阅读说明：本技术 针对癌症特异性抗原对t淋巴细胞的筛选 (Screening of T lymphocytes against cancer specific antigens ) 是由 Y·纳卡穆拉 J·H·帕克 S·吉村幸子 引地哲郎 于 2018-10-05 设计创作，主要内容包括：本文提供了鉴定识别TCR的癌症特异性抗原的方法和具有抗原特异性细胞毒活性的TCR工程化T细胞。本文提供了通过本文所述的方法产生的工程化T淋巴细胞。本文提供了治疗受试者的癌症的方法,所述方法包括施用本文所述的工程化T淋巴细胞。本文提供了通过本文所述的方法产生的抗体或其片段。本文提供了治疗受试者的癌症的方法,所述方法包括向受试者施用本文所述的抗体。在一些实施方案中,提供了本文中的治疗性组合物(例如工程化淋巴细胞、抗体等)和方法作为试剂盒或系统的一部分。(Provided herein are methods of identifying cancer specific antigens that recognize TCRs and TCR-engineered T cells having antigen-specific cytotoxic activity. Provided herein are engineered T lymphocytes produced by the methods described herein. Provided herein are methods of treating cancer in a subject, comprising administering an engineered T lymphocyte described herein. Provided herein are antibodies or fragments thereof produced by the methods described herein. Provided herein are methods of treating cancer in a subject, comprising administering to the subject an antibody described herein. In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, etc.) and methods herein are provided as part of a kit or system.)

1. A method, the method comprising:

(a) stimulating target lymphocytes with a stimulatory peptide comprising a candidate antigen sequence;

(b) capturing immunocompetent lymphocytes with a T Cell Receptor (TCR) that binds to the candidate peptide, wherein the capturing comprises contacting the immunocompetent lymphocytes with a capture reagent that displays a Major Histocompatibility Complex (MHC) bound to a capture peptide comprising the candidate antigen sequence; and

(c) sequencing all or a portion of the TCR of the captured immunocompetent lymphocytes.

2. The method of claim 1, wherein the target lymphocyte is obtained from a healthy donor.

3. The method of claim 1, wherein the target lymphocyte is CD8⁺Cytotoxic lymphocytes.

4. The method of claim 1, wherein the stimulation is performed in vitro.

5. The method of claim 1, wherein the capture reagent is an MHC multimer.

6. The method of claim 5, wherein the MHC multimer is an MHC dextrorotatory multimer.

7. The method of claim 1, wherein the sequencing comprises a next generation sequencing technique.

8. The method of claim 1, wherein the portion of the TCR that is sequenced comprises TCR-a and/or TCR- β chains.

9. The method of claim 8, wherein the portion of the TCR that is sequenced comprises one or more Complementarity Determining Regions (CDRs) of the TCR-a and/or TCR- β chains.

10. The method of claim 9, wherein the portion of the TCR that is sequenced comprises the CDR3 of the TCR-a and/or TCR- β chains.

11. The method of claim 1, wherein the target lymphocyte is a population of target lymphocytes, wherein the stimulatory peptide is one peptide in a population of stimulatory peptides comprising different candidate antigen sequences; and is

Wherein the capturing comprises contacting the population of immunocompetent lymphocytes with a capture reagent that displays a Major Histocompatibility Complex (MHC) bound to a population of capture peptides comprising the candidate antigen sequence.

12. A cancer specific antigen that recognizes a TCR identified by the method of one of claims 1-11.

13. A therapeutic antibody that binds to the cancer specific antigen that recognizes a TCR of claim 12.

14. The therapeutic antibody of claim 13, wherein the therapeutic antibody is an antibody fragment.

15. The method of one of claims 1-11, the method further comprising:

(d) generating an engineered lymphocyte that displays all or a portion of the TCR of the captured immunocompetent lymphocyte, wherein the engineered lymphocyte recognizes an antigen presenting cell that displays MHC bound to the peptide comprising the candidate antigen sequence.

16. The method of claim 15, wherein the engineered lymphocyte is CD8⁺Cytotoxic lymphocytes.

17. The method of claim 15, wherein generating engineered lymphocytes displaying all or a portion of the TCR of the captured immunocompetent lymphocytes further comprises:

(i) cloning a nucleic acid sequence encoding the portion of the TCR of the captured immunocompetent lymphocyte into a vector;

(ii) introducing the vector into a host lymphocyte; and

(iii) culturing the host lymphocyte under conditions such that the portion of the TCR of the captured immunocompetent lymphocyte is expressed and displayed on the engineered lymphocyte.

18. The method of claim 17, wherein the portion of the TCR comprises the TCR-a and/or TCR- β chains.

19. The method of claim 18, wherein the portion of the TCR comprises one or more Complementarity Determining Regions (CDRs) of the TCR-a and/or TCR- β chains.

20. The method of claim 19, wherein the portion of the TCR that is sequenced comprises the CDR3 of the TCR-a and/or TCR- β chains.

21. The method of claim 20, wherein the portion of the TCR sequenced comprises an amino acid sequence selected from the group consisting of SEQ id nos 45-132.

22. The method of claim 21, wherein the engineered lymphocyte exhibits a TCR comprising α and β chains comprising a pair of amino acid sequences selected from the group consisting of seq id nos: 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130, and 131 and 132 of SEQ ID NOs.

23. The method of claim 17, wherein the vector is introduced into a host lymphocyte from a healthy donor host.

24. The method of claim 17, wherein the vector is introduced into host lymphocytes from a cancer patient to be treated with the engineered lymphocytes.

25. An engineered lymphocyte produced by the method of one of claims 15-24.

26. The engineered lymphocyte of claim 25, wherein said engineered lymphocyte is CD8⁺Cytotoxic lymphocytes.

27. A method of treating cancer in a subject, the method comprising administering to a subject the engineered CD8 of claim 25⁺A lymphocyte.

28. The method of one of claims 1-11, the method further comprising:

(d) generating a therapeutic antibody comprising all or a portion of the sequence of the TCR of the captured immunocompetent lymphocyte.

29. The method of claim 28, wherein the portion of the TCR comprises the TCR-a and/or TCR- β chains.

30. The method of claim 29, wherein the portion of the TCR comprises one or more Complementarity Determining Regions (CDRs) of the TCR-a and/or TCR- β chains.

31. The method of claim 30, wherein the portion of the TCR that is sequenced comprises the CDR3 of the TCR-a and/or TCR- β chains.

32. The method of claim 31, wherein the portion of the TCR that is sequenced comprises an amino acid sequence selected from the group consisting of SEQ id nos 45-132.

33. The method of claim 32, wherein the therapeutic antibody comprises a CDR3 comprising an amino acid sequence pair selected from the group consisting of seq id nos: 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130, and 131 and 132 of SEQ ID NOs.

34. The method of claim 28, wherein the antibody is an antibody fragment.

35. An antibody produced by the method of one of claims 28-34.

36. A method of treating cancer in a subject, the method comprising administering to the subject the antibody of claim 35.

Technical Field

Provided herein are methods of identifying cancer-specific antigens that recognize T cell receptors and T cell receptor engineered T cells having antigen-specific cytotoxic activity.

Background

Cancer immunotherapy treats cancer by boosting the patient's own anti-tumor immune response. In particular, the success of immune checkpoint inhibitors highlights the importance of anti-cancer immune activity in cancer patients. However, only a few patients exhibit clinical benefit from anti-immune checkpoint therapy, while 70-80% of cancer patients do not derive benefit or benefit minimally from such therapy. Therefore, it is important and urgent to identify the resistance mechanism of immunotherapy and develop a method to further enhance and improve immune response (reference 1; incorporated by reference in its entirety). Although Cytotoxic T Lymphocytes (CTLs) play a critical role in cancer immunotherapy, the identification of the T Cell Receptor (TCR) of CTLs and their target cancer-specific antigens is difficult and time consuming.

Disclosure of Invention

Provided herein are methods of identifying cancer-specific antigens that recognize TCRs and TCR-engineered T cells having antigen-specific cytotoxic activity.

In some embodiments, provided herein are methods comprising: (a) stimulation of target lymphocytes with stimulatory peptides comprising candidate antigen sequences (e.g., CD 8)⁺Cytotoxic T lymphocytes); (b) capture of immunocompetent lymphocytes (e.g., CD 8) with T Cell Receptors (TCR) bound to candidate peptides⁺Cytotoxic T lymphocytes), wherein the capturing comprises contacting the immunocompetent T lymphocytes with a capture reagent that displays a Major Histocompatibility Complex (MHC) bound to a capture peptide comprising a candidate antigen sequence; and (c) sequencing all or a portion of the TCR of the captured immunocompetent T lymphocytes.

In some embodiments, the target lymphocytes are obtained from a healthy donor. In some embodiments, the target lymphocyte is CD8⁺Cytotoxic T lymphocytes. In some embodiments, the stimulation is performed in vitro (e.g., in cell culture).

In some embodiments, the peptide comprising the candidate antigen sequence is all or a fragment of an oncogenic antigen and a neoantigen. In some embodiments, the candidate antigen sequences are all or a fragment of oncogenic antigens and neoantigens.

In some embodiments, the capture reagent is an MHC multimer. In some embodiments, the MHC multimer is an MHC dextro-multimer (dextramer).

In some embodiments, sequencing comprises next generation sequencing techniques. In some embodiments, the portion of the TCR that is sequenced comprises TCR-a and/or TCR- β chains. In some embodiments, the portion of the TCR that is sequenced comprises one or more Complementarity Determining Regions (CDRs) of the TCR-a and/or TCR- β chains. In some embodiments, the portion of the TCR that is sequenced comprises CDR3 of the TCR-a and/or TCR- β chains.

In some embodiments, the target lymphocyte (e.g., CD 8)⁺Cytotoxic T lymphocytes) are a population of target lymphocytes, wherein the stimulatory peptide is one peptide in a population of stimulatory peptides comprising different candidate antigen sequences; and wherein said capturing comprises contacting immunocompetent T lymphocytes (e.g., CD 8)⁺Cytotoxic T lymphocytes) The population is contacted with a capture reagent that displays a Major Histocompatibility Complex (MHC) bound to a population of capture peptides comprising the candidate antigen sequence.

In some embodiments, provided herein are cancer specific antigens that recognize TCRs identified by the methods described herein (e.g., SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, etc. SEQ ID NO.

In some embodiments, provided herein are methods comprising: (a) stimulation of target lymphocytes with stimulatory peptides comprising candidate antigen sequences (e.g., CD 8)⁺Cytotoxic T lymphocytes); (b) capturing immunocompetent T lymphocytes (e.g., CD 8) with T Cell Receptors (TCR) that bind to candidate peptides⁺Cytotoxic T lymphocytes), wherein the capturing comprises contacting the immunocompetent T lymphocytes with a capture reagent that displays a Major Histocompatibility Complex (MHC) bound to a capture peptide comprising a candidate antigen sequence; (c) sequencing all or a portion of the TCR of the captured immunocompetent T lymphocytes; and further comprising: (d) engineered T lymphocytes (e.g., CD 8) that produce all or a portion of a TCR displaying captured immunocompetent T lymphocytes⁺Cytotoxic T lymphocytes), wherein the engineered T lymphocytes recognize antigen presenting cells displaying MHC bound to a peptide comprising a candidate antigen sequence.

In some embodiments, the engineered T lymphocyte is CD8⁺Cytotoxic T lymphocytes.

In some embodiments, the productEngineered T lymphocytes that display all or a portion of the TCR of captured immunocompetent T lymphocytes (e.g., CD 8)⁺Cytotoxic T lymphocytes) include: (i) cloning a nucleic acid sequence encoding a portion of the TCR of the captured immunocompetent T lymphocyte into a vector; (ii) introduction of vectors into host T lymphocytes (e.g., CD 8)⁺Cytotoxic T lymphocytes), and (iii) culturing under conditions that allow expression and display of portions of the TCR of captured immunocompetent T lymphocytes on engineered T lymphocytes, in some embodiments, portions of the TCR comprise TCR- α and/or TCR- β chains in some embodiments, portions of the TCR comprise one or more Complementarity Determining Regions (CDRs) of TCR- α and/or TCR- β chains in some embodiments, portions of the TCR that are sequenced comprise TCR- α and/or cdrd 3 of TCR-5 chains in some embodiments, portions of the TCR that are sequenced comprise amino acid sequences selected from the group consisting of SEQ ID NOs: 45-132 in some embodiments, engineered T lymphocytes display TCR: 45 and 46 chains (e.g., CDR3) comprising pairs of amino acid sequences selected from the group consisting of SEQ ID NOs: 45 and 46, 47 and 48, 49 and 50, 51 and 52, and 56, and 75, and 70, 99 and 70, 80 and 70, and 70.

In some embodiments, provided herein are engineered T lymphocytes (e.g., CD 8) produced by the methods described herein⁺Cytotoxic T lymphocytes). In some embodiments, the engineered T lymphocyte is CD8⁺Cytotoxic T lymphocytes.

In some embodiments, provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject an engineered T lymphocyte (e.g., CD 8) described herein⁺Cytotoxic T lymphocytes).

In some embodiments, provided herein are methods comprising: (a) stimulation of target lymphocytes with stimulatory peptides comprising candidate antigen sequences (e.g., CD 8)⁺Cytotoxic T lymphocytes); (b) capturing immunocompetent T lymphocytes (e.g., CD 8) with T Cell Receptors (TCR) that bind to candidate peptides⁺Cytotoxic T lymphocytes), wherein the capturing comprises contacting the immunocompetent T lymphocytes with a capturing reagent that displays Major Histocompatibility Complex (MHC) bound to a capture peptide comprising a candidate antigen sequence, (c) sequencing all or a portion of the TCR of the captured immunocompetent T lymphocytes, and further comprising (d) generating a therapeutic antibody comprising all or a portion of the sequence of the TCR of the captured immunocompetent T lymphocytes, in some embodiments, a portion of the TCR comprises TCR- α and/or TCR- β chains, in some embodiments, a portion of the TCR comprises one or more Complementarity Determining Regions (CDRs) of the TCR- α and/or TCR- β chains, in some embodiments, the portion of the TCR sequenced comprises TCR- α and/or TCR- β chains, CDRs 3, in some embodiments, the portion of the TCR sequenced comprising amino acid sequences selected from the group consisting of SEQ ID NO:45-132, amino acid sequences, 75, 70, 75, 70, 75, 119, 75, 99, 119, and 70, 119, 99 and 70, 75, 99 and 70, 75 and 70, 76 and 70, 75, 26 and 70, 75 and 70, 76 and 70, 26 and 70, 76 and 70, 26 and 70, 76 and 70, 76 and β, 76 and.

In some embodiments, provided herein are antibodies produced by the methods described herein. In some embodiments, the antibody is an antibody fragment.

In some embodiments, provided herein are methods of treating cancer in a subject, comprising administering to the subject an antibody described herein.

Embodiments herein are described as utilizing CD8⁺Cytotoxic lymphocytes as target cells and/or for the production of engineered CD8⁺Cytotoxic lymphocytes. However, in other embodiments within the scope herein, the target cells and/or engineered lymphocytes described herein may instead comprise CD4⁺Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells as target cells.

Drawings

Figure 1A-C. induction of FOXM1 and UBE 2T-derived peptide-specific CTLs and cytotoxic activity of established CTLs: (a) FOXM1 and UBE 2T-specific CTLs were confirmed to produce IFN- γ only when exposed to C1R-a24 cells stimulated with FOXM1 or UBE 2T-specific peptides, R/S ratio indicating responsive Cell (CTL)/stimulated cell (C1R-a24 cells) ratio (B, C) FOXM1(B) and UBE 2T-specific CTLs (C) exerted significant cell killing effect on HLA-a 24: 02-positive SW480 cells, but not on HLA-a 24: 02-negative HCC1143 cells or BT549 cells: (2 × 10) two CTLs (2 × 10: 10)⁵One cell/well) with cancer cells (2 × 10)⁴Individual cells/well) were incubated together for 5 hours.

