag 660

aacccccgca accacttccg ctgtcaagtc cagttctacg ggctctcgga gaatgacgag 720

tggacccagg atagggccaa acccgtcacc cagatcgtca gcgccgaggc ctggggtaga 780

gcagactgtg gctttacctc ggtgtcctac cagcaagggg tcctgtctgc caccatcctc 840

tatgagatcc tgctagggaa ggccaccctg tatgctgtgc tggtcagcgc ccttgtgttg 900

atggccatgg tcaagagaaa ggatttctga 930

<210> 18

<211> 930

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CO-TCR β

<400> 18

atgggctgta gactgctgtg ttgtgctgtg ctttgtctgc ttggagctgt gcctatcgat 60

acagaggtga cccagacacc taagcatctg gtgatgggca tgaccaacaa gaagagcctg 120

aagtgtgaac agcacatggg ccatagggcc atgtactggt acaagcagaa ggccaagaaa 180

cctcctgagc tgatgttcgt gtacagctac gagaagctga gcatcaacga gagcgtgccc 240

agcagatttt ctcctgagtg ccctaatagc tctctgctga atctgcacct gcatgctctg 300

cagcctgagg attctgctct gtacctgtgt gcttcttctc acggatctct gaatacagag 360

gccttcttcg gccagggcac aagactgaca gtggttgagg atctgaacaa ggtgttcccc 420

ccagaggtgg ccgtgttcga gccttctgag gccgagatct cccacaccca gaaagccacc 480

ctcgtgtgcc tggccaccgg ctttttcccc gaccacgtgg aactgtcttg gtgggtcaac 540

ggcaaagagg tgcactccgg cgtgtgcacc gatccccagc ctctgaaaga acagcccgcc 600

ctgaacgaca gccggtactg cctgagcagc agactgagag tgtccgccac cttctggcag 660

aacccccgga accacttcag atgccaggtg cagttctacg gcctgagcga gaacgacgag 720

tggacccagg acaggccaa gcccgtgaca cagatcgtgt ctgccgaagc ctggggcaga 780

gccgattgcg gctttacctc cgtgtcctat cagcagggcg tgctgagcgc cacaatcctg 840

tacgagatcc tgctgggcaa ggccaccctg tacgccgtgc tggtgtctgc cctggtgctg 900

atggccatgg tcaagcggaa ggacttctga 930

<210> 19

<211> 831

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A WT-TCR α

<400> 19

atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60

agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120

ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180

ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240

agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300

cagactgggg actcagctat ctacttctgt gcagagaccc cggggtatgg tggtgctaca 360

aacaagctca tctttggaac tggcactctg cttgctgtcc agccaaatat ccagaaccct 420

gaccctgccg tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc 480

accgattttg attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca 540

gacaaaactg tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg 600

agcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac 660

accttcttcc ccagcccaga aagttcctgt gatgtcaagc tggtcgagaa aagctttgaa 720

acagatacga acctaaactt tcaaaacctg tcagtgattg ggttccgaat cctcctcctg 780

aaagtggccg ggtttaatct gctcatgacg ctgcggctgt ggtccagctg a 831

<210> 20

<211> 831

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CO-TCR α

<400> 20

atgaagacct ttgccggctt cagcttcctg tttctgtggc tgcagctgga ttgtatgagc 60

agaggcgaag atgtggaaca gagcctgttc ctgagcgtta gagagggcga tagctctgtg 120

atcaattgca cctacaccga tagcagcagc acctacctgt actggtacaa gcaggagcct 180

ggagctggat tacagctgct gacatacatc ttcagcaaca tggacatgaa gcaggaccag 240

aggctgaccg tgctgctgaa caagaaggac aagcacctgt ctctgagaat tgccgataca 300

cagacaggcg atagcgccat ctacttctgt gccgagacac ctggctatgg aggagctacc 360

aataagctga ttttcggcac aggcacactg ttagctgtgc agcccaacat ccagaatccc 420

gatcctgctg tgtaccagct gcgggacagc aagagcagcg acaagagcgt gtgcctgttc 480

accgacttcg acagccagac caacgtgtcc cagagcaagg acagcgacgt gtacatcacc 540

gataagtgcg tgctggacat gcggagcatg gacttcaaga gcaacagcgc cgtggcctgg 600

tccaacaaga gcgacttcgc ctgcgccaac gccttcaaca acagcattat ccccgaggac 660

acattcttcc caagccccga gagcagctgc gacgtgaagc tggtggaaaa gagcttcgag 720

acagacacca acctgaactt ccagaacctc agcgtgatcg gcttccggat cctgctgctg 780

aaggtggccg gcttcaacct gctgatgacc ctgcggctgt ggtccagctg a 831

<210> 21

<211> 1841

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CO-TCR β-P2A-TCR

α

<400> 21

ggcgcgccac catgggctgt agactgctgt gttgtgctgt gctttgtctg cttggagctg 60

tgcctatcga tacagaggtg acccagacac ctaagcatct ggtgatgggc atgaccaaca 120

agaagagcct gaagtgtgaa cagcacatgg gccatagggc catgtactgg tacaagcaga 180

aggccaagaa acctcctgag ctgatgttcg tgtacagcta cgagaagctg agcatcaacg 240

agagcgtgcc cagcagattt tctcctgagt gccctaatag ctctctgctg aatctgcacc 300

tgcatgctct gcagcctgag gattctgctc tgtacctgtg tgcttcttct cacggatctc 360

tgaatacaga ggccttcttc ggccagggca caagactgac agtggttgag gatctgaaca 420

aggtgttccc cccagaggtg gccgtgttcg agccttctga ggccgagatc tcccacaccc 480

agaaagccac cctcgtgtgc ctggccaccg gctttttccc cgaccacgtg gaactgtctt 540

ggtgggtcaa cggcaaagag gtgcactccg gcgtgtgcac cgatccccag cctctgaaag 600

aacagcccgc cctgaacgac agccggtact gcctgagcag cagactgaga gtgtccgcca 660

ccttctggca gaacccccgg aaccacttca gatgccaggt gcagttctac ggcctgagcg 720

agaacgacga gtggacccag gacagagcca agcccgtgac agatcgtg tctgccgaag 780

cctggggcag agccgattgc ggctttacct ccgtgtccta tcagcagggc gtgctgagcg 840

ccacaatcct gtacgagatc ctgctgggca aggccaccct gtacgccgtg ctggtgtctg 900

ccctggtgct gatggccatg gtcaagcgga aggacttcgg ttccggagcc acgaacttct 960

ctctgttaaa gcaagcagga gacgtggaag aaaaccccgg tcccatgaag acctttgccg 1020

gcttcagctt cctgtttctg tggctgcagc tggattgtat gagcagaggc gaagatgtgg 1080

aacagagcct gttcctgagc gttagagagg gcgatagctc tgtgatcaat tgcacctaca 1140

ccgatagcag cagcacctac ctgtactggt acaagcagga gcctggagct ggattacagc 1200

tgctgacata catcttcagc aacatggaca tgaagcagga ccagaggctg accgtgctgc 1260

tgaacaagaa ggacaagcac ctgtctctga gaattgccga tacacagaca ggcgatagcg 1320

ccatctactt ctgtgccgag acacctggct atggaggagc taccaataag ctgattttcg 1380

gcacaggcac actgttagct gtgcagccca acatccagaa tcccgatcct gctgtgtacc 1440

agctgcggga cagcaagagc agcgacaaga gcgtgtgcct gttcaccgac ttcgacagcc 1500

agaccaacgt gtcccagc aaggacagcg acgtgtacat caccgataag tgcgtgctgg 1560

acatgcggag catggacttc aagagcaaca gcgccgtggc ctggtccaac aagagcgact 1620

tcgcctgcgc caacgccttc aacaacagca ttatccccga ggacacattc ttcccaagcc 1680

ccgagagcag ctgcgacgtg aagctggtgg aaaagagctt cgagacagac accaacctga 1740

acttccagaa cctcagcgtg atcggcttcc ggatcctgct gctgaaggtg gccggcttca 1800

acctgctgat gaccctgcgg ctgtggtcca gctgagtcga c 1841

<210> 22

<211> 309

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A Protein CO-TCR β

- Contains signal peptide

<400> 22

Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala

1 5 10 15

Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met

20 25 30

Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His

35 40 45

Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu

50 55 60

Met Phe Val Tyr Ser Tyr Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro

65 70 75 80

Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His

85 90 95

Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser His Gly Ser Leu Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg

