Selection of t cell receptors

Abstract

To provide methods to separately isolate antigen-binding T cells and antigen-activated T cells derived from a starting population of peripheral blood mononuclear cells, and to identify overlapping T cell receptor clonotypes.SOLUTION: Antigens include personal and shared neoantigens, as well as cancer-testis antigens. T cell receptor clonotypes can further be used to develop cancer treatment modalities. Certain embodiments can provide, for example, a method for selection of T cell receptor clonotypes (for example, rare T cell receptor clonotypes, such as T cell receptor clonotypes with a frequency of less than 1 per 10,000,000 T cells in a sample of PBMCs).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 812,572, filed Mar. 1, 2019, which is hereby incorporated by reference in its entirety. Sequence Listing

[0002] This application is electronically filed in ASCII format and includes a sequence listing which is hereby incorporated by reference in its entirety. A copy of the ASCII, created on Feb. 26, 2020, is 14560-001-228 _ SEQ _ named LISTING.txt, which is 106,016 bytes in size.

[0003] The present disclosure relates to the identification of antigen-specific T cell receptors.

Background Art

[0004] Cancer is associated with a failure of the immune surveillance mechanism that provides T cells with the ability to detect and destroy clones of transformed cells that have the potential to grow into tumors. Research has focused on developing methods for engineering helper T cells that recognize cancer-specific antigens and can provide an effective functional response. Current approaches involve efforts to culture and isolate antigen-specific and functionally responsive T cells, from which the T cell receptor sequences are identified and can be used to engineer therapeutically effective T cell lines.

[0005] Such T cells are usually rare in starting peripheral blood mononuclear cell (PBMC) samples - in some cases less than 1 in 10,000,000. As a result, current approaches often employ extensive stimulation (e.g., in vitro priming), proliferation, and enrichment steps, all of which are time-consuming, costly, and have a low success rate. The low success rate is known to be due to the fact that T cell stimulation downregulates the expression of the T cell receptor, making its detection more difficult. In addition, T cells are stimulated by the target antigen at concentrations that are thought to be much higher than the concentrations expressed by cancer cells, resulting in the selection of T cell receptors that cannot function even when the antigen is at a physiologically appropriate concentration.

[0006] The present disclosure overcomes such disadvantages, in particular, by providing a method for identifying rare antigen-specific and functional T cells that avoids one or more limitations of in vitro priming and enables the pre-identification of T cell receptor candidates for the development of therapeutically effective T cell lines when the antigen is at a physiologically appropriate concentration. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0007] Certain embodiments can provide, for example, a method for selecting a T cell receptor clonotype (e.g., a rare T cell receptor clonotype, such as a T cell receptor clonotype having a frequency of less than 1 per 10,000,000 T cells in a PBMC sample). In certain embodiments, for example, the method can include analyzing a mixture of T cells to identify antigen-binding T cells and antigen-activated T cells for a predetermined type of antigen (e.g., a neoantigen selected from a library of shared tumor neoantigens or individualized neoantigens selected from individual tumor cells). In certain embodiments, for example, the method can include identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the antigen-activated T cells.

[0008] A. In certain embodiments, for example, the analyzing step may include analyzing a first portion of the mixture to identify antigen-binding T cells and separately analyzing a second portion of the mixture to identify antigen-activated T cells. In certain embodiments, for example, analyzing the first portion of the mixture may include detecting one or more T cells bound to a P-loaded major histocompatibility complex (MHC) protein, where P is a predetermined type of antigen. In certain embodiments, for example, the P-loaded MHC may be bound to magnetic beads. In certain embodiments, for example, detecting one or more T cells bound to the P-loaded MHC protein may include isolating one or more T cells bound to the P-loaded MHC protein by magnetic separation. In certain embodiments, for example, the P-loaded MHC protein may be bound to a fluorophore. In certain embodiments, for example, one or more T cells bound to the P-loaded MHC protein may be detected and isolated by fluorescence flow cytometry. In certain embodiments, for example, detecting one or more T cells bound to the P-loaded MHC protein may include passing one or more T cells bound to the P-loaded MHC protein through a fluorescence flow cytometry device. In certain embodiments, for example, the MHC protein may be an MHC class I protein. In certain embodiments, for example, the P-loaded MHC protein may be present in a P-loaded MHC protein multimer. In certain embodiments, for example, separately analyzing the second portion of the mixture to identify antigen-activated T cells may include detecting one or more T cells expressing one or more activation markers. In certain embodiments, for example, detecting one or more T cells expressing one or more activation markers may include isolating one or more T cells expressing one or more activation markers by magnetic separation. In certain embodiments, for example, detecting one or more T cells expressing one or more activation markers may include passing one or more T cells expressing one or more activation markers through a fluorescence flow cytometry device.In certain embodiments, for example, the method may exclude in vitro priming.

[0009] B. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on the tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen) observed in some tumors across multiple subjects. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncoviral protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on the tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor.In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determined using one or more of the machine learning methods, software, and / or systems disclosed in the INCORPORATED REFERENCES.

[0010] C. In certain embodiments, for example, at least a portion of at least one T cell receptor sequence can include at least one T cell receptor clonotype. In certain embodiments, for example, at least a portion of at least one T cell receptor sequence can include at least one T cell receptor alpha chain, at least one T cell receptor beta chain, or at least a pair of T cell receptor alpha and T cell receptor beta chains. In certain embodiments, for example, the step of identifying can include sequencing at least one binding T cell at the single cell level. In certain embodiments, for example, the step of identifying can include sequencing at least one functional T cell at the single cell level. In certain embodiments, for example, at least a portion of at least one T cell receptor sequence can include at least one CDR3 sequence.

[0011] D. In certain embodiments, for example, at least one antigen-binding T cell and at least one antigen-activated T cell can together be less than 1000 T cells (e.g., less than 100, less than 10, less than 5, less than 3, or 2) per 1,000,000 T cells present in the mixture of T cells.

[0012] E. In certain embodiments, for example, the method can further include the step of preparing a mixture of T cells, including: i) isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs; and ii) expanding the at least one isolated T cell. In certain embodiments, for example, at least two T cells can bind to a predetermined type of antigen (i.e., at least one T cell can be at least two T cells), in which case, expanding can include polyclonally expanding at least two T cells. In certain embodiments, for example, at least one antigen-binding T cell and at least one antigen-activated T cell can together be less than 1000 T cells (e.g., less than 100, less than 10, less than 5, less than 3, or 2) per 10,000,000 T cells present in the population of PBMCs. In certain embodiments, for example, the mixture of T cells can be the product of in vitro priming.

[0013] Certain embodiments can provide, for example, a method for selecting shared receptor sequences within lymphocytes. In certain embodiments, for example, the method can include analyzing a mixture of lymphocytes to identify stimulated lymphocytes and costimulated lymphocytes for a predetermined type of antigen. In certain embodiments, for example, the method can include identifying at least a portion of at least one receptor sequence that is shared by at least one stimulated lymphocyte and at least one costimulated lymphocyte.

[0014] A. In certain embodiments, for example, a mixture of stimulated and co-stimulated lymphocytes can be T cells. In certain embodiments, for example, a mixture of stimulated and co-stimulated lymphocytes can be B cells. In certain embodiments, for example, a mixture of stimulated and co-stimulated lymphocytes can be natural killer cells.

[0015] B. In certain embodiments, for example, the analyzing step may include analyzing a first portion of the mixture to identify stimulated lymphocytes and separately analyzing a second portion of the mixture to identify co-stimulated lymphocytes. In certain embodiments, for example, analyzing the first portion of the mixture may include detecting one or more stimulated lymphocytes bound to a protein, where the protein comprises a predetermined type of antigen (e.g., can be incorporated into or complexed with it). In certain embodiments, for example, the protein may be bound to magnetic beads. In certain embodiments, for example, detecting one or more stimulated lymphocytes bound to the protein may include isolating one or more stimulated lymphocytes bound to the protein by magnetic separation. In certain embodiments, for example, the protein may be bound to a fluorophore. In certain embodiments, for example, one or more stimulated lymphocytes bound to the protein can be detected and isolated by fluorescence flow cytometry. In certain embodiments, for example, detecting one or more stimulated lymphocytes bound to the protein may include passing one or more stimulated lymphocytes bound to the protein through a fluorescence flow cytometry device. In certain embodiments, for example, separately analyzing the second portion of the mixture to identify co-stimulated lymphocytes may include detecting one or more stimulated lymphocytes expressing one or more markers. In certain embodiments, for example, detecting one or more stimulated lymphocytes expressing one or more markers may include isolating one or more stimulated lymphocytes expressing one or more markers by magnetic separation. In certain embodiments, for example, detecting one or more stimulated lymphocytes expressing one or more markers may include passing one or more stimulated lymphocytes expressing one or more markers through a fluorescence flow cytometry device. In certain embodiments, for example, the method may exclude priming with professional antigen-presenting cells (e.g., in vitro priming).