Fig. 2A-b. production of TCR-engineered T cells against FOXM1 and UBE 2T. (A) The distribution of TCRA and TCRB CDR3 clonotypes of FOXM1 and UBE2T specific CTLs is presented in a pie chart with CDR3 sequences. Black indicates the CDR3 clonotype portion with a reading frequency below 1%. This population contained only one clonotype that was dominant for TCRA and TCRB. (B) Transduction efficiency was examined by staining for CD8 and TCRv β 8(FOXM1TCR engineered T cells) or TCRv β 13(UBE2T TCR engineered T cells). Flow cytometry shows FOXM1 or UBE2T-TCR engineered T cells.

Figures 3A-h. cytotoxic activity of TCR-engineered T cells against FOXM1 and UBE 2T. (A, B) TCR-engineered T cells against FOXM1(A) and UBE2T (B) against HLA-A24: 02 positiveTime course of cancer cell viability in co-culture with FOXM1(C) and UBE2T TCR-engineered T cells (D) two sorted TCR-engineered T cells (4 × 10)⁵One cell/well) with cancer cells (2 × 10)⁴Individual cells/well) were incubated for 20 hours (E, F) recognition of TCR-engineered T cells against FOXM1 and UBE2T in an ELISPOT assay stimulated with C1R-a24 cells pulsed with or without FOXM1(E) or UBE 2T-specific peptides (F) — sorted TCR-engineered T cells (5 × 10)⁴Individual cells/well) and stimulated cells (2 × 10) treated with peptide pulses⁴Individual cells/well) were co-incubated in 96-well plates for 20 hours at 37 ℃. (G, H) levels of secreted protein of granzyme B and perforin from primary specific CTL or TCR-engineered T cells at 0, 2.5 and 5 hours after co-culture with cancer cells.

FIG. 4 FOXM1 and UBE2T protein expression in cancer cells. Expression of endogenous FOXM1 and UBE2T proteins in HLA-a 24:02 positive or negative cancer cell lines was examined by western blot analysis (western blotting).

Definition of

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the embodiments described herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Thus, in the context of the embodiments described herein, the following definitions apply.

As used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an engineered lymphocyte" is a reference to one or more engineered lymphocytes and equivalents thereof known to those skilled in the art, and so forth.

As used herein, the term "comprising" and its grammatical variants indicates the presence of the recited features, elements, method steps, etc. and does not exclude the presence of further features, elements, method steps, etc. Conversely, the term "consisting of … …" and its language variants indicates the presence of the recited features, elements, method steps, etc., and excludes any non-recited features, elements, method steps, etc., except for the impurities normally associated therewith. The phrase "consisting essentially of … …" means that the recited features, elements, method steps, etc. are present and that any additional features, elements, method steps, etc. that do not materially affect the basic properties of the composition, system, or method. Many of the embodiments herein are described using the open "inclusive" language. Such embodiments encompass closed "consisting of … …" and/or "consisting essentially of … …" embodiments that may be alternatively claimed or described using such language.

As used herein, "immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver, including antibodies, cytokines, and complements, to selectively target, bind to, destroy and/or eliminate from a subject an invading pathogen, a cell or tissue infected with a pathogen, or a cancer cell or other abnormal cell. Some embodiments herein include generating an immune response in a subject to treat cancer.

As used herein, the term "immunotherapy" refers to the treatment or prevention of a disease or condition by methods that include inducing, enhancing, inhibiting, or otherwise altering an immune response. Some embodiments herein include immunotherapy.

As used herein, the terms "adoptive immunotherapy" and "adoptive cell transfer" refer to the transfer of immunocompetent cells (e.g., TCR-engineered T cells) for the treatment of cancer or infectious diseases (June, C.H. eds., 2001, cancer chemother and Biotherapy: Principles and Practice, Lippincott Williams & Wilkins, Baltimore; Vonderheide et al, 2003, Immun. research 27: 1-15; incorporated by reference in its entirety). Some embodiments herein include adoptive immunotherapy.

As used herein, the term "cancer vaccine" refers to a composition (e.g., a tumor antigen) that elicits a specific immune response. The response is elicited from the subject's own immune system by administration of the cancer vaccine.

As used herein, the term "innate immune cell" refers to an immune cell that is naturally present in the immune system of a subject. Illustrative examples include, but are not limited to, T cells, NK cells, NKT cells, B cells, and dendritic cells. Some embodiments herein include eliciting a response in a subject to a subject's innate immune cells.

As used herein, the term "engineered immune cell" refers to an immune cell (e.g., T cell, NK cell, NKT cell, B cell, dendritic cell, etc.) that is genetically modified. Some embodiments herein include producing and/or administering engineered immune cells.

As used herein, the term "T cell receptor" ("TCR") refers to a molecular complex found on the surface of a T cell (T lymphocyte) that is responsible for recognizing antigen fragments bound to the Major Histocompatibility Complex (MHC) of an antigen presenting cell. The binding between TCR and antigenic peptide has relatively low affinity and is degenerate: that is, many TCRs recognize the same antigenic peptide, and many antigenic peptides are recognized by the same TCR. TCRs are heterodimers consisting of two distinct protein chains. In 95% of human T cells, the TCR consists of α (α) and β (β) chains (encoded by TRA and TRB, respectively), whereas in 5% of T cells, the TCR consists of γ and (γ /) chains (encoded by TRG and TRD, respectively). When the TCR is engaged with antigenic peptides and MHC, T lymphocytes are activated by signal transduction. Some embodiments herein include producing an engineered TCR, making a cell displaying the engineered TCR, and/or administering the cell displaying the engineered TCR to a subject to treat cancer.

As used herein, the term "human leukocyte antigen" ("HLA") refers to a Major Histocompatibility Complex (MHC) protein in humans or to a complex of genes encoding said human MHC protein.

The term "antibody" as used herein refers to whole antibody molecules or fragments thereof (e.g., such as Fab, Fab 'and F (ab')₂A fragment of (a) which may be a polyclonal or monoclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, or the like. As used herein, an antibody or other entity "specifically recognizes" or "specifically binds" an antigen or epitope, it preferentially recognizes the antigen in a complex mixture of proteins and/or macromolecules, and binds the antigen or epitope with substantially higher affinity than other entities that do not display the antigen or epitope. In this regard, "substantially higher affinity" means that the affinity is sufficiently high to enable detection of an antigen or epitope that is distinct from the entity using a desired assay or measurement instrument. Typically, it is meant to have at least 10⁷M^-1(e.g. in>10⁷M^-1、>10⁸M^-1、>10⁹M^-1、>10¹⁰M^-1、>10¹¹M^-1、>10¹²M^-1、>10¹³M^-1Etc.) of a binding constant (K)_a) Binding affinity of (4). In certain such embodiments, the antibody is capable of binding to different antigens so long as the different antigens comprise the particular epitope. In certain instances, for example, homologous proteins from different species may comprise the same epitope. Some embodiments herein include the generation and/or administration of antibodies that bind to oncogenic antigens and/or neoantigens.

As used herein, the term "antibody fragment" refers to a portion of a full-length antibody, including at least a portion of an antigen-binding or variable region. Antibody fragments include, but are not limited to, Fab ', F (ab')₂Fv, scFv, Fd, diabodies and other antibody fragments that retain at least a portion of the variable region of an intact antibody. See, e.g., Hudson et al (2003) nat. Med.9: 129-; incorporated by reference herein in its entirety. In certain embodiments, antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies produced by recombinant DNA techniques (e.g., papain digestion and pepsin digestion of antibodies) or by chemical polypeptide synthesis.For example, a "Fab" fragment comprises one light chain, and one heavy chain C_H1And a variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. The "Fab'" fragment contains one light chain and is contained at C_H1And C_H2One heavy chain of an additional constant region extending between the domains. Interchain disulfide bonds may form between the two heavy chains of a Fab 'fragment, thereby forming an "F (ab')₂"molecule. An "Fv" fragment comprises the variable regions from the heavy and light chains, but lacks the constant regions. Single chain fv (scfv) fragments comprise the heavy and light chain variable regions connected by a flexible linker, forming a single polypeptide chain with an antigen binding region. Exemplary single chain antibodies are discussed in detail in WO 88/01649 and U.S. Pat. nos. 4,946,778 and 5,260,203; incorporated by reference herein in its entirety. In some cases, a single variable region (e.g., a heavy chain variable region or a light chain variable region) is capable of recognizing and binding an antigen. The skilled artisan will appreciate other antibody fragments. Some embodiments herein include the generation and/or administration of antibody fragments that bind to oncogenic antigens and/or neoantigens.

As used herein, the term "monoclonal antibody" refers to an antibody that is a member of a substantially homogeneous population of antibodies that specifically bind to the same epitope. In certain embodiments, the monoclonal antibody is secreted by the hybridoma. In certain such embodiments, the hybridomas are produced according to certain methods known to those of skill in the art. See, e.g., Kohler and Milstein (1975) Nature 256: 495-499; incorporated by reference herein in its entirety. In certain embodiments, monoclonal antibodies are produced using recombinant DNA methods (see, e.g., U.S. patent No.4,816,567). In certain embodiments, a monoclonal antibody refers to an antibody fragment isolated from a phage display library. See, e.g., Clackson et al (1991) Nature 352: 624-; and Marks et al (1991) J.mol.biol.222: 581-597; incorporated by reference herein in its entirety. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and does not limit the method by which the antibody is produced to a particular method. For various other monoclonal antibody production techniques, see, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.); incorporated by reference herein in its entirety. Some embodiments herein include the generation and/or administration of monoclonal antibodies that bind to oncogenic antigens and/or neoantigens.

The term "antigen binding site" refers to a portion of an antibody that is capable of specifically binding an antigen. In certain embodiments, the antigen binding site is provided by one or more antibody variable regions.

The term "epitope" refers to any polypeptide determinant capable of specifically binding to an immunoglobulin or a T cell or B cell receptor. In certain embodiments, the epitope is a region of an antigen to which an antibody specifically binds. In certain embodiments, an epitope may comprise a chemically active surface grouping of molecules, such as a group of amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In certain embodiments, an epitope may have particular three-dimensional structural characteristics (e.g., a "conformational" epitope) and/or particular charge characteristics.

An epitope is defined as "identical" to another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may comprise the same epitope. In certain embodiments, the same epitope may have different primary amino acid sequences. Antibodies are said to bind to an epitope if they compete for specific binding to the same epitope.

As used herein, the term "sequence identity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same composition of monomeric subunits. The term "sequence similarity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are amino acids that share the same biophysical characteristics and can be grouped into families (see above). "percent sequence identity" (or "percent sequence similarity") is calculated by: (1) comparing the two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a designated window, etc.); (2) determining the number of positions containing the same (or similar) monomers (e.g., the same amino acid is present in both sequences, similar amino acids are present in both sequences) to yield the number of matched positions; (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., length of longer sequence, length of shorter sequence, designated window); and (4) multiplying the result by 100 to obtain the percentage of sequence identity or the percentage of sequence similarity. For example, if peptides a and B are both 20 amino acids long and have the same amino acid at all but position 1, then peptide a and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., are both acidic), then peptide a and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to amino acids of a portion of peptide C, then peptides C and D have 70% sequence identity, but peptide D has 93.3% sequence identity over the optimal comparison window for peptide C. To calculate "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is considered a mismatch at that position. In some embodiments, a peptide or polypeptide herein comprises minimal sequence identity to a base sequence.

The term "effective dose" or "effective amount" refers to an amount of an agent, such as an antibody, that causes a reduction in symptoms or produces a desired biological result in a patient. In certain embodiments, an effective dose or effective amount is sufficient to treat or reduce the symptoms of a disease or disorder.

As used herein, the terms "administration" and "administering" refer to the act of administering a drug, prodrug, or other agent or therapeutic agent to a subject or to cells, tissues, and organs in vivo, in vitro, or ex vivo. Exemplary routes of administration to the human body can be through the subarachnoid space of the brain, or spinal cord (intrathecal), eye (ocular), oral (oral), skin (topical or transdermal), nose (nasal), lung (inhalation), oral mucosa (buccal), ear, rectum, vagina, by injection (e.g., intravenous, subcutaneous, intratumoral, intraperitoneal, etc.), and the like.

Unless otherwise indicated, the term "treatment" encompasses both therapeutic and prophylactic/preventative measures. Persons in need of treatment include, but are not limited to, individuals already suffering from the particular condition as well as individuals at risk of developing the particular condition or disorder (e.g., individuals with a genetic or epigenetic predisposition; based on age, sex, lifestyle, etc.). The term "treatment" refers to the administration of an agent to a subject for therapeutic and/or prophylactic/preventative purposes.

"therapeutic agent" refers to an agent that can be administered in vivo to elicit a therapeutic and/or prophylactic/preventative effect.

"therapeutic antibody" refers to an antibody that can be administered in vivo to elicit a therapeutic and/or prophylactic/preventative effect.

As used herein, the terms "co-administration" and "co-administration" refer to the administration of at least two agents or therapies to a subject. In some embodiments, co-administration of two or more agents or therapies is concurrent. In other embodiments, the first dose/therapy is administered before the second dose/therapy. Those skilled in the art will appreciate that the formulation and/or route of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration is readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the dose of the respective agent or therapy administered is lower than the dose appropriate for its administration alone. Thus, co-administration is particularly desirable in embodiments where co-administration of an agent or therapy reduces the necessary dose of a potentially harmful (e.g., toxic) agent and/or when co-administration of two or more agents sensitizes a subject to the beneficial effects of one agent via co-administration of another agent.

As used herein, the term "pharmaceutical composition" refers to a combination of an active agent (e.g., a binding agent) and an inert or active carrier, such that the composition is particularly suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo. As used herein, the term "pharmaceutically acceptable" or "pharmacologically acceptable" refers to a composition that does not substantially produce an adverse reaction, such as a toxic, allergic, or immune response, when administered to a subject.

As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, including, but not limited to, phosphate buffered saline solutions, water, emulsions (e.g., oil/water or water/oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The composition may also include stabilizers and preservatives. See, e.g., Martin, Remington's Pharmaceutical Sciences, 15 th edition, mack pub. co., Easton, Pa. (1975), incorporated herein by reference in its entirety, for examples of carriers, stabilizers, and adjuvants.

As used herein, the term "healthy donor" refers to a mammal, e.g., a human, that does not suffer from any form of cancer and/or the cells/tissues used in embodiments herein do not show any signs of cancer (e.g., cancer morphology, cancer biomarkers, etc.).

Detailed Description

Provided herein are methods of identifying cancer specific antigens that recognize TCRs and TCR-engineered T cells having antigen-specific cytotoxic activity.

Cytotoxic T Lymphocytes (CTLs) that recognize cancer-specific antigens (oncogenic antigens and neoantigens) that are pre-existing in tumors or the blood circulation play a key role in achieving a beneficial clinical response to cancer immunotherapy. For example, a higher number of somatic mutations may increase the probability of generating a larger number of immunogenic neo-antigens that can be recognized by lymphocytes with high cytolytic activity, which may be further substantiated by immune checkpoint inhibitors (references 2-5; incorporated by reference in their entirety). Furthermore, a higher expression level of programmed death-ligand 1(PD-L1) is upregulated in cancer cells, programmed death-ligand 1 interacts with programmed death-1 (PD-1) in T cells, and is a biomarker of good clinical response (references 4, 6-8; incorporated by reference in its entirety). Furthermore, Tumor Infiltrating Lymphocytes (TILs) in patients responding to adoptive TIL metastasis therapy include CTLs that target neoantigens and oncogenic antigens (shared antigens) (reference 9; incorporated by reference in its entirety).