115 120 125

Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala

130 135 140

Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro

180 185 190

Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu

195 200 205

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn

210 215 220

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

225 230 235 240

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

245 250 255

Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln

260 265 270

Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala

275 280 285

Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val

290 295 300

Lys Arg Lys Asp Phe

305

<210> 23

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A protein CO-TCR

α-Signal peptide

<400> 23

Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu

1 5 10 15

Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser

20 25 30

Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser

35 40 45

Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu

50 55 60

Gln Leu Leu Thr Tyr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln

65 70 75 80

Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg

85 90 95

Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu

100 105 110

Thr Pro Gly Tyr Gly Gly Ala Thr Asn Lys Leu Ile Phe Gly Thr Gly

115 120 125

Thr Leu Leu Ala Val Gln Pro Asn Ile Gln Asn Pro Asp Pro Ala Val

130 135 140

Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe

145 150 155 160

Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp

165 170 175

Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe

180 185 190

Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys

195 200 205

Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro

210 215 220

Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu

225 230 235 240

Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg

245 250 255

Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg

260 265 270

Leu Trp Ser Ser

275

<210> 24

<211> 607

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A protein

Vβ-P2A-Vα

<400> 24

Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala

1 5 10 15

Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met

20 25 30

Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His

35 40 45

Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu

50 55 60

Met Phe Val Tyr Ser Tyr Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro

65 70 75 80

Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His

85 90 95

Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser His Gly Ser Leu Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg

115 120 125

Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala

130 135 140

Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr

145 150 155 160

Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser

165 170 175

Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro

180 185 190

Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu

195 200 205

Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn

210 215 220

His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu

225 230 235 240

Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu

245 250 255

Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln

260 265 270

Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala

275 280 285

Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val

290 295 300

Lys Arg Lys Asp Phe Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys

305 310 315 320

Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Lys Thr Phe Ala

325 330 335

Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu Asp Cys Met Ser Arg

340 345 350

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

355 360 365

Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

370 375 380

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

385 390 395 400

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu

405 410 415

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln

420 425 430

Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Thr Pro Gly Tyr Gly

435 440 445

Gly Ala Thr Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Leu Ala Val

450 455 460

Gln Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp

465 470 475 480

Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser

485 490 495

Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp

500 505 510

Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala

515 520 525

Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn

530 535 540

Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

545 550 555 560

Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu

565 570 575

Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys

580 585 590

Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

595 600 605

<210> 25

<211> 939

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CO-TCR β

<400> 25

atgggacctg gactgttatg ttgggttctg ctgtgtctgc ttggagctgg atctgtggaa 60

acaggcgtta cacagtcccc tacacacctg atcaagacaa gaggacagca ggtgaccctg 120

agatgttcta gccagtctgg ccacaataca gtgagctggt atcagcaggc tttaggacag 180

ggaccccagt tcatcttcca gtactaccgg gaggaagaga atggcagagg caattttcca 240

cccagattta gcggcctgca gttccccaat tacagcagcg agctgaacgt gaatgccctt 300

gaactggacg attctgctct gtacctgtgt gccagctctt ttgctggcgg aagaagcgat 360

acccagtatt ttggacctgg aaccagactg acagtgctgg aggacctgaa gaacgtgttc 420

cccccagagg tggccgtgtt cgagcctagc gaggccgaga tcagccacac ccagaaagcc 480

accctcgtgt gcctggccac cggcttttac cccgaccacg tggaactgtc ttggtgggtc 540

aacggcaaag aggtgcacag cggcgtctgc accgaccccc agcccctgaa agagcagccc 600

gccctgaacg acagccggta ctgtctgagc agcagactga gagtgtccgc caccttctgg 660

cagaaccccc ggaaccactt cagatgccag gtgcagttct acggcctgag cgagaacgac 720

gagtggaccc aggacgggc caagcccgtg accgatcg tgtctgctga ggcctggggc 780

agagccgatt gcggcttcac cagcgagagc taccagcagg gcgtgctgag cgccaccatc 840

ctgtacgaga tcctgctggg caaggccacc ctgtacgccg tgctggtgtc cgccctggtg 900

ctgatggcca tggtcaagcg gaaggacagc cggggctga 939

<210> 26

<211> 831

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CO-TCR α

<400> 26

atggagacac tgctgggact actgattctg tggctgcaac tgcaatgggt gagcagcaaa 60

caggaggtta cccagattcc tgctgctctg tctgttcctg aaggcgagaa tctggtgctg 120

aactgcagct tcacagatag cgccatctac aacctgcagt ggttcagaca ggatcctgga 180

aaaggcctga caagcctgct gctgattcag agctctcaga gagagcagac atctggaaga 240

ctgaatgcta gcctggacaa gtctagcggc agaagcaccc tgtatattgc cgcctctcaa 300

cctggagatt ctgccacata cctgtgtgct gttaggctgc tgtttgccca aggaggaagc 360

gagaaactgg tgtttggaaa gggcacaaag ctgaccgtga atccctacat ccagaaccct 420

gatcctgccg tgtaccagct gcgggacagc aagagcagcg acaagagcgt gtgcctgttc 480

accgacttcg acagccagac caacgtgtcc cagagcaagg acagcgacgt gtacatcacc 540

gataagtgcg tgctggacat gcggagcatg gacttcaaga gcaacagcgc cgtggcctgg 600

tccaacaaga gcgacttcgc ctgcgccaac gccttcaaca acagcattat ccccgaggac 660

acattcttcc caagccccga gagcagctgc gacgtgaagc tggtggaaaa gagcttcgag 720

acagacacca acctgaactt ccagaacctc agcgtgatcg gcttccggat cctgctgctg 780

aaggtggccg gcttcaacct gctgatgacc ctgcggctgt ggtccagctg a 831

<210> 27

<211> 1843

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B-TCR β-P2A-TCR

α

<400> 27

caccatggga cctggactgt tatgttgggt tctgctgtgt ctgcttggag ctggatctgt 60

ggaaacaggc gttacacagt cccctacaca cctgatcaag acaagaggac agcaggtgac 120

cctgagatgt tctagccagt ctggccacaa tacagtgagc tggtatcagc aggctttagg 180

acagggaccc cagttcatct tccagtacta ccgggaggaa gagaatggca gagcaattt 240

tccacccaga tttagcggcc tgcagttccc caattacagc agcgagctga acgtgaatgc 300

ccttgaactg gacgattctg ctctgtacct gtgtgccagc tcttttgctg gcggaagaag 360

cgatacccag tattttggac ctggaaccag actgacagtg ctggaggacc tgaagaacgt 420

gttcccccca gagtggccg tgttcgagcc tagcgaggcc gagatcagcc acaccgaa 480

agccaccctc gtgtgcctgg ccaccggctt ttaccccgac cacgtggaac tgtcttggtg 540

ggtcaacggc aaagaggtgc acagcggcgt ctgcaccgac ccccagcccc tgaaagagca 600

gcccgccctg aacgacagcc ggtactgtct gagcagcaga ctgagagtgt ccgccacctt 660

ctggcagaac ccccggaacc acttcagatg ccaggtgcag ttctacggcc tgagcgagaa 720

cgacgagtgg acccaggacc gggccaagcc cgtgacccag atcgtgtctg ctgaggcctg 780

gggcagagcc gattgcggct tcaccagcga gagctaccag cagggcgtgc tgagcgccac 840

catcctgtac gagatcctgc tgggcaaggc caccctgtac gccgtgctgg tgtccgccct 900

ggtgctgatg gccatggtca agcggaagga cagccggggc ggttccggag ccacgaactt 960

ctctctgtta aagcaagcag gagacgtgga agaaaacccc ggtcccatgg agacactgct 1020

gggactactg attctgtggc tgcaactgca atgggtgagc agcaaacagg aggttaccca 1080

gattcctgct gctctgtctg ttcctgaagg cgagaatctg gtgctgaact gcagcttcac 1140

agatagcgcc atctacaacc tgcagtggtt cagacaggat cctggaaaag gcctgacaag 1200

cctgctgctg attcagagct ctcagagaga gcagacatct ggaagactga atgctagcct 1260

ggacaagtct agcggcagaa gcaccctgta tattgccgcc tctcaacctg gagattctgc 1320

cacatacctg tgtgctgtta ggctgctgtt tgcccaagga ggaagcgaga aactggtgtt 1380

tggaaagggc acaaagctga ccgtgaatcc ctacatccag aaccctgatc ctgccgtgta 1440

ccagctgcgg gacagcaaga gcagcgacaa gagcgtgtgc ctgttcaccg acttcgacag 1500

ccagaccaac gtgtcccaga gcaaggacag cgacgtgtac atcaccgata agtgcgtgct 1560

ggacatgcgg agcatggact tcaagagcaa cagcgccgtg gcctggtcca acaagagcga 1620

cttcgcctgc gccaacgcct tcaacaacag cattatcccc gaggacacat tcttcccaag 1680

ccccgagagc agctgcgacg tgaagctggt ggaaaagagc ttcgagacag acccaacct 1740

gaacttccag aacctcagcg tgatcggctt ccggatcctg ctgctgaagg tggccggctt 1800

caacctgctg atgaccctgc ggctgtggtc cagctgagtc gac 1843

<210> 28

<211> 312

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B Protein CO-TCR β

- Contains signal peptide

<400> 28

Met Gly Pro Gly Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Ser Val Glu Thr Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys

20 25 30

Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Ser Gln Ser Gly His

35 40 45

Asn Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe

50 55 60

Ile Phe Gln Tyr Tyr Arg Glu Glu Asn Gly Arg Gly Asn Phe Pro

65 70 75 80

Pro Arg Phe Ser Gly Leu Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn

85 90 95

Val Asn Ala Leu Glu Leu Asp Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Phe Ala Gly Gly Arg Ser Asp Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly

305 310

<210> 29

<211> 276

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B protein CO-TCR

α-Signal peptide

<400> 29

Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp

1 5 10 15

Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val

20 25 30

Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala

35 40 45

Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr

50 55 60

Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg

65 70 75 80

Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile

85 90 95

Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg

100 105 110

Leu Leu Phe Ala Gln Gly Gly Ser Glu Lys Leu Val Phe Gly Lys Gly

115 120 125

Thr Lys Leu Thr Val Asn Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val

130 135 140

Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe

145 150 155 160

Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp

165 170 175

Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe

180 185 190

Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys

195 200 205

Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro

210 215 220

Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu

225 230 235 240

Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg

245 250 255

Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg

260 265 270

Leu Trp Ser Ser

275

<210> 30

<211> 610

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B protein CO-TCR

β-P2A-TCR α

<400> 30

Met Gly Pro Gly Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala

1 5 10 15

Gly Ser Val Glu Thr Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys

20 25 30

Thr Arg Gly Gln Gln Val Thr Leu Arg Cys Ser Ser Gln Ser Gly His

35 40 45

Asn Thr Val Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe

50 55 60

Ile Phe Gln Tyr Tyr Arg Glu Glu Asn Gly Arg Gly Asn Phe Pro

65 70 75 80

Pro Arg Phe Ser Gly Leu Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn

85 90 95

Val Asn Ala Leu Glu Leu Asp Asp Ser Ala Leu Tyr Leu Cys Ala Ser

100 105 110

Ser Phe Ala Gly Gly Arg Ser Asp Thr Gln Tyr Phe Gly Pro Gly Thr

115 120 125

Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val

130 135 140

Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala

145 150 155 160

Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu

165 170 175

Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp

180 185 190

Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys

195 200 205

Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg

210 215 220

Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp

225 230 235 240

Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala

245 250 255

Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln

260 265 270

Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys

275 280 285

Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met

290 295 300

Val Lys Arg Lys Asp Ser Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser

305 310 315 320

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu

325 330 335

Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp Val Ser

340 345 350

Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu

355 360 365

Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr

370 375 380

Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu

385 390 395 400

Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn

405 410 415

Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala

420 425 430

Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Leu Leu

435 440 445

Phe Ala Gln Gly Gly Ser Glu Lys Leu Val Phe Gly Lys Gly Thr Lys

450 455 460

Leu Thr Val Asn Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln

465 470 475 480

Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp

485 490 495

Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr

500 505 510

Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser

515 520 525

Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn

530 535 540

Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro

545 550 555 560

Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp

565 570 575

Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu

580 585 590

Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp

595 600 605

Ser Ser

610

<210> 31

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28

<400> 31

Ser Leu Leu Phe Leu Leu Phe Ser Leu

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln530-538

<400> 32

Val Leu Pro Leu Thr Val Ala Glu Val

1 5

<210> 33

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B CDR3 α

<400> 33

Cys Ala Val Asn Asp Ala Ser Lys Ile Ile Phe

1 5 10

<210> 34

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B CDR3 β

<400> 34

Cys Ala Ser Ser Phe Thr Ser Gly Ser Tyr Glu Gln Tyr Phe

1 5 10

<210> 35

<211> 11

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B CDR3 α

<400> 35

Cys Ala Gly Gly Gly Asn Thr Pro Leu Val Phe

1 5 10

<210> 36

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B CDR3 β

<400> 36

Cys Ala Ser Ser Ser Pro Asp Arg Leu Gly Glu Gln Tyr Phe

1 5 10

<210> 37

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CDR3 α

<400> 37

Cys Ala Glu Thr Pro Gly Tyr Gly Gly Ala Thr Asn Lys Leu Ile Phe

1 5 10 15

<210> 38

<211> 14

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CDR3 β

<400> 38

Cys Ala Ser Ser His Gly Ser Leu Asn Thr Glu Ala Phe Phe

1 5 10

<210> 39

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CDR3 α

<400> 39

Cys Ala Val Arg Leu Leu Phe Ala Gln Gly Gly Ser Glu Lys Leu Val

1 5 10 15

Phe

<210> 40

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CDR3 β

<400> 40

Cys Ala Ser Ser Phe Ala Gly Gly Arg Ser Asp Thr Gln Tyr Phe

1 5 10 15

<210> 41

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

P2A-1, a self-cleaving peptide

<400> 41

ggaagtggag ctacgaattt ttctttatta aaacaagcag gagatgttga ggagaatccc 60

ggtcca 66

<210> 42

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

Self-cleaving peptide P2A-2

<400> 42

agcggcgcca ccaacttcag cctgctgaaa caggccggcg acgtggaaga gaaccctggc 60

ct

<210> 43

<211> 65

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

Self-cleaving peptide P2A-3

<400> 43

gaagcggcgc cacaaatttc agcctgctga agcaggccgg cgacgtggaa gagaaccctg 60

gccct                                                                                                                                  

<210> 44

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

Self-cleaving peptide P2A-4

<400> 44

ggctccggcg ccaccaactt ttcactgctg aaacaggctg gggatgtgga agaaaatccc 60

ggccca 66

<210> 45

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

P2A-5 is a self-cleaving peptide.