[0016] C. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on a tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on a tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determined using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0017] D. In certain embodiments, for example, at least a portion of at least one receptor sequence can include at least one receptor clonotype. In certain embodiments, for example, at least a portion of at least one receptor sequence can include at least one receptor alpha chain, at least one receptor beta chain, or at least a pair of receptor alpha and receptor beta chains. In certain embodiments, for example, the identifying step can include sequencing at least one of the stimulated lymphocytes at the single cell level. In certain embodiments, for example, the identifying step can include sequencing at least one of the co-stimulated lymphocytes at the single cell level. In certain embodiments, for example, at least a portion of at least one receptor sequence can include at least one antigen recognition sequence.

[0018] E. In certain embodiments, for example, at least one of the stimulated lymphocytes and at least one of the co-stimulated lymphocytes can together be less than 1000 T cells per 1,000,000 T cells (e.g., less than 100, less than 10, less than 5, less than 3, or 2) present in the mixture of lymphocytes.

[0019] F. In certain embodiments, for example, the method may further include the step of preparing a mixture of lymphocytes, which includes: i) isolating at least one lymphocyte that binds to a predetermined type of antigen from a population of PBMCs, and ii) expanding the at least one isolated lymphocyte. In certain embodiments, for example, at least two lymphocytes may be capable of binding to a predetermined type of antigen (i.e., the at least one lymphocyte may be at least two lymphocytes), in which case expanding may include polyclonally expanding the at least two lymphocytes. In certain embodiments, for example, at least one of the stimulated lymphocytes and at least one of the co-stimulated lymphocytes may be less than 1000 T cells (e.g., less than 100, less than 10, less than 5, less than 3, or 2) per 10,000,000 lymphocytes present in the population of PBMCs. In certain embodiments, for example, the mixture of lymphocytes may be a product primed (e.g., in vitro priming) using professional antigen-presenting cells.

[0020] Certain embodiments may provide, for example, a method for selecting a T cell receptor clonotype. In certain embodiments, for example, the method may include the step of analyzing a mixture of naive T cells to identify antigen-binding T cells and functional T cells for a predetermined type of antigen. In certain embodiments, for example, the method may include the step of identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the functional T cells.

[0021] Certain embodiments may provide, for example, a method for selecting a T cell receptor. In certain embodiments, for example, the method comprises the step of binding at least a first antigen-binding T cell to one of at least a first predetermined type of antigen, which may include contacting a first plurality of T cells (e.g., a first plurality of T cells containing at least the first antigen-binding T cell) with one of the first predetermined type of antigen. In certain embodiments, for example, the method comprises the step of activating at least a first functional T cell, which may include contacting a second plurality of T cells (e.g., a second plurality of T cells containing at least the first functional T cell) with a plurality of cells presenting one of at least a second predetermined type of antigen (e.g., a plurality of cells presenting a predetermined type of antigen at a physiologically appropriate concentration). In certain embodiments, for example, the method may comprise the step of identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0022] A. In certain embodiments, for example, a plurality of cells presenting at least one of a second predetermined type of antigen can present a plurality of predetermined types of antigens within a predetermined concentration range (or a single predetermined concentration value). In certain embodiments, for example, a plurality of cells presenting at least one of a second predetermined type of antigen can be prepared by pulsing a plurality of cells with an amount of a predetermined type of antigen (e.g., to form a plurality of P-loaded cells where P is a predetermined type of antigen). In certain embodiments, for example, a plurality of cells presenting at least one of a second predetermined type of antigen can be prepared by pulsing a plurality of cells with a solution containing a predetermined type of antigen for a predetermined period, and the solution contains the predetermined type of antigen at a concentration of from 0.000001 μM to 100 μM, such as from 0.000001 μM to 0.00001 μM, such as from 0.00001 μM to 0.0001 μM, 0.0001 μM to 0.001 μM, 0.001 to 0.01 μM, 0.01 to 0.1 μM, 0.0001 μM to 100 μM, 0.001 μM to 100 μM, 0.01 μM to 10 μM, 0.1 μM to 10 μM, 1 μM to 100 μM, 1 μM to 50 μM, 1 μM to 25 μM, 5 μM to 25 μM, 10 μM to 100 μM, or 10 μM to 30 μM. In certain embodiments, for example, the solution can contain the predetermined type of antigen at a concentration of less than 100 μM, such as less than 75 μM, less than 50 μM, less than 25 μM, less than 10 μM, or less than 1 μM. In any of the above embodiments, for example, the predetermined period can be from 1 hour to 36 hours, such as from 6 hours to 24 hours, 6 hours to 12 hours, 12 hours to 24 hours, or the predetermined period can be from 9 hours to 18 hours. In any of the above embodiments, for example, the predetermined period can be at least 1 hour, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, or the predetermined period can be at least 24 hours. In any of the above embodiments, for example, the predetermined period can be less than 168 hours, less than 72 hours, less than 36 hours, less than 24 hours, or the predetermined period can be less than 12 hours.In certain embodiments, for example, a predetermined concentration range (or a predetermined concentration value) can be based on the predicted concentration of a predetermined type of antigen within a tumor (e.g., the predicted concentration of a predetermined type of antigen expressed on the surface of the tumor).

[0023] B. In certain embodiments, for example, the binding can include binding at least a first binding T cell to a P-loaded MHC protein, where P is a predetermined type of antigen. In certain embodiments, for example, the MHC protein can be an MHC class I protein. In certain embodiments, for example, the P-loaded MHC protein can be present in a P-loaded MHC protein multimer.

[0024] C. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be derived from a common population of PBMCs. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be derived from one or more healthy donors. In certain embodiments, for example, one or more healthy donors can be at least partially human leukocyte antigen (HLA)-compatible with the subject. In certain embodiments, for example, one or more healthy donors can be at least partially HLA-compatible with the subject with respect to the presentation of a given type of antigen. In certain embodiments, for example, one or more healthy donors can be HLA-A-compatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-B-compatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-C-compatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-DP-compatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-DQ-compatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-DR-compatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, or compatible with two or more combinations of the foregoing with respect to the subject. In certain embodiments, for example, one or more healthy donors can be at least partially HLA-incompatible with the subject. In certain embodiments, for example, one or more healthy donors can be completely HLA-incompatible with the subject. In certain embodiments, for example, one or more healthy donors can be selectively HLA-incompatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-B-incompatible with the subject. In certain embodiments, for example, one or more healthy donors can be HLA-C-incompatible with the subject.In certain embodiments, for example, one or more healthy donors may be incompatible with the subject with respect to HLA-DP. In certain embodiments, for example, one or more healthy donors may be incompatible with the subject with respect to HLA-DQ. In certain embodiments, for example, one or more healthy donors may be incompatible with the subject with respect to HLA-DR. In certain embodiments, for example, one or more healthy donors may be incompatible with the subject with respect to HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, or combinations of two or more of the foregoing.

[0025] In certain embodiments, for example, one or more healthy donors may be at least partially HLA-compatible with respect to the presentation of a given type of antigen with respect to a predicted HLA (e.g., an HLA predicted in combination with a given type of antigen, such as an HLA predicted for a given type of cancer, by one of the machine learning models and / or by a method disclosed in this specification or in one of the incorporated references that present a given type of antigen). In certain embodiments, for example, the predicted HLA may be selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, or combinations of two or more of the foregoing.

[0026] In certain embodiments, for example, one or more healthy donors may be at least partially HLA-incompatible with respect to the presentation of a given type of antigen with respect to a predicted HLA (e.g., an HLA predicted in combination with a given type of antigen, such as an HLA predicted for a given type of cancer, by one of the machine learning models and / or by a method disclosed in this specification or in one of the incorporated references that present a given type of antigen). In certain embodiments, for example, the predicted HLA may be selectable from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, or combinations of two or more of the foregoing.

[0027] D. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) naive CD8 + T cells. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) naive T cells. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) memory T cells. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) CD8 + T cells. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) CD4 + T cells. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) CD4 + CD8 + T cells. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) CD4 - CD8 + T cells. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells can be (or can include or be derived from) CD4 + CD8 - T cells. (or can include or be derived from).