To enhance CTL-mediated anti-tumor immune responses to further improve the clinical outcome of cancer immunotherapy, embodiments herein utilize cancer-specific antigens, oncogenic antigens, and neoantigens as vaccines to activate antigen-specific CTLs in cancer patients. Oncogenic antigens are immunogenic peptides derived from oncogenic proteins that are highly expressed in cancer cells but not in normal organs except the testis or fetal organs (reference 10; incorporated by reference in its entirety). Immunogenic peptide epitopes derived from oncogenic antigens have been expected to induce oncogenic antigen-specific CTLs and improve the prognosis of cancer patients (references 11-13; incorporated by reference in its entirety). Neo-antigens are immunogenic peptides derived from non-synonymous mutations in cancer cells (reference 10; incorporated by reference in its entirety). Given some evidence that neoantigen-specific T cells show superior clinical outcomes (references 14-15; incorporated by reference in its entirety), neoantigen vaccines offer an option to further activate anti-cancer immune responses in patients. However, this vaccine approach does not work for patients with large tumor burden, as induction of sufficient numbers of anti-tumor T cells with vaccine therapy often proceeds very slowly and takes several months. Thus, the identification of cancer antigen-specific T Cell Receptors (TCRs), the generation of TCR-engineered T cells using autologous T lymphocytes, and the infusion of such genetically engineered T cells with/without anti-immune checkpoint antibodies provide an attractive option for patients with advanced tumors in which the host immune system is often significantly suppressed. Preclinical studies and recent clinical trials have shown encouraging results, namely that oncogenic antigen/neoantigen-specific TCR-engineered T cells are more effective against large-sized solid tumors (references 16-18; incorporated by reference in its entirety). Provided herein are rapid screening methods for identifying TCR sequences that recognize novel antigens and rapid preparation of personalized TCR-engineered T cell therapies.

Experiments were conducted during the development of embodiments herein to establish a rapid screening method for detecting oncogenic/neoantigen specific TCRs. Stimulation of candidate peptides in vitro from healthDonor's CD8⁺After T lymphocytes, CD8 was sorted using HLA class I dextromultimers under each peptide⁺T cells, and determining the TCR sequences of these cells. Monoclonal or low clonal expansion of unique T cells is achieved by stimulating epitope peptides. TCR cDNA was cloned and TCR-engineered T cells were generated. By this method, two antigen-specific CD8+ T cell clones were generated; two T cell clones recognizing oncogenic antigens derived from FOXM1 and UBE2T exhibited strong cytotoxic activity against HLA-matched cancer cells expressing the target protein, but did not exhibit cytotoxic activity against HLA-mismatched cancer cells. The methods described herein allow for the rapid identification of cancer specific antigens that recognize TCRs after obtaining the antigenic peptides. The methods allow for the rapid development of personalized T cell immunotherapies for the treatment of cancer. This document provides a channel for identifying cancer specific antigens that recognize TCRs and for establishing TCR-engineered T cells with antigen-specific cytotoxic activity that integrates in vitro neoantigen stimulation of T cells, dextrorotatory multimer sorting, and TCR sequencing using next generation sequencers.

Experiments were conducted during the development of embodiments herein to develop a channel from screening putative oncogenic antigen/neoantigen derived peptides to the induction of specific T cells from Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors and the establishment of antigen specific TCR engineered T cells. Throughout the channel, peptides derived from immunogenic oncogenic antigens/neoantigens were identified as useful in cancer vaccines, and oncogenic antigen/neoantigen-specific TCRs were identified, which led to the establishment of antigen-specific TCR-engineered T cells to observe cytotoxic activity against HLA-matched cancer cells.

As a source of PBMCs, PBMCs from healthy donors allow detection of candidates for oncogenic antigen/neoantigen-specific CTLs as they have different T cell lineages from cancer patients. T cells obtained from healthy donors broaden the reactivity of neoantigen-specific T cells and are able to target neoantigens that have not been recognized by the patient's own immune system (reference 30; incorporated by reference in its entirety). In some embodiments, upon identification of a cancer specific antigen that recognizes a TCR, TCR-engineered T cells are established from autologous T cells from the patient and infused as adoptive cell transfer therapy.

TCR-engineered T cells generated using PBMCs from HLA-a 24:02 positive healthy donors using the methods described herein recognize only HLA-a 24:02 restricted peptides and show significant cytotoxic activity against HLA-a 24:02 matched cancer cells. Considering that TCR-engineered T cells targeting HLA-a 02:01 restricted peptides derived from NY-ESO-1 using autologous PBMCs show encouraging clinical responses in myeloma patients (reference 21; incorporated by reference in its entirety), it is noted that TCR-engineered T cells from healthy donors herein also exert cytotoxic activity against HLA-a matched cancer cells. These results demonstrate the feasibility of preparing TCR-engineered T cells from healthy donors, for example, in situations where it is not possible to obtain autologous T cells from the patient.

The channels described herein provide personalized immunotherapy that responds to both oncogenic and neoantigens. Given that some oncogenic antigens are often overexpressed in many cancer types, TCR-engineered T cell therapies targeting oncogenic antigens are reasonable because the TCRs identified that recognize specific oncogenic antigens can be widely used in patients with the same HLA genotype. For example, elevated FOXM1 or UBE2T expression in tumor tissue is associated with poor survival in patients with breast, colon, and prostate cancer (references 31-34; incorporated by reference in their entirety). Thus, in some embodiments, FOXM1 and UBE 2T-specific TCR-engineered T cells described herein can be used in adoptive transfer therapy. Given that the clinical benefits of current Chimeric Antigen Receptor (CAR) T cell therapies and TCR-engineered T cell therapies are limited to hematologic malignancies (references 35-36; incorporated by reference in its entirety), the avenues presented herein for oncogenic antigen-specific TCR-engineered T cells provide another adoptive cell transfer therapy for solid tumors. In contrast, neoantigens have greater specificity for cancer cells and are considered attractive immune targets, although their presentation depends on somatic mutations of cancer cells. Given that metastatic therapy of neoantigen-specific TILs has shown encouraging clinical outcomes not only for melanoma, but also for solid tumors (references 14-15; incorporated by reference in its entirety), TCR-engineered T cells against neoantigens described herein provide a therapy for the clinical setting.

In some embodiments, provided herein are methods for identifying sequences of immunologically active TCRs, the methods comprising stimulating target lymphocytes (e.g., CD 8) with a stimulatory peptide comprising a candidate antigen sequence⁺Cytotoxic T lymphocytes). In some embodiments, the stimulatory peptides are fragments of proteins expressed on cancer and/or tumor cells. In some embodiments, the stimulatory peptides are fragments of cancer-specific antigens and/or tumor-specific antigens. In some embodiments, the target T lymphocyte is obtained from any suitable source (e.g., donor, cell culture, etc.). In some embodiments, the target T lymphocyte is obtained from a healthy donor. In some embodiments, the target T lymphocyte is CD8⁺Cytotoxic T lymphocytes. In some embodiments, the stimulation is performed in vitro (e.g., in cell culture). In some embodiments, the type of cell culture is determined by the type of target T lymphocyte. Conditions and methods suitable for culturing and stimulating T lymphocytes with stimulatory peptides are known in the art.

In some embodiments, the target lymphocyte (e.g., CD 8)⁺Cytotoxic T lymphocytes) are a population of target T lymphocytes, and the stimulatory peptide is one peptide in a population of stimulatory peptides comprising different candidate antigen sequences; and wherein said capturing comprises contacting immunocompetent T lymphocytes (e.g., CD 8)⁺Cytotoxic T lymphocytes) population is contacted with a capture reagent that displays a Major Histocompatibility Complex (MHC) bound to a population of capture peptides comprising a candidate antigen sequence.

In some embodiments, the target lymphocyte is CD8⁺Cytotoxic lymphocytes, CD4⁺Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, and the like.

In some embodiments, the stimulatory peptides comprising the candidate antigen sequence are all or a fragment of an oncogenic antigen and a neoantigen. In some embodiments, the candidate antigen sequences are all or a fragment of oncogenic antigens and neoantigens. In some embodiments, the stimulatory peptides comprise random amino acid sequences, and the methods herein allow for the identification of peptides capable of eliciting an immune response. In some embodiments, the stimulatory peptides comprise an amino acid sequence selected from the group consisting of: SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 and 44.

In some embodiments, after stimulating target T lymphocytes with a stimulatory peptide, immunocompetent T lymphocytes (e.g., CD 8) having a T Cell Receptor (TCR) that binds to the stimulatory peptide are captured⁺Cytotoxic T lymphocytes). In some embodiments, capturing comprises contacting the immunocompetent T lymphocytes with a capture reagent that displays a Major Histocompatibility Complex (MHC) bound to a capture peptide comprising a candidate antigen sequence. In some embodiments, the capture reagent displays a peptide comprising the sequence of one or more peptides of the stimulatory peptides. In some embodiments, the peptide is added to the T lymphocyte in a form that binds to the MHC I complex. In some embodiments, the capture reagent is an MHC multimer. In some embodiments, the MHC multimer is an MHC dextrorotatory multimer. For example, peptides can be presented to T lymphocytes that bind to MHC dextrorotatory multimers. In some embodiments, the MHC dextrorotatory multimer is a fluorescently labeled MHC multimer bound to the dextrose backbone. The use of multimeric MHC structures has the advantage of presenting multiple copies of the peptide, thereby increasing the likelihood of capture.

In some embodiments, after capturing the immunocompetent T lymphocytes, all or a portion of the TCR of the captured immunocompetent T lymphocytes is sequenced. In some embodiments, sequencing comprises next generation sequencing techniques. Next generation sequencing techniques are described in more detail below. In some embodiments, the portion of the TCR that is sequenced comprises TCR-a and/or TCR- β chains. In some embodiments, the portion of the TCR that is sequenced comprises one or more Complementarity Determining Regions (CDRs) of the TCR-a and/or TCR- β chains. In some embodiments, the portion of the TCR that is sequenced comprises CDR3 of the TCR-a and/or TCR- β chains.

In some embodiments, provided herein are cancer specific antigens (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.) that recognize a TCR identified by the methods described herein. In some embodiments, cancer-specific antigens identified by the methods described herein are used as therapeutic agents, e.g., cancer vaccines. Delivery systems for cancer vaccines can include, for example, liposomes, systems made of cholesterol, cholesterol hemisuccinate, or alpha-tocopherol (e.g., vitamin E), or other amphipathic molecules that can link or insert modified or synthetic neoantigens. In some embodiments, the cancer vaccine comprises a cancer specific antigen identified by the methods herein, or a variant thereof. In some embodiments, the cancer specific antigen is provided as a fusion peptide. In some embodiments, a plurality of sequences identified in the methods herein are incorporated. In some embodiments, the peptides used in the cancer vaccine are 10-80 amino acids long (e.g., 10, 20, 30, 40, 50, 60, 70, 80, or a range therebetween).

In some embodiments, provided herein are therapeutic antibodies that bind to a cancer specific antigen that recognizes a TCR described herein. In some embodiments, a therapeutic antibody herein is an antibody fragment. Antibodies and antibody fragments useful for the treatment of cancer are well known in the art. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the therapeutic antibody binds to an antigen comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1-44. In some embodiments, the therapeutic antibody comprises a CDR3 sequence comprising an amino acid sequence pair selected from the group consisting of seq id nos: 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78, 79 and 80, 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130, and 131 and 132 of SEQ ID NOs.

In some embodiments, provided herein are engineered T lymphocytes (e.g., CD 8) for generating all or a portion of a TCR displaying captured immunocompetent T lymphocytes⁺Cytotoxic T lymphocytes), wherein the engineered T lymphocytes recognize antigen presenting cells displaying MHC bound to a peptide comprising a candidate antigen sequence. In some embodiments, the engineered lymphocyte is CD8⁺Cytotoxic lymphocytes, CD4⁺Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, and the like. In some embodiments, the TCR sequences of immunocompetent T lymphocytes are used to prepare nucleic acids and/or vectors encoding TCRs that will recognize a target oncogenic antigen or neoantigen. In some embodiments, such nucleic acids and/or vectors are transformed, transfected, and/or otherwise placed into T lymphocytes to produce engineered T lymphocytes. Nucleic acids, vectors and methods for achieving these objectives are known in the art and are described herein. In some embodiments, the engineered T lymphocyte is CD8⁺Cytotoxic T lymphocytes. In some embodiments, engineered T lymphocytes (e.g., CD 8) displaying all or a portion of the TCR of the captured immunocompetent T lymphocytes are generated⁺Cytotoxic T lymphocytes) include: (i) cloning a nucleic acid sequence encoding a portion of the TCR of the captured immunocompetent T lymphocyte into a vector; (ii) introduction of vectors into host T lymphocytes (e.g., CD 8)⁺Cytotoxic T lymphocytes); and (iii) in the region of the TCR contacting the captured immunocompetent T lymphocytesIn some embodiments, a portion of the TCRs comprises TCR- α and/or TCR- β chains in some embodiments, a portion of the TCRs comprises TCR- α and/or one or more Complementarity Determining Regions (CDRs) of TCR- β chains in some embodiments, a portion of the TCRs that are sequenced comprises TCR- α and/or TCR- β chains cdrd 3. in some embodiments, a portion of the TCRs that are sequenced comprise an amino acid sequence selected from the group consisting of SEQ ID NOs 45-132 in some embodiments, engineered T lymphocytes exhibit TCRs comprising α and β chains selected from the group consisting of SEQ ID NOs 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, and 70, and 127, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66 and 68, and 70, and 70 and 72 and 75 and 70, and 70, and 99 and 70, and 80 and 99, and 70, and 100 and 70, and 100 and 103 and 114 to 103, 103 and 70, 103 and 103 to 103.

In some embodiments, provided herein are engineered T lymphocytes (e.g., CD 8) for generating Chimeric Antigen Receptors (CARs)⁺In certain embodiments, the antigen binding domain is a single-chain variable fragment (scFv) comprising heavy and light chain variable regions that specifically bind to a desired antigen (e.g., variable regions identified by the methods herein.) in some embodiments, the CAR further comprises a transmembrane domain (e.g., a T-cell transmembrane domain (e.g., a CD28 transmembrane domain)) and a signaling domain comprising one or more Immunoreceptor Tyrosine Activation Motifs (ITAMs) (e.g., a T-cell receptor signaling domain (e.g., a TCR ξ chain). in some embodiments, the CAR comprises a CAR that recognizes an MHC-presenting cell displaying an MHC that binds to a peptide comprising a candidate antigen sequenceContaining one or more co-stimulatory domains (e.g., a domain that provides a second signal to stimulate T cell activation). The invention is not limited by the type of co-stimulus domain. In some embodiments, the engineered lymphocyte is CD8⁺Cytotoxic lymphocytes, CD4⁺Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, and the like. In some embodiments, the TCR sequences of immunocompetent T lymphocytes are used to prepare a CAR that will recognize a target oncogenic antigen or neoantigen. In some embodiments, nucleic acids and/or vectors encoding such CARs are transformed or transfected into T cells, and/or the CARs are otherwise placed into T lymphocytes to produce engineered T lymphocytes. Nucleic acids, vectors and methods for achieving these objectives are known in the art and are described herein. In some embodiments, the engineered T lymphocyte is CD8⁺In some embodiments, the CAR comprises an antigen binding region comprising the sequence of TCR- α and/or TCR- β chains identified in the methods herein in some embodiments, the CAR comprises one or more Complementarity Determining Regions (CDRs) of TCR- α and/or TCR- β chains in some embodiments, a portion of the TCR that is sequenced comprises the CDRs 3 of TCR- α and/or TCR- β chains in some embodiments, a portion of the TCR that is sequenced comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 45-132 in some embodiments, engineered T lymphocytes display TCRs comprising pairs of amino acid sequences α and β chains selected from the group consisting of TCR: SEQ ID NOs 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66 and 68, 67 and 70, and 99, 99 and 70, and 99, and 70 and 75, and 75 and 99, and 70, and 80 and 103 and 70, and 103, and 103 and 70, and 103 and 122, and 70, and 103 and 122, and 103 and 122, and 103, and 103, and 103, 103 and 103, and 103, andin host T lymphocytes of cancer patients treated with baryte therapy.