<400> 45

ggcagcggcg ccaccaactt tagcctgctg aaacaggctg gcgacgtgga agagaacccc 60

ggACCT

<210> 46

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

P2A-6 is a self-cleaving peptide.

<400> 46

ggctctggcg ccaccaactt tagcctgctg aaacaggctg gcgacgtgga agagaacccc 60

ggACCT

<210> 47

<211> 63

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence: *Melilothorax micranthum* virus 2A T2A

<400> 47

ggaagcggag aggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctgga 60

ct

<210> 48

<211> 69

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Equine rhinitis A virus E2A

<400> 48

ggaagcggac agtgtactaa ttatgctctc ttgaaattgg ctggagatgt tgagagcaac 60

cctggacct                                                                                                                    

<210> 49

<211> 75

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Foot-and-mouth disease virus 2

F2A

<400> 49

ggaagcggag tgaaacagac tttgaatttt gaccttctca agttggcggg agacgtggag 60

tccaaccctg gacct                                                                                                                    

<210> 50

<211> 630

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Human MSLN, allotype 1

<400> 50

Met Ala Leu Pro Thr Ala Arg Pro Leu Leu Gly Ser Cys Gly Thr Pro

1 5 10 15

Ala Leu Gly Ser Leu Leu Phe Leu Leu Phe Ser Leu Gly Trp Val Gln

20 25 30

Pro Ser Arg Thr Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu

35 40 45

Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg

50 55 60

Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu

65 70 75 80

Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu

85 90 95

Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro

100 105 110

Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro

115 120 125

Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile

130 135 140

Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln

145 150 155 160

Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu

165 170 175

Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu

180 185 190

Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu

195 200 205

Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg

210 215 220

Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro Pro Ser Thr Trp

225 230 235 240

Ser Val Ser Thr Met Asp Ala Leu Arg Gly Leu Leu Pro Val Leu Gly

245 250 255

Gln Pro Ile Ile Arg Ser Ile Pro Gln Gly Ile Val Ala Ala Trp Arg

260 265 270

Gln Arg Ser Ser Arg Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile

275 280 285

Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser

290 295 300

Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys

305 310 315 320

Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met

325 330 335

Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu

340 345 350

Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val

355 360 365

Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile

370 375 380

Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu

385 390 395 400

Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu

405 410 415

Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln

420 425 430

Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr

435 440 445

Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser

450 455 460

Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln

465 470 475 480

Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn

485 490 495

Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro

500 505 510

Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu

515 520 525

Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val

530 535 540

Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala

545 550 555 560

Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln

565 570 575

Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn

580 585 590

Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr

595 600 605

Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu

610 615 620

Leu Ala Ser Thr Leu Ala

625 630

<210> 51

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 1

<400> 51

Ala Leu Leu Phe Leu Leu Phe Ser Leu

1 5

<210> 52

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 2

<400> 52

Ser Ala Leu Phe Leu Leu Phe Ser Leu

1 5

<210> 53

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 3

<400> 53

Ser Leu Ala Phe Leu Leu Phe Ser Leu

1 5

<210> 54

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 4

<400> 54

Ser Leu Leu Ala Leu Leu Phe Ser Leu

1 5

<210> 55

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 5

<400> 55

Ser Leu Leu Phe Ala Leu Phe Ser Leu

1 5

<210> 56

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 6

<400> 56

Ser Leu Leu Phe Leu Ala Phe Ser Leu

1 5

<210> 57

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 7

<400> 57

Ser Leu Leu Phe Leu Leu Ala Ser Leu

1 5

<210> 58

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 8

<400> 58

Ser Leu Leu Phe Leu Leu Phe Ala Leu

1 5

<210> 59

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 Alanine Variant 9

<400> 59

Ser Leu Leu Phe Leu Leu Phe Ser Ala

1 5

<210> 60

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln20-28 TCR epitopes coexist

Sequence

<220>

<221> Variant

<222> 1,2,7,8,9

<223> Xaa = any amino acid, optionally alanine

<400> 60

Xaa Xaa Leu Phe Leu Leu Xaa Xaa Xaa

1 5

<210> 61

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln530-538 TCR epitope

There is a total sequence of 1

<220>

<221> Variant

<222> 3,5,6,9

<223> Xaa = any amino acid, optionally alanine

<400> 61

Val Leu Xaa Leu Xaa Xaa Ala Glu Xaa

1 5

<210> 62

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Msln530-538 TCR epitope

Total sequence 2

<220>

<221> Variant

<222> 1,5,9

<223> Xaa = any amino acid, optionally alanine

<400> 62

Xaa Leu Pro Leu Xaa Val Ala Glu Xaa

1 5

<210> 63

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence EHF peptide

<400> 63

Val Leu Leu Leu Ser Leu Ala Glu Ile

1 5

<210> 64

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence YN010 peptide

<400> 64

Val Leu Ala Leu Trp Glu Ala Glu Val

1 5

<210> 65

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence MLEC peptide

<400> 65

Val Leu Val Leu Lys Phe Ala Glu Val

1 5

<210> 66

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence CHPF2 peptide

<400> 66

Val Leu Pro Leu Leu Val Ala Glu Ala

1 5

<210> 67

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence ULK1 peptide

<400> 67

Val Leu Tyr Leu Lys Val Ala Glu Leu

1 5

<210> 68

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence OR2J2 peptide

<400> 68

Val Leu Ala Leu Gly Ile Ala Glu Cys

1 5

<210> 69

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence DNHD1 peptide

<400> 69

Val Leu Glu Leu Leu Leu Ala Glu Leu

1 5

<210> 70

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence AL1L1 peptide

<400> 70

Val Leu Glu Leu Thr Glu Ala Glu Leu

1 5

<210> 71

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence GALP peptide

<400> 71

Val Leu Leu Leu Ser Leu Ala Glu Thr

1 5

<210> 72

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence OR2J1 peptide

<400> 72

Val Leu Ala Leu Gly Thr Ala Glu Cys

1 5

<210> 73

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence ADPRH peptide

<400> 73

Val Met His Leu Ala Thr Ala Glu Ala

1 5

<210> 74

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence CHPF2 peptide

<400> 74

Val Leu Pro Leu Ile Val Ala Glu Ala

1 5

<210> 75

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence LRCH3 peptide

<400> 75

Asp Leu Pro Leu Arg Val Ala Glu Ile

1 5

<210> 76

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence IN80B peptide

<400> 76

Met Leu Pro Leu Pro Val Ala Glu Gly

1 5

<210> 77

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence CEAM3 peptide

<400> 77

Ser Met Pro Leu Ser Val Ala Glu Gly

1 5

<210> 78

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B CDR1 β

<400> 78

Ser Gly His Asp Tyr

1 5

<210> 79

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequences Meso20-3B, Meso20-16B CDR2 β