[0028] E. In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more tumor cells. In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more dendritic cells. In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more antigen-presenting cells (e.g., one or more professional antigen-presenting cells). In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more artificial antigen-presenting cells. In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more macrophages. In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more monocytes. In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more B cells. In certain embodiments, for example, the plurality of cells presenting at least one of a second predetermined type of antigen may include one or more of the plurality of cells presenting at least one of a second predetermined type of antigen and may express a predetermined type of antigen.

[0029] F. In certain embodiments, for example, the method may further include detecting the binding by flow cytometry (e.g., fluorescence flow cytometry). In certain embodiments, for example, one of the first predetermined type of antigen may bind to magnetic beads, in which case the method may further include detecting at least the first antigen-binding T cells by magnetic separation. In certain embodiments, for example, the method may further include detecting the activation by flow cytometry (e.g., fluorescence flow cytometry). In certain embodiments, for example, the method may further include detecting at least the first functional T cells by magnetic separation.

[0030] G. In certain embodiments, for example, the method may further include a step of detecting activation, including detecting one or more biomarkers. In certain embodiments, for example, the one or more biomarkers may include CD137. In certain embodiments, for example, the method may further include a step of detecting activation, including detecting the presence of one or more molecules that suggest activation of T cells. In certain embodiments, for example, the one or more molecules may include interferon gamma. In certain embodiments, for example, the method may further include a step of detecting activation, including detecting the proliferation of T cells. In certain embodiments, for example, the step of activating at least the first functional T cells may be T cells present in a second plurality of T cells. In certain embodiments, for example, the step of activating at least the first functional T cells may be T cells formed by the proliferation of one of the T cells present in a second plurality of T cells.

[0031] H. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on the tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on the tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be individualized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0032] Certain embodiments can provide, for example, a method for selecting a T cell receptor. In certain embodiments, for example, the method comprises binding at least a first antigen-binding T cell present in a first plurality of T cells to one of at least a first class I P-MHC protein multimer, wherein P is a predetermined type of antigen, and contacting the first plurality of T cells with one of the first class I P-MHC protein multimers. In certain embodiments, for example, the method comprises activating at least a first functional T cell present in a second plurality of T cells, comprising contacting the second plurality of T cells with a plurality of cells presenting one of at least a first class II P-MHC protein multimer. In certain embodiments, for example, the method can include identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0033] Certain embodiments may provide, for example, a method for selecting a T cell receptor. In certain embodiments, for example, the method comprises binding at least a first antigen-binding T cell present in a first plurality of T cells to one of at least a first class I P-MHC protein multimer, where P is an antigen of a predetermined type, and contacting the first plurality of T cells with one of the first class I P-MHC protein multimers. In certain embodiments, for example, the method comprises activating at least a first functional T cell present in a second plurality of T cells, comprising contacting the second plurality of T cells with a plurality of cells presenting at least the first class I P-MHC protein. In certain embodiments, for example, the method may comprise identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0034] Certain embodiments may provide, for example, a method for selecting a T cell receptor (e.g., the method excludes any in vitro priming methods disclosed herein or in one of the incorporated references). In certain embodiments, for example, the method comprises isolating a first T cell from a plurality of T cells, where the first T cell is bound to a P-loaded MHC protein and P is an antigen of a predetermined type. In certain embodiments, for example, the method comprises further isolating a second T cell from a plurality of T cells, where the second T cell expresses at least one biomarker indicative of activation by an antigen of a predetermined type. In certain embodiments, for example, the method may comprise matching at least a portion of the T cell receptor sequence of the first T cell with at least a portion of the T cell receptor sequence of the second T cell.

[0035] A. In certain embodiments, for example, the method may further comprise inducing a plurality of T cells from at least two T cells that are individually bound to at least two P-loaded MHC proteins. In certain embodiments, for example, the inducing step may comprise expanding at least a first T cell and at least a second T cell. In certain embodiments, for example, expanding may comprise polyclonally expanding at least a first T cell and at least a second T cell. In certain embodiments, for example, at least the first T cell and at least the second T cell may be in a mixed state during expansion. In certain embodiments, for example, at least the first T cell and at least the second T cell may be separated from each other prior to expansion.

[0036] B. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12 amino acids). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on a tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on a tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0037] Certain embodiments can provide, for example, a method for detecting a functional T cell receptor clonotype. In certain embodiments, for example, the method can include isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs. In certain embodiments, for example, the method can include a step of forming a plurality of cognate T cells, including the step of expanding the at least one isolated T cell. In certain embodiments, for example, the method can include a step of activating at least a first functional T cell, including contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic for a predetermined type of antigen. In certain embodiments, for example, the method can include a step of confirming that at least the first functional T cell is configured to bind to a P-loaded MHC protein, where P is a predetermined type of antigen.

[0038] A. In certain embodiments, for example, the forming step may include indirect T cell receptor cross-linking. In certain embodiments, for example, the forming step may be limited to a single polyclonal expansion. In certain embodiments, for example, the forming step may include multiple polyclonal expansions. In certain embodiments, for example, at least one of the multiple polyclonal expansions may be followed by isolating at least one additional T cell that binds to a predetermined type of antigen.

[0039] B. In certain embodiments, for example, at least the first functional T cells may have a dissociation constant of less than 50 μM for the P-loaded MHC protein. In certain embodiments, for example, at least the first functional T cells may have a half-life of 0.01 seconds to 100 seconds (e.g., 2 seconds to 10 seconds) for the P-loaded MHC protein. In certain embodiments, for example, the predetermined type of antigen may be a tumor-associated peptide antigen, but at least the first functional T cells have: i) a dissociation constant of less than 50 μM for the P-loaded MHC protein; and ii) a half-life of 0.01 seconds to 100 seconds (e.g., 2 seconds to 10 seconds) for the P-loaded MHC protein.

[0040] C. In certain embodiments, for example, at least one of the multiple activators may be antigenic for a predetermined type of antigen. In certain embodiments, for example, negative selection can be performed on at least one T cell.

[0041] D. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on the tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on the tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be individualized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0042] Certain embodiments can provide, for example, a method for detecting antigen-binding T cells. In certain embodiments, for example, the method can include isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs. In certain embodiments, for example, the method can include forming a plurality of cognate T cells, including the step of expanding the at least one isolated T cell. In certain embodiments, for example, the method can include binding at least a first binding T cell to at least a first binder, including contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of binders, wherein at least one of the plurality of binders includes a predetermined type of antigen. In certain embodiments, for example, the method can include confirming that the at least first binding T cell is configured to be activated by a cell presenting a predetermined type of antigen.

[0043] A. In certain embodiments, for example, cells that can present a given type of antigen can be antigen-presenting cells. In certain embodiments, for example, the antigen-presenting cells can be professional antigen-presenting cells.

[0044] B. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on a tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on a tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0045] Certain embodiments can provide, for example, a method for selecting a T cell receptor specific for a given type of antigen. In certain embodiments, for example, the method can include isolating a first plurality of T cells, wherein at least a portion of the first plurality of T cells is bound to a plurality of P-loaded MHC proteins, and P is a given type of antigen. In certain embodiments, for example, the method can further include isolating a second plurality of T cells, wherein in the presence of a plurality of activators, at least a portion of the second plurality of T cells upregulates one or more activation signaling molecules (and / or expresses one or more activation markers), where at least one of the plurality of activators is immunogenic for a given type of antigen. In certain embodiments, for example, the method can include identifying at least a portion of at least one T cell receptor sequence common to both at least a portion of the first plurality of T cells and at least a portion of the second plurality of T cells.

[0046] A. In certain embodiments, for example, at least a portion of at least one T cell receptor sequence can be present in at least 0.005% of a combination of at least a portion of a first plurality of T cells and at least a portion of a second plurality of T cells. In certain embodiments, for example, at least one of a plurality of activators can be antigenic for a given type of antigen.

[0047] B. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on a tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on a tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0048] Certain embodiments can provide, for example, a method for selecting a T cell receptor specific for a given type of antigen. In certain embodiments, for example, the method can include isolating a first plurality of T cells, wherein at least a portion of the first plurality of T cells express one or more first activation markers in the presence of a plurality of first activators. In certain embodiments, for example, the method can further include isolating a second plurality of T cells, wherein at least a portion of the second plurality of T cells upregulate one or more second activation markers (and / or one or more activation signaling molecules) in the presence of a plurality of second activators. In certain embodiments, for example, the method can include identifying: a) at least a portion of at least one T cell receptor sequence in common; and b) at least one of at least a portion of a first plurality of T cells and at least a portion of a second plurality of T cells having a dissociation constant less than a threshold for a P-loaded MHC protein, where P is the given type of antigen.