In some embodiments, the methods herein are useful for generating engineered lymphocytes, e.g., CD4⁺Helper lymphocytes, NK cells, NKT cells, B cells, dendritic cells, and the like.

In some embodiments, the nucleic acid (e.g., TCR cDNA) is sequenced. Nucleic acid molecules can be sequenced by a variety of techniques. Analysis may identify the sequence of all or a portion of the nucleic acid. Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing, as well as "next generation" sequencing techniques. In some embodiments, the RNA is reverse transcribed to cDNA prior to sequencing.

A number of DNA sequencing techniques are known in the art, including fluorescence-based sequencing methods (see, e.g., Birren et al, Genome Analysis: Analyzing DNA,1, Cold Spring Harbor, N.Y.; incorporated herein by reference in its entirety). In some embodiments, automated sequencing techniques known in the art are utilized. In some embodiments, the systems, devices, and methods employ parallel sequencing of separate amplicons (PCT publication No: WO2006084132 to Kevin McKernan et al, incorporated herein by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (see, e.g., U.S. Pat. No.5,750,341 to Macevicz et al and U.S. Pat. No.6,306,597 to Macevicz et al, both of which are incorporated herein by reference in their entirety). Additional examples of sequencing techniques include Church polymerase cloning (polony) technique (Mitra et al, 2003, Analytical Biochemistry 320, 55-65; Shendire et al, 2005Science 309,1728- .

A group of methods known as "next generation sequencing techniques" have emerged as alternatives to Sanger and dye terminator sequencing methods (Voelkerding et al, Clinical chem.,55:641-658, 2009; MacLean et al, NatureRev. Microbiol.,7: 287-296; each of which is incorporated herein by reference in its entirety). Next Generation Sequencing (NGS) methods share common features of large-scale parallel high-throughput strategies, with the goal of lower cost and faster speed compared to earlier sequencing methods. NGS methods can be broadly divided into cases where template amplification is desired and cases where template amplification is not desired.

Sequencing techniques that can be used in embodiments herein include, for example, Helicos True Single molecule sequencing (tSMS) (Harris T.D. et al (2008) Science 320:106-⁶Template/cm²At a density of (a). The flow cell is then loaded into a sequencer and a laser illuminates the surface of the flow cell revealing the position of each template. The CCD camera can locate the position of the template on the flow cell surface. The template fluorescent label is then cleaved and washed away. The sequencing reaction is initiated by the introduction of a DNA polymerase and a fluorescently labeled nucleotide. oligo-T nucleic acids were used as primers. The polymerase incorporates labeled nucleotides into the primer in a template-directed manner. The polymerase and unincorporated nucleotides are removed. Templates that have directed incorporation of fluorescently labeled nucleotides are detected by imaging the flow cell surface. After imaging, the cleavage step removes the fluorescent label and the process is repeated with additional fluorescently labeled nucleotidesUntil the desired read segment length is achieved. Sequence information was collected at each nucleotide addition step. Further descriptions of tSMS are shown, for example, in Lapidus et al (U.S. Pat. No.7,169,560), Lapidus et al (U.S. patent application No. 2009/0191565), Quake et al (U.S. Pat. No.6,818,395), Harris (U.S. Pat. No.7,282,337), Quake et al (U.S. patent application No. 2002/0164629), and Braslaysky et al, PNAS (USA),100: 3960-.

Another example of a DNA sequencing technique that may be used in embodiments herein is 454 sequencing (Roche) (Margulies, M et al 2005, Nature 437, 376) -380; incorporated by reference in its entirety). 454 sequencing involves two steps. In the first step, the DNA is cleaved into fragments of approximately 300-800 base pairs and the fragments are blunt-ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors are used as primers for fragment amplification and sequencing. The fragments are ligated to DNA capture beads, e.g. streptavidin coated beads, using e.g. adaptor B containing a 5' -biotin tag. The fragments attached to the beads were PCR amplified within the droplets of the oil-in-water emulsion. The results are multiple copies of the clonally amplified DNA fragments per bead. In the second step, the beads are captured in wells (picoliter size). Pyrophosphoric acid sequencing was performed simultaneously on each DNA fragment. One or more nucleotides are added to generate an optical signal, which is recorded by a CCD camera in the sequencing instrument. The signal intensity is proportional to the number of nucleotides incorporated. Pyrosequencing utilizes a pyrophosphate group (PPi) which is released upon addition of a nucleotide. ATP sulfurylase converts PPi to ATP in the presence of adenosine 5' phosphate sulfate. Luciferase uses ATP to convert luciferin to oxyluciferin and this reaction generates light that is detected and analyzed.

Another example of a DNA sequencing technique that can be used in embodiments herein is the SOLiD technique (applied biosystems). In SOLiD sequencing, genomic DNA is sheared into fragments and adaptors are ligated to the 5 'and 3' ends of the fragments, generating a library of fragments. Alternatively, the internal adaptor may be introduced by: adaptors are ligated to the 5 'and 3' ends of the fragments, the fragments are circularized, the circularized fragments are digested to produce internal adaptors, and adaptors are ligated to the 5 'and 3' ends of the resulting fragments to produce end-paired libraries. The clonal bead population is then prepared in a microreactor containing beads, primers, templates, and PCR components. After PCR, the template is denatured and the beads are enriched to isolate beads with extended template. The template on the selected beads is subjected to a 3' modification that allows binding to the slide. The sequence can be determined by successive hybridization and ligation of partially random oligonucleotides to a centrally defined base (or base pair) identified by a particular fluorophore. After recording the color, the ligated oligonucleotides are cleaved and removed, and the process is repeated.

Another example of a DNA sequencing technique that can be used in embodiments herein is Ion Torrent sequencing (U.S. patent application nos. 2009/0026082, 2009/0127589, 2010/0035252, 2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559), 2010/0300895, 2010/0301398, and 2010/0304982; incorporated by reference in its entirety). In Ion Torrent sequencing, the DNA was cleaved into fragments of approximately 300-800 base pairs and the fragments were blunt-ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors are used as primers for fragment amplification and sequencing. The fragments may be attached to the surface and at a resolution such that the fragments are individually resolvable. Addition of one or more nucleotides releases protons (H)⁺) This signal can be detected and recorded in the sequencing instrument. The signal intensity is proportional to the number of nucleotides incorporated.

Another example of a DNA sequencing technique that can be used in embodiments herein is Illumina sequencing. Illumina sequencing is based on the amplification of DNA using fold-back PCR and anchor primers on a solid surface. The genomic DNA is fragmented and adapters are added to the 5 'and 3' ends of the fragments. DNA fragments attached to the surface of the flow cell channel were extended and subjected to bridge amplification. The fragments become double stranded and denature the double stranded molecules. Multiple cycles of solid phase amplification followed by denaturation can create millions of clusters of approximately 1,000 copies of single-stranded DNA molecules of the same template in each channel of the flow cell. Primers, DNA polymerase and four fluorophore-labeled reversibly terminated nucleotides were used for sequential sequencing. After nucleotide incorporation, the fluorophore is excited using a laser, and an image is captured and the identity of the first base is recorded. The 3' terminator and fluorophore from each incorporated base were removed and the incorporation, detection and identification steps were repeated.

Another example of a DNA sequencing technique that can be used in embodiments herein is Single Molecule Real Time (SMRT) technology by Pacific Biosciences. In SMRT, each of the four DNA bases is linked to one of four different fluorescent dyes. These dyes are linked via phosphate. A single DNA polymerase is immobilized with a single molecule of template single-stranded DNA at the bottom of a Zero Mode Waveguide (ZMW). A ZMW is a closed structure that is capable of observing the incorporation of a single nucleotide by a DNA polymerase against a background of fluorescent nucleotides that diffuse rapidly (in microseconds) outside the ZMW. Incorporation of nucleotides into the growing strand takes several milliseconds. During this time, the fluorescent label is excited and a fluorescent signal is generated and the fluorescent label is cleaved off. The detection of the corresponding fluorescence of the dye indicates which base was incorporated. The process is repeated.

Another example of a DNA sequencing technique that may be used in embodiments herein relates to nanopore sequencing (Soni GV and Meller A. (2007) Clin Chem 53: 1996-. Nanopores are small pores having a diameter of about 1 nanometer. The nanopore is immersed in a conducting fluid and an electrical potential is applied to it, creating a slight current due to ionic conduction through the nanopore. The amount of current flowing is sensitive to the size of the nanopore. As a DNA molecule passes through a nanopore, each nucleotide on the DNA molecule obstructs the nanopore to a different degree. Thus, a change in the current through the nanopore as the DNA molecule passes through the nanopore represents one read of the DNA sequence.

Another example of a DNA sequencing technique that can be used in embodiments herein involves sequencing DNA using a chemically sensitive field effect transistor (chemFET) array (e.g., as described in U.S. patent application publication No. 20090026082; incorporated by reference in its entirety). In one example of such a technique, a DNA molecule may be placed in a reaction chamber and a template molecule may be hybridized to a sequencing primer that binds to a polymerase. Incorporation of one or more triphosphate groups into new nucleic acid strands at the 3' end of the sequencing primer can be detected by current change with chemfets. The array can have a plurality of chemFET sensors. In another example, a single nucleic acid can be attached to a bead, and the nucleic acid can be amplified on the bead, and individual beads can be transferred to individual reaction chambers on a chemFET array, wherein each chamber has a chemFET sensor, and the nucleic acid can be sequenced.

In some embodiments, other sequencing techniques known in the art (e.g., NGS techniques) or alternatives or combinations of the above techniques may be used in embodiments herein.

Certain embodiments herein include detecting one or more biomarkers (e.g., detecting cytokines (e.g., IFN- γ) to detect and/or quantify an immune response). In some embodiments of the method, the method further comprises isolating one or more biomarkers from the biological sample or the in vitro culture (e.g., detecting a cytokine (e.g., IFN- γ) to detect and/or quantify the immune response). In some embodiments, an agent that binds to a biomarker is provided. Such agents are selected from antibodies, antibody fragments, aptamers, and the like.

In some embodiments, the detection method comprises an enzyme/substrate combination that produces a detectable signal corresponding to the level of the biomarker (e.g., using ELISA, western blot, isoelectric focusing techniques). Generally, enzymes catalyze the chemical alteration of a chromogenic substrate, which can be measured using a variety of techniques, including spectrophotometry, fluorescence, and chemiluminescence. Suitable enzymes include, for example, luciferase, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like. In some embodiments, the detection method is a combination of fluorescence, chemiluminescence, a radionuclide or an enzyme/substrate combination that produces a measurement signal. In some embodiments, multimodal signaling has unique and advantageous features in biomarker assay formats.

In some embodiments, the presence/level of a biomarker is detected using any analytical method, including singleplex aptamer assays, multiplex aptamer assays, singleplex or multiplex immunoassays, expression profiling, mass spectrometry, histological/cytological methods, and the like, as discussed below.

In some embodiments, a suitable immunoassay is used to detect/quantify the biomarker (e.g., detect a cytokine (e.g., IFN- γ) to detect and/or quantify the immune response). Immunoassays are based on the reaction of an antibody to its corresponding target or analyte, and can detect the analyte in a sample, depending on the particular assay format. To improve the specificity and sensitivity of immunoreactivity-based assays, monoclonal antibodies and fragments thereof are often used because they specifically recognize epitopes. Polyclonal antibodies have also been successfully used in various immunoassays because of the increased affinity of these antibodies for the target as compared to monoclonal antibodies. Immunoassays for a large number of biological sample matrices have been designed. Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results.

Many immunoassay formats have been designed. For detecting analytes, ELISA or EIA may be quantitative. This method relies on the attachment of a label to the analyte or antibody, and the label comprises an enzyme, either directly or indirectly. ELISA tests can be formatted for direct, indirect, competitive or sandwich detection of analytes. Other methods depend on, for example, the radioisotope (I)¹²⁵) Or a fluorescent label. Additional techniques include, for example, agglutination, nephelometry, turbidimetry, western blotting, immunoprecipitation, immunocytochemistry, immunohistochemistry, flow cytometry, Luminex assays, and the like (see Immunoassay: analytical Guide, edited by Brian Law, Taylor&Francis, inc, published, 2005 edition; incorporated herein by reference in its entirety).

Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescence, chemiluminescence, and Fluorescence Resonance Energy Transfer (FRET) or time-resolved-FRET (TR-FRET) immunoassay. Examples of procedures for detecting biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow for differences in size and peptide levels, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.

The method of detecting and/or quantifying the detectable label or signal-generating substance depends on the nature of the label. The product of the reaction catalyzed by the appropriate enzyme (in the case of a detectable label, see above; see above) may be, without limitation, fluorescent, luminescent or radioactive, or it may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, without limitation, x-ray films, radioactive counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, photometers, and densitometers.

Any of the methods for detection may be performed in any format that allows for any suitable preparation, processing, and analysis of the reaction. This may be, for example, in a multi-well assay plate (e.g. 96-well or 384-well) or using any suitable array or microarray. Stock solutions of the various agents can be made manually or automatically, and all subsequent pipetting, dilution, mixing, dispensing, washing, incubation, sample reading, data collection and analysis can be automated using commercially available analytical software, robotics, and detection instruments capable of detecting detectable labels.