<400> 79

Phe Asn Asn Asn Val Pro

1 5

<210> 80

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B CDR1 α

<400> 80

Thr Ser Gly Phe Tyr Gly

1 5

<210> 81

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B CDR2 α v1

<400> 81

Asn Ala Leu Asp Gly

1 5

<210> 82

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B CDR1 β

<400> 82

Ser Gly His Asn Ser

1 5

<210> 83

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B / Meso20-3B /

Meso530-11A / Meso530-11B CDR1 β consistent

<220>

<221> Variant

<222> 1

<223> Xaa = M, S, or T

<220>

<221> Variant

<222> 4

<223> Xaa = D, E, N, Q, R or K

<220>

<221> Variant

<222> 5

<223> Xaa = Y, S, A, V, I, L, or T

<400> 83

Xaa Gly His Xaa Xaa

1 5

<210> 84

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B / Meso20-3B /

Meso530 CDR2 β Consistent

<220>

<221> Variant

<222> 4

<223> Xaa = D or N

<220>

<221> Variant

<222> 5

<223> Xaa = Y, S, or T

<400> 84

Ser Gly His Xaa Xaa

1 5

<210> 85

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B CDR1 α

<400> 85

Asn Ser Ala Phe Gln Tyr

1 5

<210> 86

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B CDR2 α v1

<400> 86

Thr Tyr Ser Ser Gly

1 5

<210> 87

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CDR1 β

<400> 87

Met Gly His Arg Ala

1 5

<210> 88

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CDR2 β

<400> 88

Tyr Ser Tyr Glu Lys Leu

1 5

<210> 89

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CDR1 α

<400> 89

Ser Ser Ser Ser Thr Tyr Leu

1 5

<210> 90

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A CDR2 α

<400> 90

Ile Phe Ser Asn Met Asp Met

1 5

<210> 91

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CDR1 β

<400> 91

Ser Gly His Asn Thr

1 5

<210> 92

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CDR2 β

<400> 92

Tyr Tyr Arg Glu Glu Glu

1 5

<210> 93

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CDR1 α

<400> 93

Asp Ser Ala Ile Tyr Asn

1 5

<210> 94

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B CDR2 α

<400> 94

Ile Gln Ser Ser Gln Arg Glu

1 5

<210> 95

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B V β (mature)

<400> 95

Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Phe

85 90 95

Thr Ser Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr

<210> 96

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B V α (mature)

<400> 96

Gly Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly Ala

1 5 10 15

Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly Leu

20 25 30

Ser Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser Tyr

35 40 45

Asn Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe Leu

50 55 60

Ser Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln Met

65 70 75 80

Lys Asp Ser Ala Ser Tyr Phe Cys Ala Val Asn Asp Ala Ser Lys Ile

85 90 95

Ile Phe Gly Ser Gly Thr Arg Leu Ser Ile Arg Pro

100 105

<210> 97

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B V β (mature)

<400> 97

Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asn Ser Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Ser

85 90 95

Pro Asp Arg Leu Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr

<210> 98

<211> 109

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B V α (mature)

<400> 98

Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser Val Pro Glu Gly

1 5 10 15

Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr

20 25 30

Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met

35 40 45

Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Gly Gly Gly Asn Thr Pro

85 90 95

Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala

100 105

<210> 99

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A V β (mature)

<400> 99

Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met Gly Met Thr

1 5 10 15

Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His Arg Ala Met

20 25 30

Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu Met Phe Val

35 40 45

Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro Ser Arg Phe

50 55 60

Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His Leu His Ala

65 70 75 80

Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser His Gly

85 90 95

Ser Leu Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val

100 105 110

Val

<210> 100

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A V α (mature)

<400> 100

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

1 5 10 15

Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

20 25 30

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

35 40 45

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu

50 55 60

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln

65 70 75 80

Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Thr Pro Gly Tyr Gly

85 90 95

Gly Ala Thr Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Leu Ala Val

100 105 110

Gln Pro

<210> 101

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B V β (mature)

<400> 101

Glu Thr Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys Thr Arg Gly

1 5 10 15

Gln Gln Val Thr Leu Arg Cys Ser Ser Gln Ser Gly His Asn Thr Val

20 25 30

Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe Ile Phe Gln

35 40 45

Tyr Tyr Arg Glu Glu Asn Gly Arg Gly Asn Phe Pro Pro Arg Phe

50 55 60

Ser Gly Leu Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn Val Asn Ala

65 70 75 80

Leu Glu Leu Asp Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Phe Ala

85 90 95

Gly Gly Arg Ser Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Leu

<210> 102

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B V α (mature)

<400> 102

Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly

1 5 10 15

Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn

20 25 30

Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu

35 40 45

Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala

50 55 60

Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser

65 70 75 80

Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Leu Leu Phe

85 90 95

Ala Gln Gly Gly Ser Glu Lys Leu Val Phe Gly Lys Gly Thr Lys Leu

100 105 110

Thr Val Asn Pro

115

<210> 103

<211> 293

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B TCR β (mature)

<400> 103

Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asp Tyr Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Phe

85 90 95

Thr Ser Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

115 120 125

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

130 135 140

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

145 150 155 160

Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro

165 170 175

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser

180 185 190

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe

195 200 205

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

210 215 220

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

225 230 235 240

Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val

245 250 255

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

260 265 270

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

275 280 285

Lys Asp Ser Arg Gly

290

<210> 104

<211> 249

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-3B TCR α (mature) -

Cysteine module

<400> 104

Gly Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly Ala

1 5 10 15

Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly Leu

20 25 30

Ser Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser Tyr

35 40 45

Asn Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe Leu

50 55 60

Ser Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln Met

65 70 75 80

Lys Asp Ser Ala Ser Tyr Phe Cys Ala Val Asn Asp Ala Ser Lys Ile

85 90 95

Ile Phe Gly Ser Gly Thr Arg Leu Ser Ile Arg Pro Asn Ile Gln Asn

100 105 110

Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys

115 120 125

Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln

130 135 140

Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met

145 150 155 160

Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys

165 170 175

Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu

180 185 190

Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val

195 200 205

Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser

210 215 220

Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu

225 230 235 240

Leu Met Thr Leu Arg Leu Trp Ser Ser

245

<210> 105

<211> 293

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B TCR β (mature) -

Cysteine module

<400> 105

Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr Glu Met Gly

1 5 10 15

Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His Asn Ser Leu

20 25 30

Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr

35 40 45

Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg

50 55 60

Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln

65 70 75 80

Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala Ser Ser Ser

85 90 95

Pro Asp Arg Leu Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

115 120 125

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

130 135 140

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

145 150 155 160

Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro

165 170 175

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser

180 185 190

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe

195 200 205

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

210 215 220

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

225 230 235 240

Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val

245 250 255

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

260 265 270

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

275 280 285

Lys Asp Ser Arg Gly

290

<210> 106

<211> 250

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso20-16B TCR α (mature) -

Cysteine module

<400> 106

Gln Lys Glu Val Glu Gln Asp Pro Gly Pro Leu Ser Val Pro Glu Gly

1 5 10 15

Ala Ile Val Ser Leu Asn Cys Thr Tyr Ser Asn Ser Ala Phe Gln Tyr

20 25 30

Phe Met Trp Tyr Arg Gln Tyr Ser Arg Lys Gly Pro Glu Leu Leu Met

35 40 45

Tyr Thr Tyr Ser Ser Gly Asn Lys Glu Asp Gly Arg Phe Thr Ala Gln

50 55 60

Val Asp Lys Ser Ser Lys Tyr Ile Ser Leu Phe Ile Arg Asp Ser Gln

65 70 75 80

Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Gly Gly Gly Asn Thr Pro

85 90 95

Leu Val Phe Gly Lys Gly Thr Arg Leu Ser Val Ile Ala Asn Ile Gln

100 105 110

Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp

115 120 125

Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser

130 135 140

Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp

145 150 155 160

Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn

165 170 175

Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro

180 185 190

Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu

195 200 205

Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu

210 215 220

Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn

225 230 235 240

Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250

<210> 107

<211> 290

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A TCR β (mature) -

Cysteine module

<400> 107

Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met Gly Met Thr

1 5 10 15

Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His Arg Ala Met

20 25 30

Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu Met Phe Val

35 40 45

Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro Ser Arg Phe

50 55 60

Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His Leu His Ala

65 70 75 80

Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser His Gly

85 90 95

Ser Leu Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val

100 105 110

Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu

115 120 125

Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys

130 135 140

Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val

145 150 155 160

Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu

165 170 175

Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg

180 185 190

Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg

195 200 205

Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln

210 215 220

Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly

225 230 235 240

Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu

245 250 255

Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr

260 265 270

Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys

275 280 285

Asp Phe

290

<210> 108

<211> 255

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11A TCR α (mature)