[0049] A. In certain embodiments, for example, at least one of the plurality of first activators can be immunogenic for a given type of antigen and / or at least one of the plurality of second activators can be immunogenic for a given type of antigen. In certain embodiments, for example, at least one of the plurality of first activators can be antigenic for a given type of antigen and / or at least one of the plurality of second activators can be antigenic for a given type of antigen. In certain embodiments, for example, at least one of the plurality of first activators can contain a given type of antigen and / or at least one of the plurality of second activators can contain a given type of antigen. In certain embodiments, for example, at least one of the plurality of first activators can be a cell presenting a given type of antigen and / or at least one of the plurality of second activators can be a cell presenting a given type of antigen. In certain embodiments, for example, at least one of the plurality of first activators can contain a P-loaded MHC protein and / or at least one of the plurality of second activators can contain a P-loaded MHC protein. In certain embodiments, for example, at least one of the plurality of first activators can be a cell that endogenously expresses a given type of antigen and / or at least one of the plurality of second activators can be a cell that endogenously expresses a given type of antigen. In certain embodiments, for example, at least one of the plurality of first activators can contain a P-loaded MHC protein and / or at least one of the plurality of second activators can be a cell that endogenously expresses a given type of antigen.

[0050] B. In certain embodiments, for example, the dissociation constant can correspond to the binding between at least a portion of at least one T cell receptor sequence and a P-loaded MHC protein. In certain embodiments, for example, the threshold can be less than 1000 μM (e.g., less than 50 μM).

[0051] C. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on a tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on a tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determined using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0052] Certain embodiments can provide, for example, a method for negatively selecting T cell receptor clonotypes. In certain embodiments, for example, the method can include analyzing a mixture of T cells to identify a first antigen-binding T cell and a first antigen-activated T cell against a predetermined first type of antigen, and a second antigen-activated T cell against a predetermined second type of antigen. In certain embodiments, for example, the method can include identifying at least a portion of at least one T cell receptor sequence that is a) shared by at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells; and b) not shared with any of the second antigen-activated T cells.

[0053] A. In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids (e.g., 8 to 12). In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be presented on the tumor. In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be an individualized antigen. In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a predetermined first type of antigen and / or a predetermined second type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on the tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen.In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references. In certain embodiments, for example, a given second type of antigen may not be presented on the tumor. In certain embodiments, for example, a given second type of antigen may not be a personalized antigen. In certain embodiments, for example, a given second type of antigen may not be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen may not be a cancer / testis antigen. In certain embodiments, for example, a given second type of antigen may not be specific to a particular type of tumor. In certain embodiments, for example, a given second type of antigen may not be a tumor-associated peptide antigen. In certain embodiments, for example, a given second type of antigen may not be a neoantigen. In certain embodiments, for example, a given first type of antigen can be a first peptide, and a given second type of antigen can be a second peptide. In certain embodiments, for example, the first peptide can be expressed by a variant of the gene that expresses the second peptide.In certain embodiments, for example, the first peptide can be expressed by an allele of the gene that expresses the second peptide. In certain embodiments, for example, the second peptide can be expressed by a wild-type gene. In certain embodiments, for example, the first peptide can be a neoantigen, and the second peptide can be expressed by a related wild-type gene. In certain embodiments, for example, the first peptide and the second peptide can differ by at least 1 amino acid, for example at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids, at least 9 amino acids, at least 10 amino acids, at least 12 amino acids, or the first peptide and the second peptide can differ by at least 15 amino acids. In certain embodiments, for example, the first peptide and the second peptide can differ by 1 to 15 amino acids, for example 5 to 10 amino acids, or the first peptide and the second peptide can differ by 5 to 8 amino acids. In certain embodiments, for example, the differences can include (can be composed of) conservative substitutions. In certain embodiments, for example, the differences can include (can be composed of) radical substitutions. In certain embodiments, for example, the first peptide and the second peptide can have less than 95% sequence identity, for example less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60% sequence identity, or the first peptide and the second peptide can have less than 55% sequence identity. In certain embodiments, for example, the first peptide and the second peptide can have 55% - 95% sequence identity, for example 55% - 90%, 55% - 85%, 55% - 80%, 55% - 75% sequence identity, or the first peptide and the second peptide can have 55% - 70% sequence identity.

[0054] B. In certain embodiments, for example, the step of identifying a first antigen-activated T cell can include contacting a portion of a mixture of T cells with a cell that endogenously presents a predetermined first type of antigen (e.g., by expression, e.g., a tumor cell). In certain embodiments, for example, the step of identifying a second antigen-activated T cell can include contacting a portion of a mixture of T cells with a cell that endogenously presents a predetermined second type of antigen (e.g., by expression, e.g., a tumor cell). In certain embodiments, for example, the step of identifying a first antigen-activated T cell can include contacting a portion of a mixture of T cells with a cell loaded with a predetermined first type of antigen (e.g., an exogenous source, e.g., a professional antigen-presenting cell). In certain embodiments, for example, the step of identifying a second antigen-activated T cell can include contacting a portion of a mixture of T cells with a cell loaded with a predetermined second type of antigen (e.g., an exogenous source, e.g., a professional antigen-presenting cell).

[0055] Certain embodiments can provide, for example, a method for negatively selecting T cell clonotypes. In certain embodiments, for example, the method can include analyzing a mixture of T cells to identify a first antigen-activated T cell and a first antigen-binding T cell for a predetermined first type of antigen, and a second antigen-binding T cell for a predetermined second type of antigen. In certain embodiments, for example, the method can include identifying at least a portion of at least one T cell receptor sequence that is a) common to at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells; and b) not shared with any of the second antigen-binding T cells.

[0056] Certain embodiments may provide, for example, a method for identifying T cell activation markers. In certain embodiments, for example, the method may include contacting a first plurality of T cells with a plurality of P-presenting cells (e.g., cells expressing P or cells pulsed with P) (e.g., a plurality of P-presenting cells comprising P presented by MHC class I protein and / or MHC class II protein), wherein the first plurality of T cells comprises a plurality of P-binding T cells where P is a predetermined type of antigen. In certain embodiments, for example, the method may include measuring a plurality of expression rate profiles for at least a portion of the contacted plurality of P-binding T cells. In certain embodiments, for example, the method may include measuring a functional response to P in at least two T cells present in at least a portion of the contacted plurality of P-binding T cells. In certain embodiments, for example, the method may include dividing the contacted plurality of P-binding T cells into a plurality of T cell clusters. In certain embodiments, for example, the method may include mapping an expression rate profile to the plurality of T cell clusters to identify one of the plurality of T cell clusters comprising at least two T cells. In certain embodiments, for example, the method may include identifying activation markers (or secreted molecules suggesting activation) expressed by at least two T cells.

[0057] A. In certain embodiments, for example, P-binding T cells may be identified using a bioinformatics filter that compares at least portions of the T cell receptor sequences of at least a portion of the contacted plurality of P-binding T cells with at least portions of a predetermined T cell receptor sequence.

[0058] B. In certain embodiments, for example, the dividing step may comprise dividing the plurality of P-binding T cells that have been contacted into a plurality of groups, wherein at least one of the plurality of groups consists of T cells having at least a plurality of portions of a common T cell receptor sequence. In certain embodiments, for example, the dividing step may comprise dividing the plurality of P-binding T cells that have been contacted into a plurality of groups, wherein at least one of the plurality of groups consists of T cells having at least a plurality of portions of a T cell receptor sequence characterized by at least 70% (e.g., at least 80%, 90%, 95%, 100%) sequence identity to each other.

[0059] In certain embodiments, for example, the dividing step may comprise dividing the plurality of P-binding T cells that have been contacted into a plurality of groups, wherein at least one of the plurality of groups consists of T cells having at least a plurality of portions of a T cell receptor sequence that differ by at most one amino acid from each other (e.g., at most one amino acid is conservatively substituted). In certain embodiments, for example, the dividing step may comprise dividing the plurality of P-binding T cells that have been contacted into a plurality of groups, wherein at least one of the plurality of groups consists of T cells having at least a plurality of portions of a T cell receptor sequence that differ only by conservative substitutions. In certain embodiments, for example, the dividing step may comprise group lympocyte interaction by paratope hotspots (GLIPH). In certain embodiments, for example, at least a plurality of portions of the common T cell receptor sequence may be at least a plurality of portions of the CDR3 region. In certain embodiments, for example, at least a plurality of portions of the CDR3 region may comprise a linear amino acid sequence having a length of 6 to 35 amino acids. In certain embodiments, for example, at least a plurality of portions of the CDR3 region may not include a stem region. In certain embodiments, for example, at least a plurality of portions of the CDR3 region may include a CDR3 beta chain portion.