In some embodiments, the antigenic peptides and sequences thereof used in embodiments herein are derived from cancer or tumor cell markers. Such markers may be selected from the group including, but not limited to: epidermal growth factor receptors (EGFR, EGFR1, ErbB-1, HER 1). ErbB-2(HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligand family; insulin-like growth factor receptor (IGFR) family, IGF binding protein (IGFBP), IGFR ligand family (IGF-1R); platelet Derived Growth Factor Receptor (PDGFR) family, PDGFR ligand family; fibroblast Growth Factor Receptor (FGFR) family, FGFR ligand family, Vascular Endothelial Growth Factor Receptor (VEGFR) family, VEGF family; HGF receptor family: the TRK receptor family; the pterosin (EPH) receptor family: the AXL receptor family; the Leukocyte Tyrosine Kinase (LTK) receptor family; the TIE receptor family, angiopoietins 1, 2; a family of receptor tyrosine kinase-like orphan receptors (ROR); the Discoidin Domain Receptor (DDR) family; the RET receptor family; the KLG receptor family; the RYK receptor family; the MuSK receptor family; transforming growth factor (TGF-alpha), TGF-alpha receptor; transforming growth factor-beta (TGF-beta), TGF-beta receptor; interleukin beta receptor alpha 2 chain (IL13R alpha 2), interleukin-6 (IL-6), 1L-6 receptor, interleukin-4, IL-4 receptor, cytokine receptor, class I (erythropoietin family) and class II (interferon/1L-10 family) receptors, Tumor Necrosis Factor (TNF) family, TNF-alpha, Tumor Necrosis Factor (TNF) receptor superfamily (TNTRSF), death receptor family, TRAIL-receptor; cancer-testis (CT) antigen, lineage specific antigen, differentiation antigen, alpha-actinin-4, ARTC1, breakpoint cluster region-Eibelson (Bcr-abl) fusion product, B-RAF, caspase-5 (CASP-5), caspase-8 (CASP-8), beta-catenin (CTNNB1), cell division cycle 27(CDC27), cyclin-dependent kinase 4(CDK4), CDKN2A, COA-1, dek-can fusion protein, EFTUD-2, elongation factor 2(ELF2), Ets variant gene 6/acute myeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein, GPFN, NMB, low density lipid receptor/GDP-L fucose beta-D2-alpha-LR-L glycosyltransferase (LDL) fucosyl transferase/fucosyl transferase (LDH-D2-L-D2-alpha-LR-T-fucosyl transferase (LDH) fusion protein, HLA-A2, MLA-A11, heat shock protein 70-2 mutation (HSP70-2M), KIAA0205, MART2, melanoma extensive mutation 1, 2, 3(MUM-1, 2, 3), Prostatic Acid Phosphatase (PAP), neo-PAP, myosin class 1, NFYC, OGT, OS-9, pml-RAR alpha fusion protein, PRDX5, PTPRK, K-ras (KRAS2), N-ras (NRAS), HRAS, RBAF600, SIRT12, SNRPD1, SYT-SSX1 or-SSX 2 fusion protein, triose phosphate isomerase, BAGE-1, BAGE-2, 3, 4, 5, GAGE-1, 2,3, 4, 5,6, 7, 8, GnT-V (abnormal N-acetyl HEK V, MGAT5 transferase), HN-MEL, KK-1, KM-LAG, KM-1, LAG, and so, CTL recognition antigens on melanoma (CAMEL), MAGE-A1 (MAGE-1). MAGE-A2, MAGE-A3, MAGE-A4, MAGE-AS, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10. MAGE-All, MAGE-A12, MAGE-3, MAGE-B1, MAGE-B2, MAGE-B5. MAGE-B6, MAGE-C1, MAGE-C2, mucin 1(MUC1), MART-1/Melan-A (Melan-A) (MLANA), gp100/Pme117(S1LV), Tyrosinase (TYR), TRP-1, HAGE, NA-88, NY-ESO-1/LAGE-2, SAGE, Sp17.SSX-1, 2,3, 4, TRP2-1NT2, carcinoembryonic antigen (CEA), vasopressin 4, mammaglobin-A, OA1, Prostate Specific Antigen (PSA), prostate specific membrane antigen, TRP-1/, 75.TRP-2 lipophilin, an inducer protein not present in melanoma 2 (AIM-2). BING-4, CPSF, cyclin D1, epithelial cell adhesion molecule (Ep-CAM), EpbA3, fibroblast growth factor-5 (FGF-5), glycoprotein 250(gp250 Intestinal Carboxyl Esterase (iCE), alpha-fetoprotein (AFP), M-CSF, mdm-2, MUCI, p53(TP53), PBF, PRAME, PSMA, RAGE-1, RNF43, RU2AS, SOX10, STEAP1, survivin (BIRCS), human telomerase reverse transcriptase (hTERT), telomerase, Wilms' tumor gene (WT 1), SYDT 1, BRDT, SPANX, XAGE, ADAM2, PAGE-5, LIP1, CTAGE-1, CAGE, BOTES-85, HOM-6315, HOMA-3523, SAGE-3527, BESPHCC 6335, SAR-11, SAGE 6335, SAGE 6335, BEHCC-11, SAGE 6335, SAGE, BEHCRD, BEHCET 6335, SAC-11, SAGE 6335, SAGE, BEHCET 6335, BEHCET, SAC 6335, SAC, SAGE, SAC, Estrogen Receptor (ER), Androgen Receptor (AR), CD40, CD30, CD20, CD19, CD33, CD4, CD25, CD3, cancer antigen 72-4(CA 72-4), cancer antigen 15-3(CA 15-3), cancer antigen 27-29(CA 27-29), cancer antigen 125(CA 125), cancer antigen 19-9(CA 19-9), beta-human chorionic gonadotropin, 1-2 microglobulin, squamous cell carcinoma antigen, neuron-specific enolase, heat shock protein gp96.GM2, sargrastim, CTLA-4, 707 alanine proline (707-AP), adenocarcinoma antigen recognized by T cell 4 (ART-4), carcinoembryonic peptide-1 (CAP-1), calcium activated chloride channel 2(CLCA2), cyclophilin B (Cyp-B), Human signet ring tumor-2 (HST-2), etc. In some embodiments, the antigenic peptides and sequences thereof used in embodiments herein are derived from cell surface markers that are specific for or displayed predominantly on (e.g., recognized by antibodies and/or immune cells) cancer/tumor cells.

In some embodiments, provided herein are T lymphocytes engineered to express an immunologically active TCR. In some embodiments, provided herein are T lymphocytes engineered to express an immunologically active CAR. The engineered cells can be produced by any suitable method in the art. In some embodiments, T lymphocytes are engineered to express/display an immunologically active TCR obtained by the methods described herein (e.g., stimulating, capturing, sequencing). In some embodiments, the T lymphocytes are engineered to express/display an immunologically active CAR obtained by the methods described herein (e.g., stimulating, capturing, sequencing).

In some embodiments, provided herein are nucleic acids and nucleic acid sequences encoding an immunologically active TCR (or immunologically active CAR) as described above and cells having such nucleic acids. In some embodiments, the nucleic acid molecule is a recombinant nucleic acid molecule. In some embodiments, the nucleic acid molecule is synthetic. The nucleic acid encoding the immunologically active TCR and portions thereof may comprise DNA, RNA, PNA (peptide nucleic acid) and hybrids thereof.

In some embodiments, the nucleic acid encoding the immunologically active TCR, and portions thereof, comprises one or more regulatory sequences. For example, promoters, transcription enhancers, and/or sequences that allow for the induction of expression of the polynucleotides of the disclosure may be employed. In some embodiments, the nucleic acid molecule is transcribed from an appropriate vector comprising a chimeric gene that allows transcription of the nucleic acid molecule in a cell.

In some embodiments, the nucleic acid molecule is a recombinantly produced chimeric nucleic acid molecule comprising any of the above nucleic acid molecules, alone or in combination. In some embodiments, the nucleic acid molecule is part of a vector.

In some embodiments, provided herein are vectors comprising a nucleic acid molecule described herein (e.g., encoding an immunologically active TCR and portions thereof). Many suitable vectors are known to those skilled in molecular biology, and the choice of vector will depend on the desired function and includes plasmids, cosmids, viruses, bacteriophages and other vectors commonly used in genetic engineering. Various plasmids and vectors can be constructed using methods well known to those skilled in the art; see, e.g., the techniques described in: sambrook et al (1989) and Ausubel, Current Protocols in Molecular Biology, Green publishing associates and Wiley Interscience, N.Y. (1989), (1994); incorporated by reference in its entirety. Alternatively, the polynucleotides and vectors of the present disclosure are reconstituted in liposomes for delivery to target cells. Cloning vectors can be used to isolate individual sequences of DNA. The relevant sequences may be transferred to an expression vector where expression of the particular polypeptide is desired. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322, and pGBT 9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, pOP13 CAT.

In some embodiments, the vector comprises a nucleic acid sequence that is a regulatory sequence operably linked to a nucleic acid sequence encoding an immunologically active TCR, and portions thereof. Such regulatory sequences (control elements) are known to the skilled person and may include promoters, splicing cassettes, translation initiation codons and insertion sites for introducing the insert into the vector. In a particular embodiment, the nucleic acid molecule is operably linked to said expression control sequence allowing expression in eukaryotic or prokaryotic cells.

In some embodiments, the vector is a viral vector, such as a lentiviral vector or an adeno-associated vector.

In some embodiments, the nucleic acids and/or vectors are used in a cell to express an encoded polypeptide (e.g., an immunologically active TCR, portions thereof, etc.) in the cell. A nucleic acid molecule or vector comprising a DNA sequence encoding any of the immunologically active TCRs described herein is introduced into a cell, which in turn produces a polypeptide. The nucleic acid molecules and vectors described can be designed to be introduced directly or via liposomes or viral vectors (e.g., adenovirus, retrovirus) into cells.

In light of the above, provided herein are methods of obtaining vectors, particularly plasmids, cosmids, viruses, and phages commonly used in genetic engineering, comprising nucleic acid molecules encoding the polypeptide sequences described herein (e.g., immunologically active TCRs and portions thereof). In some embodiments, the vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses may be used to deliver the polynucleotides and/or vectors to a target cell population. Recombinant vectors can be constructed using methods well known to those skilled in the art. Well known methods for transferring vectors into host cells vary depending on the type of cellular host.

In some embodiments, provided herein are cells comprising a host cell transformed or transfected with a vector as defined above (e.g., encoding an immunologically active TCR described herein). The host cell may be produced by introducing at least one of the above-described vectors or at least one of the above-described nucleic acid molecules into the host cell. The presence of at least one vector or at least one nucleic acid molecule in the host may mediate the expression of genes encoding the above-described immunologically active TCRs and portions thereof. The nucleic acid molecule or vector introduced into the host cell may be incorporated into the host genome or it may be maintained extrachromosomally.

In some embodiments, provided herein are methods comprising culturing a host cell as defined above under conditions that allow for the introduction of nucleic acids and/or vectors. In some embodiments, provided herein are methods comprising culturing a host cell as defined above under conditions that allow for expression of the construct (e.g., comprising an immunologically active TCR, or a portion thereof). In particular embodiments, the cultured cells are provided to a subject (e.g., a subject from which the original cells were obtained, a second subject, etc.). Conditions for culturing cells with expression constructs are known in the art.

In some embodiments, the lymphocytes for engineering according to embodiments herein are from any suitable source. For example, the source of the lymphocytes is a subject (e.g., a subject to be treated, a healthy subject, etc.). Lymphocytes can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, the particular type of lymphocyte (e.g., a cytotoxic T cell) desired for the embodiments described herein is obtained by an appropriate method. In some embodiments, lymphocytes expressing a particular marker are obtained by known methods (e.g., cell sorting). In some embodiments, the cells are cultured after isolation. In some embodiments, the cells are engineered using the methods described herein.

In some embodiments, the compositions herein (e.g., engineered lymphocytes, antibodies, vaccines, nucleic acid molecules, vectors, etc.), alone or in any combination, are administered using standard delivery systems and methods and, in at least some aspects, along with a pharmaceutically acceptable carrier or excipient. In the case of a nucleic acid molecule or vector, it can be stably incorporated into the genome of the subject.

In some embodiments, methods and compositions relating to preventing, treating, or ameliorating cancer are provided, the methods comprising the step of administering to a subject in need thereof an effective amount of a composition herein (e.g., engineered lymphocytes, antibodies, vaccines, nucleic acid molecules, vectors, etc.) as encompassed herein and/or produced by a method as encompassed herein. When cells are administered, the engineered cells are administered to the treatment site or may be concentrated at the treatment site (e.g., cell type, tissue type, etc.).

Non-limiting examples of cancers that can be treated with the compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods described herein include (but are not limited to): cancer cells from the bladder, blood, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. Furthermore, the cancer may in particular belong to the following histological types, although it is not limited to these: neoplasm, malignant; cancer and tumor; carcinoma, undifferentiated; giant cell and spindle cell cancers; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphatic epithelial cancer; basal cell carcinoma; hair matrix cancer; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinomas, malignant; cholangiocellular carcinoma; hepatocellular carcinoma; mixed hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma within adenomatous polyps; adenocarcinoma, familial polyposis coli; a solid cancer; carcinoid tumor, malignant; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; a cancer of the chromophobe; eosinophilic carcinoma; eosinophilic adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinomas; non-encapsulated sclerosing cancer; adrenocortical carcinoma; endometrioid carcinoma; skin appendage cancer; hyperhidrosis carcinoma; sebaceous gland cancer; cerumen adenocarcinoma; mucoepidermoid carcinoma; cystic carcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory cancer; paget's disease of the breast; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma with squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecal cell tumor, malignant; granulosa cell tumor, malignant; and blastoma (robustoma), malignant; sertoli cell carcinoma (sertoli cell carcinoma); leydig cell tumor (leydig cell tumor), malignant; lipocytoma, malignant; paraganglioma, malignant; external paraganglioma of mammary gland, malignant; pheochromocytoma; glomus; malignant melanoma; melanotic melanoma-free; superficial diffusible melanoma; malignant melanoma within a giant pigmented nevus; epithelial-like cell melanoma; blue nevus, malignant; a sarcoma; fibrosarcoma; fibrohistiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; miller tube mixed tumor (mullerian mixed tumor); nephroblastoma; hepatoblastoma; a carcinosarcoma; phyllomas, malignant; brenner tumor (brenner tumor), malignant; phylloid tumors, malignant; synovial sarcoma; mesothelioma, malignant; clonal cell tumors; an embryonic carcinoma; teratoma, malignancy; ovarian thyroid tumor, malignant; choriocarcinoma; middle kidney tumor, malignant; angiosarcoma; vascular endothelioma, malignant; kaposi's sarcoma; vascular endothelial cell tumor, malignant; lymphangioleiomyosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumors, malignant; amelogenic cell dental sarcoma; ameloblastoma, malignant; an amelogenic fibrosarcoma; pineal tumor, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; primary plasma astrocytoma; fibroid astrocytoma; an astrocytoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectoblastoma; cerebellar sarcoma; nodal cell neuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumors; meningioma, malignant; neurofibrosarcoma; schwannoma, malignant; granulocytic tumors, malignant; malignant lymphoma; hodgkin's disease; hodgkin's lymphoma; granuloma-like; malignant lymphoma, small lymphocytic; large cell diffuse malignant lymphoma; malignant lymphoma, follicular; mycosis fungoides; other non-hodgkin's lymphoma designated; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cellular leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; primitive megakaryocytic leukemia; myeloid sarcoma; and hairy cell leukemia. In some embodiments, the cancer is melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate cancer), pancreatic cancer (e.g., adenocarcinoma), breast cancer, colon cancer, gallbladder cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other malignancies. In some embodiments, the cancer is a solid tumor cancer.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are employed with one or more combination therapies for treating cancer. In some embodiments, one or more chemotherapies and/or immunotherapies are co-administered with the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein. In some embodiments, one or more chemotherapeutic agents and/or immunotherapy are provided as a combination therapy, with or without (known) synergistic effects.