- Cysteine module

<400> 108

Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser Val Arg Glu Gly Asp

1 5 10 15

Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser Ser Ser Thr Tyr Leu

20 25 30

Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu Gln Leu Leu Thr Tyr

35 40 45

Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln Arg Leu Thr Val Leu

50 55 60

Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg Ile Ala Asp Thr Gln

65 70 75 80

Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Glu Thr Pro Gly Tyr Gly

85 90 95

Gly Ala Thr Asn Lys Leu Ile Phe Gly Thr Gly Thr Leu Leu Ala Val

100 105 110

Gln Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp

115 120 125

Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser

130 135 140

Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp

145 150 155 160

Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala

165 170 175

Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn

180 185 190

Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser

195 200 205

Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu

210 215 220

Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys

225 230 235 240

Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

245 250 255

<210> 109

<211> 293

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B TCR β (mature) -

Cysteine module

<400> 109

Glu Thr Gly Val Thr Gln Ser Pro Thr His Leu Ile Lys Thr Arg Gly

1 5 10 15

Gln Gln Val Thr Leu Arg Cys Ser Ser Gln Ser Gly His Asn Thr Val

20 25 30

Ser Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Gln Phe Ile Phe Gln

35 40 45

Tyr Tyr Arg Glu Glu Asn Gly Arg Gly Asn Phe Pro Pro Arg Phe

50 55 60

Ser Gly Leu Gln Phe Pro Asn Tyr Ser Ser Glu Leu Asn Val Asn Ala

65 70 75 80

Leu Glu Leu Asp Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Phe Ala

85 90 95

Gly Gly Arg Ser Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr

100 105 110

Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

115 120 125

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

130 135 140

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

145 150 155 160

Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro

165 170 175

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser

180 185 190

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe

195 200 205

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

210 215 220

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

225 230 235 240

Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val

245 250 255

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

260 265 270

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

275 280 285

Lys Asp Ser Arg Gly

290

<210> 110

<211> 257

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B TCR α (mature)

- Cysteine module

<400> 110

Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly

1 5 10 15

Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn

20 25 30

Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu

35 40 45

Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala

50 55 60

Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser

65 70 75 80

Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Arg Leu Leu Phe

85 90 95

Ala Gln Gly Gly Ser Glu Lys Leu Val Phe Gly Lys Gly Thr Lys Leu

100 105 110

Thr Val Asn Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu

115 120 125

Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe

130 135 140

Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile

145 150 155 160

Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn

165 170 175

Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala

180 185 190

Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu

195 200 205

Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr

210 215 220

Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu

225 230 235 240

Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser

245 250 255

Ser

<210> 111

<211> 831

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B WT TCR α

<400> 111

atggagaccc tcttgggcct gcttatcctt tggctgcagc tgcaatgggt gagcagcaaa 60

caggaggtga cgcagattcc tgcagctctg agtgtcccag aaggagaaaa cttggttctc 120

aactgcagtt tcactgatag cgctatttac aacctccagt ggtttaggca ggaccctggg 180

aaaggtctca catctctgtt gcttattcag tcaagtcaga gagagcaaac aagtggaaga 240

cttaatgcct cgctggataa atcatcagga cgtagtactt tatacattgc agcttctcag 300

cctggtgact cagccaccta cctctgtgct gtgaggctat tattcgctca gggcggatct 360

gaaaagctgg tctttggaaa gggaacgaaa ctgacagtaa acccatatat ccagaaccct 420

gaccctgccg tgtaccagct gagagactct aaatccagtg acaagtctgt ctgcctattc 480

accgattttg attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca 540

gacaaaactg tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg 600

agcaacaaat ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac 660

accttcttcc ccagcccaga aagttcctgt gatgtcaagc tggtcgagaa aagctttgaa 720

acagatacga acctaaactt tcaaaacctg tcagtgattg ggttccgaat cctcctcctg 780

aaagtggccg ggtttaatct gctcatgacg ctgcggctgt ggtccagctg a 831

<210> 112

<211> 939

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence Meso530-11B WT TCR β

<400> 112

atgggccctg ggctcctctg ctgggtgctg ctttgtctcc tggggagcagg ctcagtggag 60

actggagtca cccaaagtcc cacacacctg atcaaaacga gaggacagca agtgactctg 120

agatgctctt ctcagtctgg gcacaacact gtgtcctggt accaacaggc cctgggtcag 180

gggccccagt ttatctttca gtattatagg gaggaagaga atggcagagg aaacttccct 240

cctagattct caggtctcca gttccctaat tatagctctg agctgaatgt gaacgccttg 300

gagctggacg actcggccct gtatctctgt gccagcagct tcgcgggggg gcgatcagat 360

acgcagtatt ttggcccagg cacccggctg acagtgctcg aggacctgaa aaacgtgttc 420

ccacccgagg tcgctgtgtt tgagccatca gaagcagaga tctcccacac ccaaaaggcc 480

acactggtgt gcctggccac aggcttctac cccgaccacg tggagctgag ctggtgggtg 540

aatgggaagg aggtgcacag tggggtcagc agagacccgc agcccctcaa ggagcagccc 600

gccctcaatg actccagata ctgcctgagc agccgcctga gggtctcggc caccttctgg 660

cagaaccccc gcaaccactt ccgctgtcaa gtccagttct acgggctctc ggagaatgac 720

gagtggaccc aggatagggc caaacctgtc accgatcg tcagcgccga ggcctggggt 780

agagcagact gtggcttcac ctccgagtct taccagcaag gggtcctgtc tgccaccatc 840

ctctatgaga tcttgctagg gaaggccacc ttgtatgccg tgctggtcag tgccctcgtg 900

ctgatggcca tggtcaagag aaaggattcc agaggctag 939

<210> 113

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

P2A-1, a self-cleaving peptide

<400> 113

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 114

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence: Porcine swine virus

Self-cleaving peptide P2A-2

<400> 114

Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 115

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence: *Melilothorax micranthum* virus 2A T2A

<400> 115

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 116

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Equine rhinitis A virus E2A

<400> 116

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 117

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence Foot-and-mouth disease virus 2

F2A

<400> 117

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 118

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence - Meso20-3B CDR2 α v2

<400> 118

Asn Ala Leu Asp Gly Leu

1 5

<210> 119

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence - Meso20-16B CDR2 α v2

<400> 119

Thr Tyr Ser Ser Gly Asn

1 5

<210> 120

<211> 1833

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic sequence - Meso530-11B

CO-β-P2A-α: (Substitution Optimization)