[0060] C. In certain embodiments, for example, the splitting step may be performed using an algorithm (e.g., a statistical algorithm). In certain embodiments, for example, the algorithm may include a similarity analysis of the plurality of expression rate profiles. In certain embodiments, for example, the plurality of expression rate profiles may include expression rates for one or more activation markers that suggest a functional response to P. In certain embodiments, for example, the one or more activation markers may comprise CD137, CD69, CD25, Ki67, CD107, CD122, CD27, CD28, CD95, CD134, KLRG1, CD38, CD154, or combinations of two or more of the foregoing activation markers. In certain embodiments, for example, the algorithm may be a clustering analysis algorithm. In certain embodiments, for example, the algorithm may include a t-distributed stochastic neighbor embedding method.

[0061] D. In certain embodiments, for example, the functional response to the measured P may include the detection of one or more activation markers and / or one or more secreted molecules. In certain embodiments, for example, one or more activation markers may include CD137, CD69, CD25, Ki67, CD107, CD122, CD27, CD28, CD95, CD134, KLRG1, CD38, CD154, or a combination of two or more of the foregoing activation markers. In certain embodiments, for example, one or more secreted molecules may include one or more cytokines. In certain embodiments, for example, one or more cytokines may be interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or a combination of two or more of the foregoing. In certain embodiments, for example, one or more secreted molecules may include granzyme. In certain embodiments, for example, one or more secreted molecules may include perforin. In certain embodiments, for example, the functional response to the measured P may include the detection of T cell proliferation.

[0062] E. In certain embodiments, for example, the first plurality of T cells and the second plurality of T cells may be derived from a common starting population of PBMCs.

[0063] F. In certain embodiments, for example, a plurality of expression rate profiles can be obtained from a series of single-cell transcriptome analyses. In certain embodiments, for example, T cells included in one of a plurality of T cell clusters can express a predetermined first activation marker at an average of a second expression rate that exceeds a first expression rate threshold (e.g., greater than 0.05% (e.g., greater than 0.1%, greater than 0.5%, or between 0.05% and 0.5%)). In certain embodiments, for example, T cells included in one of a plurality of T cell clusters can express a second activation marker at an average of a second expression rate that exceeds a second expression rate threshold (e.g., greater than 0.05% (e.g., greater than 0.1%, greater than 0.5%, or between 0.05% and 0.5%)).

[0064] G. In certain embodiments, for example, the method can further include identifying a plurality of P-binding T cells by matching the T cell receptor sequences of the plurality of P-binding T cells to a predetermined T cell receptor sequence. In certain embodiments, for example, the predetermined T cell receptor sequence can be determined by sequencing a second plurality of T cells bound to a P-loaded MHC protein.

[0065] In certain embodiments, for example, the activation marker may not be expressed or may be downregulated in at least two other T cells present in another one of the plurality of T cell clusters, and the at least two other T cells do not show a functional response when measured.

[0066] H. In certain embodiments, for example, a given type of antigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 (e.g., 8 to 12) amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, a given type of antigen can be derived from a tumor (e.g., a solid tumor). In certain embodiments, for example, a given type of antigen can be presented on a tumor. In certain embodiments, for example, a given type of antigen can be an individualized antigen. In certain embodiments, for example, a given type of antigen can be a shared tumor antigen (e.g., a shared tumor neoantigen). In certain embodiments, for example, the shared tumor antigen can be a cancer / testis antigen. In certain embodiments, for example, the shared tumor antigen can be a cancer / testis-like antigen. In certain embodiments, for example, the shared tumor antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be specific to a particular type of tumor. In certain embodiments, for example, a given type of antigen can be a tumor-associated peptide antigen. In certain embodiments, for example, a given type of antigen can be a viral antigen (e.g., an oncogenic virus protein, e.g., HPV E6 and HPV E7). In certain embodiments, for example, a given type of antigen can be a neoantigen. In certain embodiments, for example, the neoantigen can be a peptide. In certain embodiments, for example, the peptide can be composed of 8 to 15 amino acids. In certain embodiments, for example, the peptide can be composed of 12 to 40 amino acids. In certain embodiments, for example, the neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, the neoantigen can be presented on a tumor. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen.In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be personalized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0067] Certain embodiments can provide, for example, methods for pre-identifying therapeutically effective antigen-binding T cells and T cell receptors for the development of antigen-activated T cells. In certain embodiments, for example, the development can include transfecting a T cell receptor into a T cell line. In certain embodiments, for example, the T cell receptor can be identified from T cells derived from one or more healthy HLA-matched donor samples. In certain embodiments, for example, the identified T cell receptor can be present at less than 1 in 10,000 of the derived T cells. In certain embodiments, for example, the derived T cells can be obtained by one or more of the enrichment step, and / or the expansion step described herein or in one of the incorporated references. In certain embodiments, for example, the identified T cell receptor can be present at less than 1 in 10,000,000 T cells present in one or more donor samples. In certain embodiments, for example, that at least a portion of the derived T cells have been antigen-activated can be determined by exposing the cells presenting the antigen at a physiological concentration (e.g., the concentration range in which the antigen is presented on tumor cells).

[0068] Certain embodiments may provide, for example, a method for selecting a T cell receptor. In certain embodiments, for example, the method may include the step of expanding a series of antigen-specific T cells (e.g., by polyclonal expansion). In certain embodiments, for example, the method may include the step of obtaining a T cell from one of a series of antigen-specific T cells, and then complexing the obtained antigen-specific T cell with a binding agent (e.g., a fluorescently labeled antigen-MHC protein multimer or a magnetically tagged antigen-MHC protein multimer), and may include the step of expanding the complexed antigen-specific T cells to form the next member of the series of antigen-specific T cells. In certain embodiments, for example, the method may further include the step of exposing the member's cognate to cells expressing the antigen at a physiologically appropriate concentration, and then detecting the activation of the cognate. Certain embodiments may provide, for example, a therapy for cancer that includes the step of administering a T cell, the T cell includes a transfected T cell receptor, and the T cell receptor includes at least a portion of a T cell receptor sequence determined by cognate sequencing.

[0069] Certain embodiments may provide, for example, a method for screening candidate antigens for an antigen-specific vaccine. In certain embodiments, for example, the method may include the step of isolating at least one T cell that binds to a candidate antigen from a population of PBMCs. In certain embodiments, for example, the method may include the step of forming a plurality of cognate T cells, which includes the step of expanding the at least one isolated T cell. In certain embodiments, for example, the method may include the step of activating at least a first functional T cell, which includes contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic for the candidate antigen.

[0070] A. In certain embodiments, for example, the candidate antigen may be a neoantigen. In certain embodiments, for example, the antigen-specific vaccine may be for the treatment of cancer (e.g., for the treatment of a cancerous tumor).

[0071] Certain embodiments may provide, for example, a method for screening candidate neoantigens for immunogenicity. In certain embodiments, for example, the method may include the step of isolating at least one T cell that binds to a candidate neoantigen from a population of PBMCs. In certain embodiments, for example, the method may include the step of forming a plurality of cognate T cells, which includes the step of expanding the at least one isolated T cell. In certain embodiments, for example, the method may include the step of activating at least a first functional T cell, which includes the step of contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic for the candidate neoantigen.

[0072] Certain embodiments may provide, for example, an artificial T cell receptor that is selective for a predetermined type of antigen (e.g., a neoantigen, e.g., an individual neoantigen or a shared neoantigen). In certain embodiments, for example, the method may include: a) analyzing a mixture of natural T cells to identify antigen-binding T cells and antigen-activating T cells for a predetermined type of antigen; and b) identifying at least a portion of at least one T cell receptor sequence that is shared by at least one of the antigen-binding T cells and at least one of the antigen-activating T cells, wherein the at least a portion of the at least one T cell receptor sequence contains at least a portion of the CDR3 region. The method may, for example, in certain embodiments, include a T cell receptor fragment (e.g., a universal backbone fragment that can be combined with a plurality of different CDR3 sequences to form a plurality of products, e.g., a plurality of therapeutic products).

[0073] Certain embodiments can provide a method for selecting a T-cell receptor clonotype, comprising, for example: i) analyzing a mixture of T cells to identify antigen-binding T cells and antigen-activated T cells for a given type of antigen; and ii) identifying at least a portion of at least one T-cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the antigen-activated T cells.

[0074] Certain embodiments can provide a method for selecting a shared receptor sequence within lymphocytes, comprising, for example: i) analyzing a mixture of lymphocytes to identify stimulated lymphocytes and co-stimulated lymphocytes for a given type of antigen; and ii) identifying at least a portion of at least one receptor sequence shared by at least one of the stimulated lymphocytes and at least one of the co-stimulated lymphocytes.