The present disclosure further contemplates co-administration regimens with other compounds that act via immune cells, such as targeted toxins or other blocking or functional antibodies or compounds. A co-administered clinical regimen may encompass co-administration simultaneously with, before, or after administration of the other component. Specific combination therapies include chemotherapy, radiation, surgery, hormonal therapy, or other immunotherapy types. Many chemotherapeutic agents are currently known in the art and may be used in combination with the compounds of the present invention. In some embodiments, the chemotherapeutic agent is selected from the group consisting of: mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with one or more chemotherapeutic agents. Chemotherapy for co-administration herein includes all classes of chemotherapeutic agents, such as alkylating agents, antimetabolites, plant alkaloids, antibiotics, hormonal agents, and other anticancer agents. Specific agents include, for example, albumin-bound paclitaxel (abraxane), altretamine (altretamine), docetaxel (docetaxel), herceptin (herceptin), methotrexate (methotrexate), norvaselin (novantrone), norrex (zoladex), Cisplatin (CDDP), carboplatin (carboplatin), procarbazine (procarbazine), mechlorethamine (meclorethamine), cyclophosphamide (cyclophosphamide), camptothecin (camptothecin), ifosfamide (ifosfamide), melphalan (melphalan), chlorambucil (chlamubuucil), busulfan (busufan), nitrosourea (nitrosulrea), actinomycin (dannomycin), daunomycin (daunorubicin), doxorabicin (doxorubin), bleomycin (bleomycin), gemcitabine (gemcitabine), etoposide (flavodoxin), mitomycin (VP 25), mitomycin (mitomycin), mitomycin (norubin), mitomycin (norubicin), norbixin (norubicin), norfloxacin (doxorubicin (norfloxacin), norfloxacin (norfloxacin), norfloxacin (mitomycin (e), norubicin (mitomycin (norubicin), norubicin (mitomycin), norubicin (mitomycin (norubicin), norubicin (norubicin), norubicin (mitomycin (norubicin), norubicin), norubicin (norubicin), norubi, Navelbine, farnesyl-protein transferase inhibitors, antiplatin, 5-fluorouracil, vincristine, vinblastine, or any analogue or derivative variant of any of the foregoing agents, and combinations thereof. In some embodiments, chemotherapy is employed prior to, during, and/or after administration of the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with radiotherapy, methods of which are known in the art. In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein employ radiotherapy before, during, and/or after administration.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with non-immune targeted therapies, e.g., agents that inhibit, e.g., WNT, p53, and/or signaling pathways. Other examples include agents that inhibit tyrosine kinases, BRAF, STAT3, c-met, regulate gene expression, induce cell death, or block angiogenesis. Examples of specific agents include imatinib mesylate (imatinib mesylate), dasatinib (dasatinib), nilotinib (nilotinib), bosutinib (bosutinib), lapatinib (lapatinib), gefitinib (gefinitib), erlotinib (erlotinib), temsirolimus (tensirolimus), everolimus (everolimus), vemurafenib (vemurafenib), crizotinib (crizotinib), vorinostat (vorinostat), romidepsin (romidepsin), bexarotene (bexarotene), alitrinin (alitrinonin), tretinoin (tretinoin), bortezomib (bortezomib), carfilzomib (carzomib), prasutrofitinib (prasufelinib), sunitinib (sunitinib), or bocacitinib (bocacitinib). In some embodiments, non-immune targeted therapy is employed before, during, and/or after administration of the engineered lymphocytes.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with immunotherapy. Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may be used as an effector of therapy, or it may complement other cells to actually effect cell killing. The antibodies may also prevent cancer immune evasion or immunosuppression. The antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin a chain, cholera toxin, pertussis toxin, etc.) and simply used as targeting agents. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts directly or indirectly with the tumor cell target. In some embodiments, immunotherapy is employed before, during, and/or after administration of the engineered lymphocytes.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with gene therapy, wherein the therapeutic polynucleotide is administered prior to, after, or simultaneously with the administration of the engineered lymphocytes described herein. A variety of expression products are contemplated, including cell proliferation inducers, cell proliferation inhibitors, or programmed cell death modulators.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are administered pre-, during, and/or post-operatively. Surgery includes resection in which all or a portion of cancerous tissue is physically removed, resected, and/or destroyed. Tumor resection refers to the physical removal of at least a portion of a tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosection, and microscopically controlled surgery (Mohs' surgery). It is further contemplated that the embodiments herein may be used in conjunction with the removal of surface cancer, pre-cancer, or concomitant amounts of normal tissue.

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are co-administered with other agents that enhance the therapeutic efficacy of the treatment.

In some embodiments, the co-administered agents are formulated as a single dose and/or composition. In some embodiments, the co-administered agents are in separate doses and/or compositions. In some embodiments where separate doses and/or compositions are administered, the doses and/or compositions are administered simultaneously, sequentially, or at intervals (e.g., <30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week or more or any suitable range in between).

In some embodiments, the therapeutic compositions (e.g., engineered lymphocytes, antibodies, vaccines, etc.) and methods herein are provided as part of a kit or system, along with one or more additional components such as instructions, administration devices, additional therapeutic agents, diagnostic agents, research agents, and the like.

Experiment of

Materials and methods

Peptides

9-mer and 10-mer peptides were synthesized by using standard solid phase synthesis methods and purified by reverse phase High Performance Liquid Chromatography (HPLC) (see tables 1 a-e). The purity (> 90%) and identity of the peptide were determined by analytical HPLC and mass spectrometry, respectively. The peptide was dissolved in dimethyl sulfoxide to 20mg/ml and stored at-80 ℃.

TABLE 1 peptide amino acid sequence for establishing peptide-specific CTL

TABLE 1a list of HLA-A24: 02 restricted peptides for establishing peptide-specific CTL

Peptide name	Amino acid sequence	SEQ ID NO
			CDCA5-A24-10-232	EWAAAMNAEF	1
CDH3-A24-10-807	DYLNEWGSRF	2
			FOXM1-A24-9-262	IYTWIEDHF	3
HJURP-A24-9-408	KWLISPVKI	4
			INHBB-A24-9-180	LYLKLLPYV	5
KIF20A-A24-10-66	KVYLRVRPLL	6
			MELK-A24-9-87_7N	EYCPGGNLF	7
NEIL3-A24-9-545	EWADLSFPF	8
			RNF43-A24-9-721	NSQPVWLCL	9
SEMA5B-A24-10-290	AYDIGLFAYF	10
			SMYD3-A24-9-197	QYCFECDCF	11
topK-A24-10-289	SYQKVIELFS	12
			UBE2T-A24-9-60	RYPFEPPQI	13
VANGL1-A24-9-443	RYLSAGPTL	14
			VEGFR1-A24-9-1084	SYGVLLWEI	15
VEGFR2-A24-9-169	RFVPDGNRI	16
			WDHD1-A24-9-844	GYSNTATEW	17
WDRPUH-A24-9-314	IYRVSFTDF	18

TABLE 1b List of HLA-A02: 01 restriction peptides for the establishment of peptide-specific CTL

TABLE 1c List of HLA-A11: 01 restriction peptides for the establishment of peptide-specific CTL

Peptide name	Amino acid sequence	SEQ ID NO
			CDCA1-A11-9-219	KTKRLNELK	36
DEPDC1v1-A11-9-627	MSQNVDMPK	37
			KIF20A-A11-9-45	VVSTSLEDK	38
MPHOSPH1-A11-10-1546	STSFEISRNK	39

TABLE 1d List of HLA-A33: 03-restricted peptides for the establishment of peptide-specific CTL

Peptide name	Amino acid sequence	SEQ ID NO
			CDCA1-A33-9-43	EVLHMIYMR	40
FOXM1-A33-9-308	WTIHPSANR	41
			MPHOSPH1-A33-9-608	EFTQYWAQR	42
VEGFR2-A33-9-114	IYVYVQDYR	43

TABLE 1e List of HLA-A03: 01 restriction peptides used to establish peptide-specific CTL

Peptide name	Amino acid sequence	SEQ ID NO
			KOC1-A03-10-120	AVVNVTYSSK	44

Cell lines

TISI (HLA-A24: 02, B lymphoblastoid cell line) was purchased from the International Histocompatibility Working Group (International Histocompatibility Working Group). T2 (HLA-A02: 01, B-lymphoblastoid cell line), EB-3(HLA-A3/Aw32, B-lymphoblastoid cell line), Jiyoye (HLA-A32, B-lymphoblastoid cell line), SW480 (HLA-A24: 02, colorectal adenocarcinoma), HCC1143 (HLA-A31: 01, breast cancer), BT549 (HLA-A02: 01, breast cancer), and C1R (lacking HLA-A and HLA-B, B-lymphoblastoid cells) were purchased from American Type Culture Collection (Rockville, Md.). All cells were cultured according to the recommendations of their respective depositors.

Production of C1R cells stably expressing HLA class I

In addition to TISI and T2, C1R cells transfected with Human Leukocyte Antigen (HLA) were used as stimulating cells. The cDNA encoding the open reading frame of HLA class I (a 24:02, a 02:01, a 11:01, a 33:03, or a 03:01) was amplified by PCR and inserted into an expression vector. C1R cells were transfected with HLA class I expression vector and cultured for 14 days in the presence of G418(Invitrogen, Carlsbad, CA). G418 resistant single cells and feeder cells were plated into 96-well cell culture plates (Corning, inc., Corning, NY) containing medium supplemented with G418 and further cultured for 30 days. Expression of transfected HLA class I on C1R cells was confirmed by flow cytometry analysis.

In vitro CTL Induction

Mononuclear globular dendritesThe somatic cells (DCs) serve as antigen presenting cells to induce Cytotoxic T Lymphocytes (CTLs) that respond to peptides presented on HLA class I. DCs were generated in vitro (reference 37; incorporated by reference in its entirety). Peripheral Blood Mononuclear Cells (PBMC) were isolated from the blood of healthy volunteers by Ficoll-Paque PLUS (GE Healthcare). Granulocyte-macrophage colony stimulating factor (R) at 1000IU/ml in AIM-V medium (Invitrogen) (AIM-V/2% HS medium) containing 2% heat-inactivated human serum&D Systems, Minneapolis, MN) and Interleukin (IL) -4 (R) at 1000IU/ml&D System) to induce DC formation, mononuclear spheroids (adherent cells in PBMC) were pulsed with 20. mu.g/ml synthetic peptide in AIM-V medium in the presence of 3. mu.g/ml of β -2-microglobulin at 37 ℃ for 3 hours after seven days of culture these peptide pulsed DCs were inactivated by X-ray irradiation (20) Gy and mixed at a ratio of 1:20 with autologous CD8+ T cells obtained from PBMC by using a CD8 positive isolation kit (Thermo Fisher Scientific, Carlsbad, CA), these cultures were set in 48-well cell culture plates (Corning), each well contained 0.5ml of AIM-V/2% HS medium 1.5 × 10⁴Peptide-pulsed DC, 3 × 10⁵CD8+ T cells and 10ng/ml IL-7 (R)&D System). The following day (day 2), IL-2(Novartis) was added to the culture at a final concentration of 20 IU/ml. On days 7 and 14, CD8+ T cells were further stimulated with autologous peptide-pulsed DCs. DCs were prepared each time in the same manner as described above. CD8+ T cells were tested for peptide-specific IFN- γ production by ELISPOT assay on day 21 (references 38-39; incorporated by reference in its entirety).

Amplification culture

After limiting dilution, CD8+ T cells were expanded using the rapid expansion method (reference 40; incorporated by reference in its entirety), EB-3 and Jiyoye were treated with mitomycin C and used as feeder cells, CD8+ T cells were combined with feeder cells (5 × 10 each)⁶Individual cells) and 40ng/ml anti-CD 3 antibody were cultured together in 25ml AIM-V/5% HS medium. The following day (day 1), 3000IU of IL-2 was added to the culture. Half the volume of the medium was changed to on day 5, 8 and 11Fresh AIM-V/5% HS medium containing 60IU/ml IL-2. CD8+ T cells were tested for peptide-specific IFN-. gamma.production by ELISA between day 14 and day 16 (references 38-39; incorporated by reference in its entirety).

Detection of peptide-specific IFN-gamma

To examine peptide-specific IFN-. gamma.production by CD8+ T cells, an ELISPOT assay or ELISA was performed.T 2, TISI, or HLA class I expressing C1R cells (1 × 10) were prepared after peptide pulsing⁴Individual cells) as the stimulating cells. CD8+ T cells were used as responder cells. IFN- γ ELISPOT assays and IFN- γ ELISA were performed according to the manufacturer's protocol (BD Biosciences, San Jose, Calif.).

Evaluation of cytotoxic Activity of CTL against cancer cells by time-lapse recording

CTL and TCR-engineered T cells were pretreated with IL-2(100U/mL) for 16 hours before the experiment, target cells were pretreated with IFN-. gamma. (100U/mL) for 48 hours before the experiment, cells were incubated with 1ug/mL Calcein AM (Dojindo, Kumamoto, Japan) for 30 minutes, 2 × 10 was washed 3 times with PBS⁴A target cell and 2 × 10⁵FOXM1/UBE2T specific CTL or 4 × 10⁵Individual TCR-engineered T cells were mixed into Lab-Tek Chamber Slide coverslips sterile 16 wells (Thermo Scientific). Time-lapse recordings were carried out by means of an inverted microscope Axio Vert.A1TL (Zeiss, Oberkochen, Germany). Living and dead cells were quantified using the ImageJ program (National Institutes of health, Bethesda, MD).

T cell receptor sequencing

TCR sequences were determined (reference 41; incorporated by reference in its entirety). Total RNA was extracted from expanded or dextrorotatory multimer positive T cells. cDNA with the common 5' -RACE adaptor was synthesized using the SMART library construction kit (Clontech, Mountain View, Calif.). Fusion PCR was performed using a forward primer corresponding to the SMART adaptor sequence and a reverse primer corresponding to the constant region of each of TCRA or TCRB to amplify TCRA or TCRB cDNA. After addition of Illumina index sequence with barcode using Nextera indexing kit (Illumina, San Diego, CA), the prepared library was sequenced by reading 300bp double ends on miseq (Illumina). The resulting sequence reads were analyzed using Tcrip software (ref. 41; incorporated by reference in its entirety). The sequence was also confirmed by sanger sequencing using the fusion PCR product as template (Thermo Scientific).

TCR engineered T cells

TCRA and TCRB sequences were codon optimized and cloned into pMP71-PRE (references 18, 42; incorporated by reference in their entirety). To maximize TCR expression, modified murine TCRA and TCRB constant domains were used. Transient retroviral supernatants were generated and donor-derived PBMCs were transduced (ref 18; incorporated by reference in its entirety). TCR expression was assessed with anti-human TCR β V antibodies. APC-conjugated anti-mouse TCR β monoclonal antibodies (H57-597, eBioscience, San Diego, CA) were stained under conditions suitable for TCR-engineered T cells against FOXM1 and UBE2T, followed by incubation with anti-APC microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions, to transduce only transduced TCR-engineered T cells. To increase the number of T cells transduced with the desired TCR not occupied by the antibody, the conditions were based on peak fluorescence intensity and the number of cells sorted by five different conditions of comparing antibody dilutions. Antibody staining at ratios of 1:2000(0.1ug/mL) and 1:4000(0.05ug/mL) was determined to be appropriate for sorting of TCR-engineered T cells against FOXM1 and UBE2T, respectively.

Results

Peptide-specific CTL

CTL clones specific for HLA-A24: 02, HLA-A02: 01, HLA-A11: 01, HLA-A33: 03 or HLA-A03: 01 restriction peptides from tables 1a-e were induced. CTLs were captured using HLA dextrorotatory multimers of each peptide, and the TCR sequences of these cells were determined (see tables 2a-e for CDR3 amino acid sequences of TCRs in peptide-specific CTLs). All 44 CTL clones were evaluated for peptide-specific IFN- γ production by ELISA assay using HLA-expressing cells with or without peptide.

TABLE 2 CDR3 amino acid sequence of TCR in peptide-specific CTL

Table 2a list of major CDR3 sequences of CTL clones specific for HLA-a 24:02 restricted peptides

Table 2b list of major CDR3 sequences of CTL clones specific for HLA-a 02:01 restriction peptides

TABLE 2c listing of the main CDR3 sequences of CTL clones specific for HLA-A11: 01 restriction peptides

Table 2d list of major CDR3 sequences of CTL clones specific for HLA-a 33:03 restricted peptides

TABLE 2e listing of the main CDR3 sequences of CTL clones specific for HLA-A03: 01 restriction peptides

Induction of FOXM 1-and UBE 2T-derived peptide-specific CTL having cytotoxic activity against cancer cells

CTL clones specific for peptides derived from FOXM1 and UBE2T were induced (references 19-20; incorporated by reference in their entirety). Highly immunogenic FOXM1 and UBE 2T-derived short peptides (e.g., IYTWIEDHF (SEQ ID NO:3) and RYPFEPPQI (SEQ ID NO:13), respectively) were identified by an Interferon (IFN) - γ Enzyme Linked Immunospot (ELISPOT) assay, which induced HLA-A24: 02 restricted CTLs from PBMCs of healthy donors. After obtaining CTL clones by limiting dilution, it was confirmed that these FOXM1 and UBE 2T-specific CTLs produced IFN- γ when exposed to HLA-a 24: 02-expressing antigen-presenting C1R cells (C1R-a24 cells) pulsed with specific peptides, whereas no or low IFN- γ production was detected in the absence of peptide stimulation to C1R-a24 cells (fig. 1A), indicating that the established FOXM1 and UBE 2T-specific CTLs specifically recognize HLA-a 24: 02-restricted peptides.