<400> 120

atgggacctg gattgctttg ttgggtgctg ctgtgtctgc tcggagccgg atctgtggaa 60

acaggcgtga cacagagccc cacacacctg atcaagacca gaggccagca agtgaccctg 120

agatgcagct ctcagagcgg ccacaatacc gtgtcctggt atcagcaggc cctcggacag 180

ggccctcagt tcatcttcca gtactacaga gaggaagaga acggcagagg caacttccca 240

cctagattca gcggcctgca gttccccaac tacagcagcg agctgaacgt gaacgccctg 300

gaactggatg acagcgccct gtacctgtgc gccagttctt ttgccggcgg aagaagcgac 360

acccagtact ttggacctgg caccagactg accgtgctgg aagatctgaa gaacgtgttc 420

ccacctgagg tggccgtgtt cgagccttct gaggccgaga tcagccacac agaaagcc 480

acactcgtgt gtctggccac cggcttctat cccgatcacg tggaactgtc ttggtgggtc 540

aacggcaaag aggtgcacag cggcgtctgt accgatcctc agcctctgaa agagcagccc 600

gctctgaacg acagcagata ctgcctgagc agcagactga gagtgtccgc caccttctgg 660

cagaacccca gaaaccactt cagatgccag gtgcagttct acggcctgag cgagaacgat 720

gagtggaccc aggattagc caagcctgtg actcagatcg tgtctgccga agcctggggc 780

agagccgatt gtggctttac cagcgagtct taccagcagg gcgtgctgtc tgccaccatc 840

ctgtatgaga tcctgctggg caaagccact ctgtacgccg tgctggtttc tgccctggtg 900

ctgatggcca tggtcaagcg gaaggatagc agaggcggaa gcggcgccac aaacttctca 960

ctgctgaaac aggccggcga cgtggaagag aatcccggac ctatggaaac actgctggga 1020

ctgctgatcc tgtggctgca gcttcagtgg gtgtccagca agcaagaagt gacccagatt 1080

cctgccgcac tgtctgtgcc tgagggcgag aatctggtcc tgaactgctc cttcaccgac 1140

agcgccatct acaacctgca gtggttcaga caggaccccg gcaagggact gacaagcctg 1200

ctgctgattc agagcagcca gagagagcag accagcggca gactgaatgc cagcctggat 1260

aagtcctccg gcagaagcac cctgtatatc gccgcttctc agcctggcga tagcgccaca 1320

tatctgtgtg ccgtgcggct gctgtttgcc caaggcggat ctgagaagct ggtgttcggc 1380

aagggcacca agctgacagt gaacccctac attcagaacc ccgatcctgc cgtgtaccag 1440

ctgagagaca gcaagagcag cgacaagagc gtgtgcctgt tcaccgactt cgacagccag 1500

accaacgtgt cccagagcaa ggacagcgac gtgtacatca ccgataagtg cgtgctggac 1560

atgcggagca tggacttcaa gagcaacagc gccgtggcct ggtccaacaa gagcgatttc 1620

gcctgcgcca acgccttcaa caacagcatt atccccgagg acacattctt cccaagtcct 1680

gagtccagct gcgacgtgaa actggtggaa aagagcttcg agacagacac caacctgaac 1740

ttccagaacc tgagcgtgat cggcttccgg atcctgctcc tgaaagtggc cggcttcaac 1800

ctgctgatga ccctgcgact gtggtccagc taa           1833

<210> 121

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence - Wide range of nuclease sequences

<400> 121

Leu Ala Gly Leu Ile Asp Ala Asp Gly

1 5

<210> 122

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence - Wide range of nuclease sequences

<400> 122

Gly Ile Tyr Tyr Ile Gly

1 5

<210> 123

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic sequence - Wide range of nuclease sequences

<220>

<221> Variant

<222> 3

<223> Xaa = D or E

<220>

<221> Variant

<222> 4

<223> Xaa = any amino acid

<400> 123

Pro Asp Xaa Xaa Lys

1 5

Claims (61)