[0075] Certain embodiments can provide a method for selecting a T-cell receptor clonotype, comprising, for example: i) analyzing a mixture of naive T cells to identify antigen-binding T cells and functional T cells for a given type of antigen; and ii) identifying at least a portion of at least one T-cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the functional T cells.

[0076] Certain embodiments can provide a method for selecting a T cell receptor, comprising: i) binding at least a first antigen-binding T cell to at least one of at least a first predetermined type of antigen, the step comprising contacting a first plurality of T cells with at least one of a first predetermined type of antigen; ii) activating at least a first functional T cell, the step comprising contacting a second plurality of T cells with a plurality of cells presenting at least one of at least a second predetermined type of antigen; and iii) identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0077] Certain embodiments can provide a method for selecting a T cell receptor, comprising: i) binding at least a first antigen-binding T cell present in a first plurality of T cells to at least one of at least a first class I P-MHC protein multimer, where P is a predetermined type of antigen, the step comprising contacting the first plurality of T cells with at least one of a first class I P-MHC protein multimer; ii) activating at least a first functional T cell present in a second plurality of T cells, the step comprising contacting the second plurality of T cells with a plurality of cells presenting at least one of at least a first class II P-MHC protein multimer; and iii) identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0078] Certain embodiments may provide a method for selecting a T cell receptor, comprising: i) binding at least a first antigen-binding T cell present in a first plurality of T cells to one of at least a first class I P-MHC protein multimer, wherein P is an antigen of a predetermined type, and contacting the first plurality of T cells with one of the first class I P-MHC protein multimers; ii) activating at least a first functional T cell present in a second plurality of T cells by contacting the second plurality of T cells with a plurality of cells presenting at least the first class I P-MHC protein; and iii) identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0079] Certain embodiments may provide a method for selecting a T cell receptor, comprising: i) isolating a first T cell from a plurality of T cells, wherein the first T cell binds to a P-loaded MHC protein and P is an antigen of a predetermined type; ii) further isolating a second T cell from the plurality of T cells, wherein the second T cell expresses at least one biomarker indicative of activation by an antigen of a predetermined type; and iii) matching at least a portion of the T cell receptor sequence of the first T cell with at least a portion of the T cell receptor sequence of the second T cell.

[0080] A particular embodiment may provide a method for detecting, for example, a functional T cell receptor clonotype, comprising: i) isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs; ii) forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell; iii) activating at least a first functional T cell, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic with respect to a predetermined type of antigen; and iv) confirming that at least the first functional T cell is configured to bind to a P-loaded MHC protein, where P is a predetermined type of antigen.

[0081] A particular embodiment may provide a method for detecting, for example, antigen-binding T cells, comprising: i) isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs; ii) forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell; iii) binding at least a first binding T cell to at least a first binder, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of binders, at least one of the plurality of binders comprising a predetermined type of antigen; and iv) confirming that at least the first binding T cell is configured to be activated by a cell presenting a predetermined type of antigen.

[0082] Certain embodiments can provide a method for selecting a T cell receptor specific for a given type of antigen, comprising: i) isolating a first plurality of T cells, wherein at least a portion of the first plurality of T cells is bound to a plurality of antigen-loaded MHC proteins, where the antigen is of a given type; ii) further isolating a second plurality of T cells, wherein in the presence of a plurality of activators, at least a portion of the second plurality of T cells upregulates one or more activation signaling molecules (and / or expresses one or more activation markers or another indicator of activation), and at least one of the plurality of activators is immunogenic for the given type of antigen; and iii) identifying at least a portion of at least one T cell receptor sequence common to at least a portion of the first plurality of T cells and at least a portion of the second plurality of T cells.

[0083] Certain embodiments can provide a method for selecting a T cell receptor specific for a given type of antigen, comprising: i) isolating a first plurality of T cells, wherein in the presence of a plurality of first activators, at least a portion of the first plurality of T cells expresses one or more first activation markers; ii) further isolating a second plurality of T cells, wherein in the presence of a plurality of second activators, at least a portion of the second plurality of T cells upregulates one or more second activation markers (and / or one or more activation signaling molecules); and iii) identifying at least one of at least a portion of the first plurality of T cells and at least a portion of the second plurality of T cells that has a dissociation constant below a threshold for: a) at least a portion of at least one common T cell receptor sequence; and b) an antigen-loaded MHC protein, where the antigen is of a given type.

[0084] Certain embodiments can provide a method for, e.g., negatively selecting T cell receptor clonotypes, comprising: i) analyzing a mixture of T cells to identify a first antigen-activated T cell and a first antigen-binding T cell against a predetermined first type of antigen, and a second antigen-activated T cell against a predetermined second type of antigen; and ii) identifying at least a portion of at least one T cell receptor sequence that is a) shared by at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells and b) not shared by any of the second antigen-activated T cells.

[0085] Certain embodiments can provide a method for, e.g., negatively selecting T cell receptor clonotypes, comprising: i) analyzing a mixture of T cells to identify a first antigen-activated T cell and a first antigen-binding T cell against a predetermined first type of antigen, and a second antigen-binding T cell against a predetermined second type of antigen; and ii) identifying at least a portion of at least one T cell receptor sequence that is a) shared by at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells and b) not shared by any of the second antigen-binding T cells.

[0086] A particular embodiment may provide a method for identifying, for example, T cell activation markers, comprising: i) contacting a first plurality of T cells with a plurality of antigen-presenting cells, wherein the first plurality of T cells comprises a plurality of antigen-binding T cells and the antigen is a predetermined type of antigen; ii) measuring a plurality of expression rate profiles for at least a portion of the contacted plurality of antigen-binding T cells; iii) measuring a functional response to the predetermined type of antigen in at least two T cells present in at least a portion of the contacted plurality of antigen-binding T cells; iv) dividing at least a portion of the contacted plurality of antigen-binding T cells into a plurality of T cell clusters; v) mapping the expression rate profiles to the plurality of T cell clusters to identify one of the plurality of T cell clusters comprising at least two T cells; and vi) identifying activation markers expressed by the at least two T cells.

[0087] A particular embodiment may provide a method for screening, for example, candidate antigens for an antigen-specific vaccine, comprising: i) isolating at least one T cell that binds to a candidate antigen from a population of PBMCs; ii) forming a plurality of cognate T cells, comprising expanding the at least one isolated T cell; and iii) activating at least a first functional T cell, comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic with respect to the candidate antigen.

[0088] Certain embodiments can provide a method for screening candidate neoantigens for immunogenicity, for example, comprising: i) isolating at least one T cell that binds to a candidate neoantigen from a population of PBMCs; ii) forming a plurality of cognate T cells, including the step of expanding the at least one isolated T cell; and iii) activating at least a first functional T cell, including contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic for the candidate neoantigen.

[0089] Certain embodiments can provide, for example: i) a) analyzing a mixture of natural T cells to identify antigen-binding T cells and antigen-activated T cells for a predetermined type of antigen; b) identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the antigen-activated T cells, wherein the at least a portion of the at least one T cell receptor sequence contains at least a portion of the CDR3 region, and thus at least a portion of the CDR3 region (e.g., at least a portion of the CDR3 beta chain) selected thereby; and ii) an artificial T cell receptor selective for a predetermined type of antigen (e.g., a neoantigen, e.g., an individual neoantigen or a shared neoantigen), including a T cell receptor fragment (e.g., a plurality of products, e.g., a universal backbone fragment combinable with a plurality of different CDR3 sequences to form a plurality of therapeutic products).

[0090] Certain embodiments can provide, for example, a method for identifying one or more viral epitopes, including the methods disclosed herein for identifying and / or selecting one or more of a T cell receptor (or a T cell receptor clonotype or shared receptor sequence).

[0091] Certain embodiments can provide, for example, a composition for organ transplantation therapy, and include the methods disclosed herein for identifying and / or selecting at least a portion of one or more T cell receptor sequences (or T cell receptor clonotypes or shared receptor sequences) determined from one or more of the T cell receptors.

[0092] Certain embodiments can provide, for example, a composition for performing cell therapy in one or more subjects, and include the methods disclosed herein for identifying and / or selecting at least a portion of one or more T cell receptor sequences (or T cell receptor clonotypes or shared receptor sequences) determined from one or more of the T cell receptors.

[0093] Certain embodiments can provide, for example, a composition for enhancing the immune system in one or more subjects, and include the methods disclosed herein for identifying and / or selecting at least a portion of one or more T cell receptor sequences (or T cell receptor clonotypes or shared receptor sequences) determined from one or more of the T cell receptors.