After examining the FOXM1 and UBE2T protein levels in cancer cell lines by western blot analysis (fig. 4), the cytotoxic activity of FOXM1 and UBE 2T-specific CTLs against several cancer cell lines was examined by a time-lapse recording system. FOXM1 and UBE 2T-specific CTLs showed very potent cytotoxic activity against SW480 cells expressing high levels of HLA-A24 and FOXM1 and UBE2T proteins. Little cytotoxicity was observed against HLA-a24 negative cancer cell lines HCC1143 and BT549 cells (fig. 1B and 1C). The results clearly indicate HLA-restricted cytotoxic activity of antigen-specific T cells against cancer cells.

Production of FOXM1 and UBE2T specific TCR engineered T cells

Subsequently, the TCRA and TCRB chains of these FOXM1 and UBE 2T-specific CTLs were sequenced by TCR lineage analysis with next generation sequencing (fig. 2A). Both CTL clones showed a monoclonal TCR lineage (fig. 2A). Dominant TCRA and TCRB CDR3 clonotypes were identified by DNA sequencing against FOXM1-CTL (CACPIMWGSNYKLTF (SEQ ID NO:49) and CASSLRVHEQYF (SEQ ID NO:50)) and UBE2T-CTL (CAMREGRNFNKFYF (SEQ ID NO:69) and CASSLSGGPNEQFF (SEQ ID NO: 70)). The cDNA information was used to construct vectors expressing the TCR, cloned into lentiviral vectors, and to generate TCR-engineered T cells that recognize FOXM1 and UBE 2T. Transduction efficiency was measured by TCRv β specific antibodies (representative staining data is shown in figure 2B). For the assay, only TCR-transduced cells were transduced.

Cytotoxic activity of FOXM1 and UBE2T specific TCR engineered T cells

It was then assessed whether the TCR-engineered T cells killed cancer cells as the original CTL clones, as shown in fig. 1B and 1C. TCR-engineered T cells directed against FOXM1 and UBE2T exerted significant killing on HLA-a24 positive SW480 cells and reduced cell viability by 47.5% and 39.3% during the initial five hours, but had no effect on HLA-a24 negative HCC1143 cells (fig. 3A-3D). TCR-engineered T cells showed peptide-specific IFN- γ production in the ELISPOT assay when co-cultured with C1R-a24 cells pulsed with the corresponding peptide (fig. 3E and 3F).

Reference to the literature

The following references are incorporated by reference in their entirety, some of which are cited above by number.

1.Postow MA，Callahan MK，Wolchok JD.Immune Checkpoint Blockade inCancer Therapy.J Clin Oncol.2015；33：1974-82.

2.Yarchoan M，Johnson BA 3rd，Lutz ER，Laheru DA，Jaffee EM.Targetingneoantigens to augment antitumour immunity.Nat Rev Cancer.2017；17：209-222.

3.Rizvi NA，Hellmann MD，Snyder A，Kvistborg P，Makarov V，Havel JJ，Lee W，Yuan J，Wong P，Ho TS，Miller ML，Rekhtman N，Moreira AL，Ibrahim F，Bruggeman C，Gasmi B，Zappasodi R，Maeda Y，Sander C，Garon EB,Merghoub T，Wolchok JD，Schumacher TN，Chan TA.Cancer immunology.Mutational landscape determinessensitivity to PD-1 blockade in non-small cell lung cancer.Science.

2015；348：124-8.

4.Topalian SL，Hodi FS，Brahmer JR，Gettinger SN，Smith DC，McDermott DF，Powderly JD，Carvajal RD，Sosman JA，Atkins MB，Leming PD，Spigel DR，Antonia SJ，Horn L，Drake CG，Pardoll DM，Chen L，Sharfman WH，Anders RA，Taube JM，McMiller TL，Xu H，Korman AJ，Jure-Kunkel M，Agrawal S，McDonald D，Kollia GD，Gupta A，WiggintonJM，Sznol M.Safety，activity，and immune correlates of anti-PD-1 antibody incancer.N Engl J Med.2012；366：2443-54.

5.Rooney MS，Shukla SA，Wu CJ，Getz G，Hacohen N.Molecular and geneticproperties of tumors associated with local immune cytolytic activity.Cell.

2015；160：48-61.

6.Tumeh PC，Harview CL，Yearley JH，Shintaku IP，Taylor EJ，Robert L，Chmielowski B，Spasic M，Henry G，Ciobanu V，West AN，Carmona M，Kivork C，Seja E，Cherry G，Gutierrez AJ，Grogan TR，Mateus C,Tomasic G，Glaspy JA，Emerson RO，Robins H，Pierce RH，Elashoff DA，Robert C，Ribas A.PD-1 blockade inducesresponses by inhibiting adaptive immune resistance.Nature.2014；515：568-71.

7.Schalper KA.Velcheti V，Carvajal D，Wimberly H，Brown J，Pusztai L，RimmDL.In situ tumor PD-L1 mRNA expression is associated with increased TILs andbetter outcome in breast carcinomas.Clin Cancer Res.2014；20：2773-82.

8.Pardoll DM.The blockade of immune checkpoints in cancerimmunotherapy.Nat Rev Cancer.2012；12：252-64.

9.

S，Pasetto A，Helman SR，Gartner JJ，Prickett TD，Howie B，RobinsHS，Robbins PF，Klebanoff CA，Rosenberg SA，Hinrichs CS.Landscape of immunogenictumor antigens in successful immunotherapy of virally induced epithelialcancer.

Science.2017；356：200-205.

10.Coulie PG，Van den Eynde BJ，van der Bruggen P，Boon T.Tumourantigens recognized by T lymphocytes：at the core of cancer immunotherapy.NatRev Cancer.2014；14：135-46.

11.Yoshitake Y，Fukuma D，Yuno A，Hirayama M，Nakayama H，Tanaka T，NagataM，Takamune Y，Kawahara K，Nakagawa Y，Yoshida R，Hirosue A，Ogi H，Hiraki A，Jono H，Hamada A，Yoshida K，Nishimura Y，Nakamura Y，Shinohara M.Phase IIclinical trialof multiple peptide vaccination for advanced head and neck cancer patientsrevealed induction of immune responses and improved OS.Clin Cancer Res.

2015；21：312-21.

12.Hazama S，Nakamura Y，Tanaka H，Hirakawa K，Tahara K，Shimizu R，OzasaH，Etoh R，Sugiura F，Okuno K，Furuya T，Nishimura T，Sakata K，Yoshimatsu K，Takenouchi H，Tsunedomi R，Inoue Y，Kanekiyo S，Shindo Y，Suzuki N，Yoshino S，Shinozaki H，Kamiya A，Furukawa H，Yamanaka T，Fujita T，Kawakami Y，Oka M.A phaseII study of five peptides combination with oxaliplatin-based chemotherapy asa first-line therapy for advanced colorectal cancer(FXV study).J Transl Med.

2014；12：108.

13.Kono K，Iinuma H，Akutsu Y，Tanaka H，Hayashi N，Uchikado Y，Noguchi T，Fujii H，Okinaka K，Fukushima R，Matsubara H，Ohira M，Baba H，Natsugoe S，Kitano S，Takeda K，Yoshida K，Tsunoda T，Nakamura Y.Multicenter，phase II clinical trialof cancer vaccination for advanced esophageal cancer with three peptidesderived from novel cancer-testis antigens.J Transl Med.2012；10：141.

14.Tran E，Turcotte S，Gros A，Robbins PF，Lu YC，Dudley ME，Wunderlich JR，Somerville RP，Hogan K，Hinrichs CS，Parkhurst MR，Yang JC,Rosenberg SA.Cancerimmunotherapy based on mutation-specific CD4+T cells in a patient withepithelial cancer.Science.2014；344：641-5.

15.Tran E Robbins PF，Lu YC，Prickett TD，Gartner JJ，Jia L，Pasetto A，Zheng Z，Ray S，Groh EM，Kriley IR，Rosenberg SA.T-Cell Transfer TherapyTargeting Mutant KRAS in Cancer.N Engl J Med.2016；375：2255-2262.

16.Johnson LA，Morgan RA，Dudley ME，Cassard L，Yang JC，Hughes MS，KammulaUS，Royal RE，Sherry RM，Wunderlich JR，Lee CC，Restifo NP，Schwarz SL，Cogdill AP，Bishop RJ，Kim H，Brewer CC，Rudy SF，VanWaes C，Davis JL，Mathur A，Ripley RT，Nathan DA，Laurencot CM，Rosenberg SA.Gene therapy with human and mouse T-cellreceptors mediates cancer regression and targets normal tissues expressingcognate antigen.Blood.2009；114：535-46.

17.Robbins PF，Morgan RA，Feldman SA，Yang JC，Sherry RM，Dudley ME，Wunderlich JR，Nahvi AV，Helman LJ，Mackall CL，Kammula US，Hughes MS，Restifo NP，Raffeld M，Lee CC，Levv CL，Li YF，El-Gamil M，Schwarz SL，Laurencot C，RosenbergSA.Tumor regression in patients with metastatic synovial cell sarcoma andmelanoma using genetically engineered lymphocytes reactivewith NY-ESO-1.JClin Oncol.2011；29：917-24.

18.Leisegang M，Engels B，Schreiber K，Yew PY，Kiyotani K，Idel C，Arina A，Duraiswamy J，Weichselbaum RR，Uckert W，Nakamura Y，Schreiber H.Eradication ofLarge Solid Tumors by Gene Therapy with a T-Cell Receptor Targeting a SingleCancer-Specific Point Mutation.Clin Cancer Res.2016；22：2734-43.

19.Osawa R，Tsunoda T，Yoshimura S，Watanabe T，Miyazawa M，Tani M，TakedaK，Nakagawa H，Nakamura Y，Yamaue H.Identification of HLA-A24-restricted novel TCell epitope peptides derived from P-cadherin and kinesin family member 20A.JBiomed Biotechnol.2012；2012：848042.

20.Yoshimura S，Tsunoda T，Osawa R，Harada M，Watanabe T，Hikichi T，Katsuda M，Miyazawa M，Tani M，Iwahashi M，Takeda K，Katagiri T，Nakamura Y，YamaueH.Identification of an HLA-A2-restricted epitope peptide derived fromhypoxia-inducible protein 2(HIG2).PLoS One.2014；9：e85267.

21.Rapoport AP,Stadtmauer EA，Binder-Scholl GK，Goloubeva O，Vogl DT，Lacey SF，Badros AZ，Garfall A，Weiss B，Finklestein J，Kulikovskaya I，Sinha SK，Kronsberg S，Gupta M，Bond S，Melchiori L，Brewer JE，Bennett AD，Gerry AB，PumphreyNJ，Williams D，Tayton-Martin HK，Ribeiro L，Holdich T，Yanovich S，Hardy N，YaredJ，Kerr N，Philip S，Westphal S，Siegel DL，Levine BL，Jakobsen BK，Kalos M，JuneCH.NY-ESO-l-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma.Nat Med.2015；21：914-921.

22.van der Bruggen P，Traversari C，Chomez P，Lurquin C，De Plaen E，Vanden Eynde B，Knuth A，Boon T.A gene encoding an antigen recognized by cytolyticT lymphocytes on a human melanoma.Science.1991；254：1643-7.

23.lshida Y，Agata Y，Shibahara K，Honjo T.Induced expression of PD-1，anovel member of the immunoglobulin gene superfamily，upon programmed celldeath.EMBO J.1992；11：3887-95.

24.Krummel MF，Allison JP.CD28 and CTLA-4 have opposing effects on theresponse of T cells to stimulation.J Exp Med.1995；182：459-65.

25.Guo C，Manjili MH，Subjeck JR，Sarkar D，Fisher PB，Wang XY.Therapeuticcancer vaccines：past，present，and future Adv Cancer Res.2013；119：421-75.

26.Hinrichs CS，Rosenberg SA.Exploiting the curative potential ofadoptive T-cell therapy for cancer.Immunol Rev.2014；257：56-71.

27.Dudley ME，Wunderlich JR，Robbins PF，Yang JC，Hwu P，SchwartzentruberDJ，Topalian SL，Sherry R，Restifo NP，Hubicki AM，Robinson MR，Raffeld M，Duray P，Seipp CA，Rogers-Freezer L，Morton KE，Mavroukakis SA，White DE，RosenbergSA.Cancer regression and autoimmunity in patients after clonal repopulationwith antitumor lymphocytes.Science.2002；298：850-4.

28.Rosenberg SA，Yang JC，Sherry RM，Kammula US，Hughes MS，Phan GQ，CitrinDE，Restifo NP，Robbins PF，Wunderlich JR，Morton KE，Laurencot CM，Steinberg SM,White DE,Dudley ME.Durable complete responses in heavily pretreated patientswith metastatic melanoma using T-cell transfer immunotherapy.Clin CancerRes.2011；17：4550-7.

29.Hodi FS，O′Day SJ，McDermott DF，Weber RW，Sosman JA，Haanen JB，Gonzalez R，Robert C，Schadendorf D，Hassel JC，Akerley W，van den Eertwegh AJ，Lutzky J，Lorigan P，Vaubel JM，Linette GP，Hogg D，Ottensmeier CH，Lebbé C，PeschelC，Quirt I，Clark JI，Wolchok JD，Weber JS，Tian J，Yellin MJ，Nichol GM，Hoos A，UrbaWJ.Improved survival with ipilimumab in patients with metastatic melanoma.NEngl J Med.2010；363：711-23.

30.E，Toebes M，Kelderman S，van Buuren MM，Yang W，van Rooij N，Donia M，

ML，Lund-Johansen F，Olweus J，Schumacher TN.Targeting of cancerneoantigens with donor-derived T cell receptor repertoires.Science.2016；352：1337-41.

31.Bektas N，Haaf At，Veeck J，Wild PJ，Lüscher-FirzlaffJ，Hartmann A,Knüchel R，Dahl E.Tight correlation between expression of the Forkheadtranscription factor FOXM1 and HER2 in human breast cancer.BMC Cancer.2008；8：42.

32.Weng W，Okugawa Y，Toden S，Toiyama Y，Kusunoki M，Goel A.FOXM1 andFOXQ1 Are Promising Prognostic Biomarkers and Novel Targets of Tumor-Suppressive miR-342 in Human Colorectal Cancer.Clin Cancer Res.2016；22：4947-4957.

33.Wen M，Kwon Y，Wang Y，Mao JH，Wei G.Elevated expression of UBE2Texhibits oncogenic properties in human prostate cancer.Oncotarget.2015；6：25226-39.

34.Yu H，Xiang P，Pan Q，Huang Y，Xie N，Zhu W.Ubiquitin-ConjugatingEnzyme E2T is an Independent Prognostic Factor and Promotes Gastric CancerProgression.

Tumour Biol.2016；37：11723-11732.

35.Maude SL，Frey N，Shaw PA，Aplenc R，Barrett DM，Bunin NJ，Chew A，Gonzalez VE，Zheng Z，Lacey SF，Mahnke YD，Melenhorst JJ，Rheingold SR，Shen A，Teachey DT，Levine BL，June CH，Porter DL，Grupp SA.Chimeric antigen receptor Tcells for sustained remissions in leukemia.N Engl J Med.2014；371：1507-17.

36.Jackson HJ，Rafiq S，Brentjens RJ.Driving CAR T-cells forwardNat RevClin Oncol.2016；13：370-83.

37.Uchida N，Tsunoda T，Wada S，Furukawa Y，Nakamura Y，Tahara H.Ringfinger protein 43 as a new target for cancer immunotherapy.Clin CancerRes.2004；10：8577-86.

38.Watanabe T，Suda T，Tsunoda T，Uchida N，Ufa K，Kato T，HasegawaS，SatohS，Ohgi S，Tahara H，Furukawa Y，Nakamura Y.Identification of immunoglobulinsupeffamily 11(IGSF11)as a novel target for cancer immunotherapy ofgastrointestinal and hepatocellular carcinomas.Cancer Sci.2005；96：498-506.