1. A binding protein comprising a T cell receptor (TCR) α chain variable domain ( _Vα ) and a TCR β chain variable domain ( _Vβ ), wherein: (a) _Vα contains complementarity-determining regions (CDRs) 1α, CDR2α, and CDR3α, respectively, of the amino acid sequences shown in SEQ ID NO: 93, 94, and 39; and (b) _Vβ comprises CDR1β, CDR2β, and CDR3β, respectively, consisting of the amino acid sequences shown in SEQ ID NO:91, 92, and 40. The binding protein can specifically bind to the Msln peptide:HLA-A*02:01 complex shown in SEQ ID NO:32. 2. The binding protein of claim 1, comprising an amino acid sequence having at least 85% identity with the amino acid sequence encoded by: (a)TRBJ2-3*01; (b)TRAV21*01 or TRAV21*02; (c)TRBV5-4*01; (d)TRAJ57*01; and/or (e)TRBD1*01 or TRBD2*02. 3. The binding protein of claim 1, wherein the _Vα comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:102, and wherein the _Vβ comprises an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO:101. 4. The binding protein of claim 3, wherein _Vα comprises or consists of the amino acid sequence shown in SEQ ID NO:102, and wherein _Vβ comprises or consists of the amino acid sequence shown in SEQ ID NO:101. 5. The binding protein according to claim 1, comprising a TCRα chain (TCRα) and a TCRβ chain (TCRβ), wherein the TCRα comprises or is composed of an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 110 or 29, and/or wherein the TCRβ comprises or is composed of an amino acid sequence having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 109 or 28. 6. The binding protein according to claim 5, wherein TCRβ and TCRα respectively comprise amino acid sequences having at least 90% identity with SEQ ID NO:109 or 28 and SEQ ID NO:110 or 29. 7. The binding protein according to claim 5, wherein TCRβ and TCRα respectively comprise amino acid sequences having at least 95% identity with SEQ ID NO:109 or 28 and SEQ ID NO:110 or 29. 8. The binding protein of claim 7, wherein TCRα comprises or consists of the amino acid sequence shown in SEQ ID NO: 110 or 29, and wherein TCRβ comprises or consists of the amino acid sequence shown in SEQ ID NO: 109 or 28. 9. The binding protein of claim 8, wherein TCRα comprises or is composed of the amino acid sequence shown in SEQ ID NO:29, and wherein TCRβ comprises or is composed of the amino acid sequence shown in SEQ ID NO:28. 10. The binding protein of claim 8, wherein TCRα comprises or is composed of the amino acid sequence shown in SEQ ID NO: 110, and wherein TCRβ comprises or is composed of the amino acid sequence shown in SEQ ID NO: 109. 11. The binding protein of claim 10, wherein TCRα consists of the amino acid sequence shown in SEQ ID NO: 110, and wherein TCRβ consists of the amino acid sequence shown in SEQ ID NO: 109. 12. The binding protein of claim 1, wherein mutagenesis of any one or more alanine residues 1, 5, or 9 of the Msln peptide shown in SEQ ID NO:32 does not eliminate or weaken the binding of the binding protein to the Msln peptide:HLA complex. 13. The binding protein of claim 1, wherein the binding protein is capable of binding a peptide:HLA complex, wherein the peptide comprises or is composed of the common amino acid sequence shown in SEQ ID NO:62. 14. The binding protein according to any one of claims 1-13, wherein the binding protein does not bind or does not specifically bind the peptide:HLA complex, wherein the peptide comprises or consists of any one or more of the amino acid sequences shown in SEQ ID NO:63-77, and wherein the HLA is optionally HLA-A:02*01. 15. The binding protein according to any one of claims 1-13, wherein the binding protein is or comprises: a TCR, an antigen-binding fragment of a TCR, a scTCR, or a CAR, wherein the TCR is optionally soluble. 16. The binding protein according to any one of claims 1-13, wherein the binding protein is human, humanized, or chimeric. 17. The binding protein according to any one of claims 1-13, wherein the binding protein is capable of binding to the mesothelin:HLA complex in the absence of or independently of CD8. 18. The binding protein according to any one of claims 1-13, wherein the binding protein has 9 μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 0.9 μM, 0.8 μM, 0.7 μM, 0.6 μM, 0.5 μM, 0.4 μM, 0.3 μM, 0.2 μM or less of the Msln peptide EC50. 19. A composition comprising a binding protein according to any one of claims 1-13 and a pharmaceutically acceptable carrier or excipient. 20. A polynucleotide encoding a binding protein according to any one of claims 1-13. 21. The polynucleotide of claim 20, wherein the polynucleotide is a codon optimized for expression in a host cell, wherein the host cell is optionally a human immune system cell. 22. The polynucleotide of claim 20, comprising a polynucleotide having at least 50% identity with the polynucleotide sequences shown in SEQ ID NO: 25 and 26. 23. The polynucleotide of claim 22, comprising a TCRα chain-encoded polynucleotide and a TCRβ chain-encoded polynucleotide having at least 85% identity with the polynucleotide sequences shown in SEQ ID NO: 25 and 26, respectively. 24. The polynucleotide of claim 22, comprising a polynucleotide encoding a self-cleaving peptide located between a TCRβ chain-encoded polynucleotide and a TCRα chain-encoded polynucleotide. 25. The polynucleotide of claim 24, wherein the polynucleotide encodes the amino acid sequence shown in SEQ ID NO:30. 26. The polynucleotide of claim 25, wherein the polynucleotide encoding the binding protein has at least 50% identity with the polynucleotide sequence shown in either SEQ ID NO: 27 or 120. 27. The polynucleotide of claim 20, wherein the encoded binding protein comprises TCRα and TCRβ, said TCRα comprising or consisting of the amino acid sequence shown in SEQ ID NO:110, and said TCRβ comprising or consisting of the amino acid sequence shown in SEQ ID NO:109. 28. The polynucleotide of claim 27, wherein the polynucleotide comprises or consists of the polynucleotide sequence shown in SEQ ID NO:120. 29. An expression vector comprising a polynucleotide according to claim 27 operably linked to an expression control sequence. 30. An expression vector comprising a polynucleotide of claim 20 operably linked to an expression control sequence. 31. The expression vector of claim 30, wherein the expression vector is capable of delivering the polynucleotide to a host cell. 32. The expression vector according to claim 31, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell. 33. The expression vector according to claim 32, wherein the immune system cells are CD4+ T cells, CD8+ T cells, CD4-CD8- double-negative T cells, γδ T cells, natural killer cells, natural killer T cells, macrophages, dendritic cells, or any combination thereof. 34. The expression vector according to claim 33, wherein the T cell is an immature T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof. 35. The expression vector according to claim 29 or 30, wherein the expression vector is a viral vector. 36. The expression vector according to claim 35, wherein the viral vector is a lentiviral vector or a γ-retroviral vector. 37. A recombinant host cell comprising the polynucleotide of claim 27, wherein the polynucleotide is heterologous to the recombinant host cell, and the recombinant host cell is capable of expressing a binding protein encoded by the polynucleotide on its cell surface. 38. A recombinant host cell comprising the polynucleotide of claim 20, wherein the polynucleotide is heterologous to the recombinant host cell, and the recombinant host cell is capable of expressing a binding protein encoded by the polynucleotide on its cell surface. 39. The recombinant host cell according to claim 38, wherein the recombinant host cell is a hematopoietic progenitor cell or a human immune system cell. 40. The recombinant host cell of claim 39, wherein the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double-negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a dendritic cell, or any combination thereof. 41. The recombinant host cell of claim 39, wherein the immune system cell is a T cell. 42. The recombinant host cell of claim 41, wherein the T cell is an naive T cell, a central memory T cell, a stem cell memory T cell, an effector memory T cell, or any combination thereof. 43. The recombinant host cell of claim 38, wherein the host cell is a T cell or NK-T cell encoding an endogenous TCR, and wherein the binding protein is able to bind to the CD3 protein more effectively than the endogenous TCR. 44. The recombinant host cell of claim 38, wherein Nur77 expression in the host cell is increased when the host cell contains a Msln peptide bound by the binding protein at a concentration of ^10⁻² μM peptide, ^10⁻¹ μM peptide, 1 μM peptide, or ^10¹ μM peptide, wherein the peptide is optionally presented to the host cell by an antigen-presenting cell. 45. The recombinant host cell of claim 38, wherein the recombinant host cell does not produce IFN-γ and/or does not exhibit activating and/or cytotoxic activity when contacted with cells expressing the following: (i)HLA-C6:02:01; (ii) HLA-B13:01:01 without HLA-B13:02:01; (iii) HLA-A3; (iv)HLA-A29; (v)HLA-B40; (vi)HLA-B44; (vii)HLA-C3; (viii)HLA-C16; (ix)HLA-A1; (x)HLA-24; (xi)HLA-B7; (xii)HLA-B57; (xiii)HLA-C7; (xiv)HLA-A11; (xv)HLA-B15; (xvi)HLA-C4; (xvii)HLA-C12; (xviii)HLA-B8; (xix)HLA-B49; (xx)HLA-B51; (xxi)HLA-C15; (xxii)HLA-A30; (xxiii)HLA-A68; (xxiv)HLA-C2; (xxv)HLA-A32; (xxvi)HLA-A33; (xxvii)HLA-B55; (xxviii)HLA-C1; (xxvix)HLA-C5; (xxix)HLA-B8; (xxx)HLA-B35; or Any combination of (xxxi)(i)-(xxx), The premise is that there is no mesothelin peptide bound by a binding protein encoded by a polynucleotide. 46. The recombinant host cell of claim 39, wherein the host cell is a T cell or NK-T cell encoding an endogenous TCR, wherein the binding protein encoded by the heterologous polynucleotide has higher cell surface expression compared to the endogenous TCR. 47. A cell composition comprising the recombinant host cell of claim 38 and a pharmaceutically acceptable carrier or excipient. 48. A cell composition comprising the recombinant host cell of claim 37 and a pharmaceutically acceptable carrier or excipient. 49. Use of the binding protein according to any one of claims 1-13, the polynucleotide according to claim 28, the expression vector according to claim 29, the recombinant host cell according to claim 37, or the cell composition according to claim 47 or 48 in the preparation of a pharmaceutical composition for treating cancer in a subject, wherein the Msln _530-538 peptide is expressed on cancer tumor cells. 50. The use according to claim 49, wherein the subject expresses HLA-A*02:01. 51. The use according to claim 50, wherein the subject does not express HLA-B13:02:01. 52. Use of the polynucleotide of claim 27 in the preparation of a pharmaceutical composition for treating cancer in a subject, wherein the Msln _530-538 peptide is expressed on cancer tumor cells. 53. Use of the recombinant host cell of claim 38 in the preparation of a pharmaceutical composition for treating cancer in a subject, wherein the Msln _530-538 peptide is expressed on cancer tumor cells. 54. The use according to claim 53, wherein the cancer is a solid tumor or a hematologic malignancy. 55. The use according to claim 53, wherein the cancer is selected from: bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, cervical cancer, colorectal cancer, desmoidoma, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic carcinoma, primary thyroid cancer, prostate cancer, kidney cancer, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, and uterine cancer. 56. The use according to claim 53, wherein the cancer is selected from: pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, mesothelioma, lung cancer, colon cancer, and renal cell carcinoma. 57. The use according to claim 53, wherein the recombinant host cells are administered parenterally or intravenously. 58. The use according to claim 57, wherein the subject is receiving or has received cytokines. 59. The use according to claim 58, wherein the cytokine comprises IL-2, IL-15, IL-21, or any combination thereof. 60. The use according to claim 58, wherein the subject further receives or has received any of the following treatments: immune checkpoint inhibitors, agonists of stimulating immune checkpoint agents, radiotherapy, antibodies, antibody-drug conjugates, Fc fusion proteins, antisense nucleotide therapy, gene therapy, vaccines, surgery, chemotherapy, or any combination thereof. 61. The use according to claim 53, wherein the cancer is colorectal adenocarcinoma.