[0094] In any of the above embodiments, a given type of antigen can be a neoantigen (e.g., a neoantigen identified using a machine learning-based model).

[0095] Compositions obtained by any of the methods described herein are also provided herein.

[0096] The present disclosure also provides a composition comprising an artificial T cell receptor selective for a predetermined type of antigen, the steps of analyzing a mixture of natural T cells to identify antigen-binding T cells and antigen-activated T cells for a predetermined type of antigen; and identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the antigen-activated T cells, wherein at least one T cell receptor sequence contains at least a portion of the CDR3 region, and selecting at least a portion of the CDR3 region thus selected; and comprising a T cell receptor fragment.

[0097] Certain embodiments provide T cells comprising an artificial T cell receptor or a fragment thereof obtained by any one of the methods described herein. In some embodiments, the T cells are used in the treatment of cancer.

[0098] Also provided herein is a kit comprising a composition obtained by any of the methods described herein.

[0099] The present disclosure also provides a kit for use in any one of the methods presented herein. [The present invention 1001] A method for selecting a T cell receptor clonotype, comprising: i) analyzing a mixture of T cells to identify antigen-binding T cells and antigen-activated T cells for a predetermined type of antigen; and ii) identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the antigen-activated T cells. A method comprising the steps of. [The present invention 1002] A method for selecting a shared receptor sequence within lymphocytes, comprising: i) analyzing a mixture of lymphocytes to identify stimulated lymphocytes and co-stimulated lymphocytes for a predetermined type of antigen; and ii) identifying at least a portion of at least one receptor sequence shared by at least one of the stimulated lymphocytes and at least one of the costimulated lymphocytes A method comprising the steps of: [Inventive concept 1003] A method for selecting a T cell receptor type, comprising: i) analyzing a mixture of naive T cells to identify antigen-binding T cells and functional T cells for a given type of antigen; and ii) identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the functional T cells A method comprising the steps of: [Inventive concept 1004] A method for selecting a T cell receptor, comprising: i) binding at least a first antigen-binding T cell to at least one of at least a first given type of antigen, comprising contacting a first plurality of T cells with at least one of the first given type of antigen; and ii) activating at least a first functional T cell, comprising contacting a second plurality of T cells with a plurality of cells presenting at least one of at least a second given type of antigen; and iii) identifying at least a portion of at least one T cell receptor sequence common to at least one of the antigen-binding T cells and at least one of the functional T cells A method comprising the steps of: [Inventive concept 1005] A method for selecting a T cell receptor, comprising: i) binding at least a first antigen-binding T cell present in a first plurality of T cells to at least one of at least a first class I p-MHC protein multimer, where p is a given type of antigen, comprising contacting the first plurality of T cells with at least one of the first class I p-MHC protein multimers; and ii) activating at least a first functional T cell present in the second plurality of T cells, the step comprising contacting the second plurality of T cells with a plurality of cells presenting at least one of at least a first class II P-MHC protein multimer; iii) identifying at least a portion of at least one T cell receptor sequence common to at least one of the antigen-binding T cells and at least one of the functional T cells; A method comprising the steps of: [Invention 1006] A method for selecting a T cell receptor, comprising: i) binding at least a first antigen-binding T cell present in a first plurality of T cells to at least one of at least a first class I P-MHC protein multimer, wherein P is an antigen of a predetermined type, the step comprising contacting the first plurality of T cells with at least one of the first class I P-MHC protein multimers; ii) activating at least a first functional T cell present in a second plurality of T cells, the step comprising contacting the second plurality of T cells with a plurality of cells presenting at least a first class I P-MHC protein; iii) identifying at least a portion of at least one T cell receptor sequence common to at least one of the antigen-binding T cells and at least one of the functional T cells; A method comprising the steps of: [Invention 1007] A method for selecting a T cell receptor, comprising: i) isolating a first T cell from a plurality of T cells, the first T cell being bound to a P-loaded MHC protein, wherein P is an antigen of a predetermined type; ii) further isolating a second T cell from the plurality of T cells, the second T cell expressing at least one biomarker indicative of activation by the antigen of the predetermined type; iii) matching at least a part of the T cell receptor sequence of the first T cell with at least a part of the T cell receptor sequence of the second T cell A method comprising the steps of: [Invention 1008] A method for detecting a functional T cell receptor clonotype, comprising: i) isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs; ii) forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell; iii) activating at least a first functional T cell, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic for the predetermined type of antigen; iv) confirming that the at least first functional T cell is configured to bind to a P-loaded MHC protein, where P is the predetermined type of antigen A method comprising the steps of: [Invention 1009] A method for detecting antigen-binding T cells, comprising: i) isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs; ii) forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell; iii) binding at least a first binding T cell to at least a first binder, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of binders, wherein the at least one of the plurality of binders comprises the predetermined type of antigen; iv) confirming that the at least first binding T cell is configured to be activated by a cell presenting the predetermined type of antigen A method comprising the steps of: [Invention 1010] A method for selecting a T cell receptor specific for a predetermined type of antigen, comprising: i) isolating a first plurality of T cells, wherein at least a portion of said first plurality of T cells is bound to a plurality of P-loaded MHC proteins, and P is said predetermined type of antigen; ii) further isolating a second plurality of T cells, wherein in the presence of a plurality of activators, at least a portion of said second plurality of T cells upregulates one or more activation signaling molecules and / or expresses one or more activation markers, and at least one of said second plurality of T cells and at least one of said plurality of activators is immunogenic for said predetermined type of antigen; iii) identifying at least a portion of at least one T cell receptor sequence common to both said at least a portion of said first plurality of T cells and said at least a portion of said second plurality of T cells; A method comprising the steps of: [Invention 1011] A method for selecting a T cell receptor specific for a predetermined type of antigen, comprising: i) isolating a first plurality of T cells, wherein in the presence of a plurality of first activators, at least a portion of said first plurality of T cells expresses one or more first activation markers; ii) further isolating a second plurality of T cells, wherein in the presence of a plurality of second activators, at least a portion of said second plurality of T cells upregulates one or more second activation markers and / or expresses one or more activation signaling molecules; iii) a) at least a portion of at least one common T cell receptor sequence, and b) a dissociation constant less than a threshold value for a P-loaded MHC protein, where P is said predetermined type of antigen, identifying at least one of said at least a portion of said first plurality of T cells and at least one of said portion of said second T cells having the same; A method comprising [Inventive concept 1012] A method for negatively selecting a T cell receptor clonotype, comprising: i) analyzing a mixture of T cells to identify a first antigen-binding T cell and a first antigen-activated T cell against a predetermined first type of antigen, and a second antigen-activated T cell against a predetermined second type of antigen; ii) a) being shared by at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells, and b) not being shared with any of the second antigen-activated T cells identifying at least a portion of at least one T cell receptor sequence; A method comprising [Inventive concept 1013] A method for negatively selecting a T cell receptor clonotype, comprising: i) analyzing a mixture of T cells to identify a first antigen-activated T cell and a first antigen-binding T cell against a predetermined first type of antigen, and a second antigen-binding T cell against a predetermined second type of antigen; ii) a) being shared by at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells, and b) not being shared with any of the second antigen-binding T cells identifying at least a portion of at least one T cell receptor sequence; A method comprising [Inventive concept 1014] A method for identifying a T cell activation marker, comprising: i) contacting a first plurality of T cells with a plurality of antigen-presenting cells, wherein the first plurality of T cells comprises a plurality of antigen-binding T cells and the antigen is a predetermined type of antigen; ii) measuring a plurality of expression rate profiles for at least a portion of the contacted plurality of antigen-binding T cells; iii) dividing at least a portion of the plurality of P-binding T cells that have been contacted into a plurality of T cell clusters; iv) measuring a functional response to P in at least two T cells present in at least a portion of the plurality of P-binding T cells that have been contacted; and v) mapping the expression rate profile to the plurality of T cell clusters to identify one of the plurality of T cell clusters that includes the at least two T cells; vi) identifying activation markers expressed by the at least two T cells; A method comprising the steps of. [Invention 1015] A method for screening a candidate antigen for an antigen-specific vaccine, i) isolating