39.Suda T，Tsunoda T，Uchida N，Watanabe T，Hasegawa S，Satoh S，Ohgi S，Furukawa Y，Nakamura Y，Tahara H.Identification ofsecernin 1 as a novelimmunotherapy target for gastric cancer using the expression profiles of cDNAmicroarray.Cancer Sci.2006；97：411-9.

40.Riddell SR，Elliott M，Lewinsohn DA，Gilbert MJ，Wilson L，Manley SA，Lupton SD，Overell RW，Reynolds TC，Corey L，Greenberg PD.T-cell mediatedrejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients.Nat Med.1996；2：216-23.

41.Fang H，Yamaguchi R，Liu X，Daigo Y，Yew PY，Tanikawa C，Matsuda K，ImotoS，Miyano S，Nakamura Y.Quantitative T cell repertoire analysis by deep cDNAsequencing of T cell receptor αandβ chains using next-generation sequencing(NGS).Oncoimmunology.2015；3：e968467.

42.Engels B，Chervin AS，Sant AJ，Kranz DM，Schreiber H.Long-termpersistence of CD4(+)but rapid disappearance of CD8(+)T cells expressing anMHC classI-restricted TCR of nanomolar affinity.Mol Ther.2012；20：652-60.

Sequence listing

<110> UNIVERSITY OF Chicago (THE UNIVERSITY OF CHICAGO)

<120> screening of T lymphocytes against cancer-specific antigens

<130>UCHI-35360/WO-1/ORD

<150>US 62/569,215

<151>2017-10-06

<160>132

<170>PatentIn version 3.5

<210>1

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>1

Glu Trp Ala Ala Ala Met Asn Ala Glu Phe

1 5 10

<210>2

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>2

Asp Tyr Leu Asn Glu Trp Gly Ser Arg Phe

1 5 10

<210>3

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>3

Ile Tyr Thr Trp Ile Glu Asp His Phe

1 5

<210>4

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>4

Lys Trp Leu Ile Ser Pro Val Lys Ile

1 5

<210>5

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>5

Leu Tyr Leu Lys Leu Leu Pro Tyr Val

1 5

<210>6

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>6

Lys Val Tyr Leu Arg Val Arg Pro Leu Leu

1 5 10

<210>7

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>7

Glu Tyr Cys Pro Gly Gly Asn Leu Phe

1 5

<210>8

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>8

Glu Trp Ala Asp Leu Ser Phe Pro Phe

1 5

<210>9

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>9

Asn Ser Gln Pro Val Trp Leu Cys Leu

1 5

<210>10

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>10

Ala Tyr Asp Ile Gly Leu Phe Ala Tyr Phe

1 5 10

<210>11

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>11

Gln Tyr Cys Phe Glu Cys Asp Cys Phe

1 5

<210>12

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>12

Ser Tyr Gln Lys Val Ile Glu Leu Phe Ser

1 5 10

<210>13

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>13

Arg Tyr Pro Phe Glu Pro Pro Gln Ile

1 5

<210>14

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>14

Arg Tyr Leu Ser Ala Gly Pro Thr Leu

1 5

<210>15

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>15

Ser Tyr Gly Val Leu Leu Trp Glu Ile

1 5

<210>16

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>16

Arg Phe Val Pro Asp Gly Asn Arg Ile

1 5

<210>17

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>17

Gly Tyr Ser Asn Thr Ala Thr Glu Trp

1 5

<210>18

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>18

Ile Tyr Arg Val Ser Phe Thr Asp Phe

1 5

<210>19

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>19

Ser Ile Ala Gly Gly Gln Ile Leu Ser Val

1 5 10

<210>20

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>20

Ser Leu Gln Lys Ala Leu His His Leu

1 5

<210>21

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>21

Thr Leu Leu Ser Ile Tyr Ile Asp Gly Val

1 5 10

<210>22

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>22

Val Val Cys Ser Lys Leu Thr Glu Val

1 5

<210>23

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>23

Ile Leu Val Val Cys Gly Tyr Ile Thr Val

1 5 10

<210>24

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>24

Leu Leu Ile Gly Ser Thr Ser Tyr Val

1 5

<210>25

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>25

Ala Leu Val Trp Leu Ile Asp Cys Ile

1 5

<210>26

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>26

Leu Leu Phe Asp Glu Tyr His Lys Leu

1 5

<210>27

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>27

Tyr Ile Tyr Asp Leu Phe Val Pro Val Ser

1 5 10

<210>28

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>28

Arg Ile Ile Cys Glu Ala His Lys Val

1 5

<210>29

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>29

Phe Gln Asn Ser Pro Pro Ala Ser Val

1 5

<210>30

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>30

Arg Leu Ala Phe Asp Ile Met Arg Val

1 5

<210>31

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>31

Thr Thr Met Trp Arg Ala Thr Thr Thr Val

1 5 10

<210>32

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>32

Ala Leu Tyr Gly Arg Ala Leu Arg Val

1 5

<210>33

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>33

Ile Thr Asp Gln Tyr Ile Tyr Met Val

1 5

<210>34

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>34

Gly Leu Gly Pro Gly Leu Leu Gly Val

1 5

<210>35

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>35

Lys Cys Leu Asp Phe Ser Leu Val Val

1 5

<210>36

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>36

Lys Thr Lys Arg Leu Asn Glu Leu Lys

1 5

<210>37

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>37

Met Ser Gln Asn Val Asp Met Pro Lys

1 5

<210>38

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>38

Val Val Ser Thr Ser Leu Glu Asp Lys

1 5

<210>39

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>39

Ser Thr Ser Phe Glu Ile Ser Arg Asn Lys

1 5 10

<210>40

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>40

Glu Val Leu His Met Ile Tyr Met Arg

1 5

<210>41

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>41

Trp Thr Ile His Pro Ser Ala Asn Arg

1 5

<210>42

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>42

Glu Phe Thr Gln Tyr Trp Ala Gln Arg

1 5

<210>43

<211>9

<212>PRT

<213> Intelligent (Homo sapiens)

<400>43

Ile Tyr Val Tyr Val Gln Asp Tyr Arg

1 5

<210>44

<211>10

<212>PRT

<213> Intelligent (Homo sapiens)

<400>44

Ala Val Val Asn Val Thr Tyr Ser Ser Lys

1 5 10

<210>45

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>45

Cys Ala Ala Leu Asp Ser Asn Tyr Gln Leu Ile Trp

1 5 10

<210>46

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>46

Cys Ala Ser Ser Lys Asn Gly Gly Ser Tyr Lys Asn Glu Gln Phe Phe

1 5 10 15

<210>47

<211>17

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>47

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

1 5 10 15

Phe

<210>48

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>48

Cys Ala Ser Ser Pro Leu Ile Asp Thr Asn Gln Pro Gln His Phe

1 5 10 15

<210>49

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>49

Cys Ala Cys Pro Ile Met Trp Gly Ser Asn Tyr Lys Leu Thr Phe

1 5 10 15

<210>50

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>50

Cys Ala Ser Ser Leu Arg Val His Glu Gln Tyr Phe

1 5 10

<210>51

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>51

Cys Ala Met Arg Glu Ala Leu Ser Tyr Asn Thr Asp Lys Leu Ile Phe

1 5 10 15

<210>52

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>52

Cys Ala Ser Arg Glu Tyr Lys Asn Glu Gln Phe Phe

1 5 10

<210>53

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>53

Cys Ala Pro Ser Gly Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe

1 5 10 15

<210>54

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>54

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

1 5 10

<210>55

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>55

Cys Ala ValIle Gly Gly Gly Ser Asn Tyr Gln Leu Ile Trp

1 5 10

<210>56

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>56

Cys Ala Ser Ser Pro Ser Pro Leu Asp Trp Glu Thr Gln Tyr Phe

1 5 10 15

<210>57

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>57

Cys Ala Gly Arg Asn Ser Gly Thr Tyr Lys Tyr Ile Phe

1 5 10

<210>58

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>58

Cys Ala Ser Ser Leu Gly Thr Pro Lys Glu Thr Gln Tyr Phe

1 5 10

<210>59

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>59

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

1 5 10 15

<210>60

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>60

Cys Ala Ser Arg Gln Gly Gly Thr Pro Leu His Phe

1 5 10

<210>61

<211>11

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>61

Cys Ala Val Arg Arg Gly Asn Gln Phe Tyr Phe

1 5 10

<210>62

<211>17

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>62

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

1 5 10 15

Phe

<210>63

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>63

Cys Ala Val Asp Met Trp Ser Gln Gly Asn Leu Ile Phe

1 5 10

<210>64

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>64

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

1 5 10

<210>65

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>65

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

1 5 10 15

<210>66

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>66

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

1 5 10 15

<210>67

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>67

Cys Ala Val Glu Ala Gly Tyr Ser Thr Leu Thr Phe

1 5 10

<210>68

<211>7

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>68

Cys Ala Ser Gly Ala Phe Phe

15

<210>69

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>69

Cys Ala Met Arg Glu Gly Arg Asn Phe Asn Lys Phe Tyr Phe

1 5 10

<210>70

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>70

Cys Ala Ser Ser Leu Ser Gly Gly Pro Asn Glu Gln Phe Phe

1 5 10

<210>71

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>71

Cys Ala Met Arg Glu Val Thr Gly Asn Gln Phe Tyr Phe

1 5 10

<210>72

<211>17

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>72

Cys Ala Ser Ser Gln Lys Ser Gly Pro Leu Lys Arg Gln Pro Gln His

1 5 10 15

Phe

<210>73

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>73

Cys Ala Val Arg Ala Gly Ala Gly Asn Met Leu Thr Phe

1 5 10

<210>74

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>74

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

1 5 10

<210>75

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>75

Cys Ala Met Ser Gln Tyr Gly Asn Lys Leu Val Phe

1 5 10

<210>76

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>76

Cys Ala Ser Ser Glu Ile Arg Asn Ala Tyr Glu Gln Tyr Phe

1 5 10

<210>77

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>77

Cys Ala Val Arg Gly Gly Ser Asn Tyr Gln Leu Ile Trp

1 5 10

<210>78

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>78

Cys Ala Ser Ser Ser Ser Ser Gly Thr Pro Trp Asn Glu Gln Phe Phe

1 5 10 15

<210>79

<211>11

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>79

Cys Ala Thr Val Asn Asp Tyr Lys Leu Ser Phe

1 5 10

<210>80

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>80

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

1 5 10 15

<210>81

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>81

Cys Leu Val Gly Asp Arg Gln Ala Gly Thr Ala Leu Ile Phe

1 5 10

<210>82

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>82

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

1 5 10 15

<210>83

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>83

Cys Ala Met Arg Glu Arg Ser Gly Gly Ser Tyr Ile Pro Thr Phe

1 5 10 15

<210>84

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>84

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

1 5 10

<210>85

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>85

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

1 5 10 15

<210>86

<211>17

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>86

Cys Ala Ser Ser Leu Phe Ala Gln Ser Ser Tyr Lys Asn Glu Gln Phe

1 5 10 15

Phe

<210>87

<211>18

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>87

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

1 5 10 15

Thr Phe

<210>88

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>88

Cys Ala Ser Ser Leu Leu Lys Asn Thr Glu Ala Phe Phe

1 5 10

<210>89

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>89

Cys Ala Val His Asp Asn Tyr Gly Gln Asn Phe Val Phe

1 5 10

<210>90

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>90

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

1 5 10

<210>91

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>91

Cys Ala Thr Ile Arg Lys Leu Thr GlyAsn Gln Phe Tyr Phe

1 5 10

<210>92

<211>18

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>92

Cys Ala Ser Ser Arg Trp Lys Gly Gln Gly Leu His Thr Gly Glu Leu

1 5 10 15

Phe Phe

<210>93

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>93

Cys Ala Met Arg Glu Gly Gln Ala Gly Thr Ala Leu Ile Phe

1 5 10

<210>94

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>94

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

1 5 10

<210>95

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>95

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

1 5 10

<210>96

<211>11

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>96

Cys Ala Ser Ser Ser Asp Arg Thr Ala Phe Phe

1 5 10

<210>97

<211>11

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>97

Cys Ala Val Asn Glu Pro Tyr Lys Leu Ser Phe

1 5 10

<210>98

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>98

Cys Ala Ser Ser Phe Thr Lys Asn Glu Gln Tyr Phe

1 5 10

<210>99

<211>11

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>99

Cys Ala Met Arg Thr Gly Gly Lys Leu Ile Phe

1 5 10

<210>100

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>100

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

1 5 10 15

<210>101

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>101

Cys Ala Glu Asn Leu Ala Arg Gly Gly Asn Lys Leu Thr Phe

1 5 10

<210>102

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>102

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

1 5 10

<210>103

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>103

Cys Ala Gly Cys Pro Phe Arg Asp Asp Lys Ile Ile Phe

1 5 10

<210>104

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>104

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

1 5 10

<210>105

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>105

Cys Ala Leu Asn Asn Ala Gly Asn Met Leu Thr Phe

1 5 10

<210>106

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>106

Cys Ala Ser Thr Leu Arg Gly Trp Ser Thr Gly Glu Leu Phe Phe

1 5 10 15

<210>107

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>107

Cys Ile Val Arg Ala Tyr Tyr Gly Gly Ala Thr Asn Lys Leu Ile Phe

1 5 10 15

<210>108

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>108

Cys Ala Ser Ser Gln Ala Arg Met Gly Asn Gly Glu Leu Phe Phe

1 5 10 15

<210>109

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>109

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

1 5 10

<210>110

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>110

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

1 5 10 15

<210>111

<211>13

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>111

Cys Ala Thr Asp Phe Asn Ala Gly Asn Met Leu Thr Phe

1 5 10

<210>112

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>112

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

1 5 10 15

<210>113

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>113

Cys Ala Ala Ser Gly Arg Ala Gly Ala Asn Asn Leu Phe Phe

1 5 10

<210>114

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>114

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

1 5 10 15

<210>115

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>115

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

1 5 10

<210>116

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>116

Cys Ala Ser Ser Leu Gly Ile Asp Ser Gly Tyr Gly Tyr Thr Phe

1 5 10 15

<210>117

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>117

Cys Ala Asp Val Ser Arg Asp Asp Lys Ile Ile Phe

1 5 10

<210>118

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>118

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

1 5 10

<210>119

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>119

Cys Ala Met Arg Glu Gly Arg Ser Glu Val Ile Phe

1 5 10

<210>120

<211>11

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>120

Cys Ala Ser Ser Ser Tyr Asn Glu Gln Phe Phe

1 5 10

<210>121

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>121

Cys Ala Glu Asn Gln Lys Gly Gly Lys Leu Ile Phe

1 5 10

<210>122

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>122

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

1 5 10 15

<210>123

<211>11

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>123

Cys Ala Gly Gln Asp Asn Asn Asp Met Arg Phe

1 5 10

<210>124

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>124

Cys Ala Ser Thr Ala Trp Gly Ala Asn Thr Glu Ala Phe Phe

1 5 10

<210>125

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>125

Cys Ala Val Asn Ala Asn Thr Asp Lys Leu Ile Phe

1 5 10

<210>126

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>126

Cys Ser Ala Trp Glu Arg Thr Ser Leu Phe Glu Gln Tyr Phe

1 5 10

<210>127

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>127

Cys Leu Val Gly Arg Asp Asn Ala Gly Asn Met Leu Thr Phe

1 5 10

<210>128

<211>12

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>128

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

1 5 10

<210>129

<211>14

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>129

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

1 5 10

<210>130

<211>16

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>130

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

1 5 10 15

<210>131

<211>18

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>131

Cys Ala Met Ser Ala Thr Glu Gly Arg Asp Asn Tyr Gly Gln Asn Phe

1 5 10 15

Val Phe

<210>132

<211>15

<212>PRT

<213> Artificial sequence (Artificial sequence)

<220>

<223> synthetic peptide

<400>132

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

1 5 10 15