at least one T cell that binds to the candidate antigen from a population of PBMCs; ii) forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell; iii) activating at least a first functional T cell, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic with respect to the candidate antigen; A method comprising the steps of. [Invention 1016] A method for screening a candidate neoantigen for immunogenicity, i) isolating at least one T cell that binds to the candidate neoantigen from a population of PBMCs; ii) forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell; iii) activating at least a first functional T cell, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic with respect to the candidate neoantigen; A method comprising [Invention 1017] Any method of Inventions 1001 - 1016, wherein the analyzing step comprises analyzing a first portion of the mixture to identify said antigen - binding T cells and separately analyzing a second portion of the mixture to identify said antigen - activated T cells. [Invention 1018] Any method of Inventions 1001 - 1017, wherein analyzing the first portion of the mixture comprises detecting one or more T cells bound to a P - loaded MHC protein, where P is said predetermined type of antigen. [Invention 1019] Any method of Inventions 1001 - 1018, wherein said P - loaded MHC protein is bound to magnetic beads. [Invention 1020] Any method of Inventions 1001 - 1019, wherein detecting said one or more T cells bound to a P - loaded MHC protein comprises isolating said one or more T cells bound to the P - loaded MHC protein by magnetic separation. [Invention 1021] Any method of Inventions 1001 - 1020, wherein said P - loaded MHC protein is bound to a fluorophore. [Invention 1022] Any method of Inventions 1001 - 1021, wherein said one or more T cells bound to the P - loaded MHC protein are detected and isolated by fluorescence flow cytometry. [Invention 1023] Any method of Inventions 1001 - 1022, wherein detecting said one or more T cells bound to a P - loaded MHC protein comprises passing said one or more T cells bound to the P - loaded MHC protein through a fluorescence flow cytometry device. [Invention 1024] Any method of Inventions 1001 - 1023, wherein said MHC protein is an MHC class I protein. [Invention 1025] Any method of the present invention 1001 to 1024, wherein the P-loaded MHC protein is present in the P-loaded MHC protein multimer. [The present invention 1026] Any method of the present invention 1001 to 1025, wherein analyzing a second portion of the mixture individually to identify the antigen-activated T cells includes detecting one or more T cells expressing one or more activation markers. [The present invention 1027] Any method of the present invention 1001 to 1026, wherein detecting the one or more T cells expressing one or more activation markers includes isolating the one or more T cells expressing the one or more activation markers by magnetic separation. [The present invention 1028] Any method of the present invention 1001 to 1027, wherein detecting the one or more T cells expressing one or more activation markers includes passing the one or more T cells expressing the one or more activation markers through a fluorescence flow cytometry device. [The present invention 1029] Any method of the present invention 1001 to 1028, wherein in vitro priming is excluded. [The present invention 1030] Any method of the present invention 1001 to 1029, wherein the predetermined type of antigen is a peptide. [The present invention 1031] Any method of the present invention 1001 to 1030, wherein the peptide is composed of 8 to 15 amino acids. [The present invention 1032] Any method of the present invention 1001 to 1031, wherein the peptide is composed of 12 to 40 amino acids. [The present invention 1033] Any method of the present invention 1001 to 1032, wherein the predetermined type of antigen is derived from a tumor (e.g., a solid tumor). [The present invention 1034] Any method of the present invention 1001 to 1033, wherein the predetermined type of antigen is presented on the tumor. [Invention 1035] Any method of the inventions 1001 to 1034, wherein the antigen of the predetermined type is a neoantigen derived from a tumor. [Invention 1036] Any method of the inventions 1001 to 1035, wherein the antigen of the predetermined type is an individualized antigen. [Invention 1037] Any method of the inventions 1001 to 1036, wherein the antigen of the predetermined type is a shared tumor antigen. [Invention 1038] Any method of the inventions 1001 to 1037, wherein the shared tumor antigen is a cancer / testis antigen. [Invention 1039] Any method of the inventions 1001 to 1038, wherein the shared tumor antigen is a cancer / testis-like antigen. [Invention 1040] Any method of the inventions 1001 to 1039, wherein the shared tumor antigen is a tumor-associated peptide antigen. [Invention 1041] Any method of the inventions 1001 to 1040, wherein the antigen of the predetermined type is specific to a particular type of tumor. [Invention 1042] Any method of the inventions 1001 to 1041, wherein the antigen of the predetermined type is a tumor-associated peptide antigen. [Invention 1043] Any method of the inventions 1001 to 1042, wherein at least a part of the at least one T cell receptor sequence comprises at least one T cell receptor clonotype. [Invention 1044] Any method of the inventions 1001 to 1043, wherein at least a part of the at least one T cell receptor sequence comprises at least one T cell receptor alpha chain, at least one T cell receptor beta chain, or at least a pair of T cell receptor alpha chain and T cell receptor beta chain. [Invention 1045] Any method of the present invention from 1001 to 1044, wherein the identifying step comprises sequencing the at least one binding T cell at the single cell level. [The present invention 1046] Any method of the present invention from 1001 to 1045, wherein the identifying step comprises sequencing the at least one functional T cell at the single cell level. [The present invention 1047] Any method of the present invention from 1001 to 1046, wherein at least a portion of the at least one T cell receptor sequence comprises at least one CDR3 sequence. [The present invention 1048] Any method of the present invention from 1001 to 1047, wherein the at least one of the antigen-binding T cells and the at least one of the antigen-activated T cells are in total less than 1000 T cells per 1,000,000 T cells present in the mixture of T cells. [The present invention 1049] The step of preparing the mixture of T cells, i) isolating at least one T cell that binds to the predetermined type of antigen from a population of PBMCs, ii) expanding the at least one isolated T cell comprising the steps of Any method of the present invention from 1001 to 1048, further comprising. [The present invention 1050] Any method of the present invention from 1001 to 1049, wherein the at least one T cell is at least two T cells, and the expanding comprises polyclonally expanding the at least two T cells. [The present invention 1051] Any method of the present invention from 1001 to 1050, wherein the at least one of the antigen-binding T cells and the at least one of the antigen-activated T cells are in total less than 1000 T cells per 10,000,000 T cells present in the population of PBMCs. [The present invention 1052] Any method of the present invention from 1001 to 1051, wherein the mixture of stimulated lymphocytes and costimulated lymphocytes is a T cell. [The present invention 1053] Any method of the present invention from 1001 to 1052, wherein the mixture of stimulated lymphocytes and costimulated lymphocytes is a B cell. [The present invention 1054] Any method of the present invention from 1001 to 1053, wherein the mixture of stimulated lymphocytes and costimulated lymphocytes is a natural killer cell. [The present invention 1055] Any method of the present invention from 1001 to 1054, wherein the step of analyzing includes analyzing a first portion of the mixture to identify the stimulated lymphocytes and individually analyzing a second portion of the mixture to identify the costimulated lymphocytes. [The present invention 1056] Any method of the present invention from 1001 to 1055, wherein analyzing the first portion of the mixture includes detecting one or more stimulated lymphocytes bound to a protein, the protein containing the predetermined type of antigen. [The present invention 1057] Any method of the present invention from 1001 to 1056, wherein the protein is bound to magnetic beads. [The present invention 1058] Any method of the present invention from 1001 to 1057, wherein detecting the one or more stimulated lymphocytes bound to the protein includes isolating the one or more stimulated lymphocytes bound to the protein by magnetic separation. [The present invention 1059] Any method of the present invention from 1001 to 1058, wherein the protein is bound to a fluorophore. [The present invention 1060] Any method of the present invention from 1001 to 1059, wherein the one or more stimulated lymphocytes bound to the protein are detected and isolated by fluorescence flow cytometry. [The present invention 1061] Detecting the one or more stimulated lymphocytes bound to the protein comprises passing the one or more stimulated lymphocytes bound to the protein through a fluorescence flow cytometry device, according to any of the methods of the present invention from 1001 to 1060. [The present invention 1062] Analyzing the second portion of the mixture individually to identify the co-stimulated lymphocytes comprises detecting one or more stimulated lymphocytes expressing one or more markers, according to any of the methods of the present invention from 1001 to 1061. [The present invention 1063] Detecting the one or more stimulated lymphocytes expressing one or more markers comprises isolating the one or more stimulated lymphocytes expressing the one or more markers by magnetic separation, according to any of the methods of the present invention from 1001 to 1062. [The present invention 1064] Detecting the one or more stimulated lymphocytes expressing one or more markers comprises passing the one or more stimulated lymphocytes expressing the one or more markers through a fluorescence flow cytometry device, according to any of the methods of the present invention from 1001 to 1063. [The present invention 1065] Priming using professional antigen-presenting cells is excluded, according to any of the methods of the present invention from 1001 to 1064. [The present invention 1066] The predetermined type of antigen is a peptide, according to any of the methods of the present invention from 1001 to 1065. [The present invention 1067] The peptide is composed of 8 to 15 amino acids, according to any of the methods of the present invention from 1001 to 1066. [The present invention 1068] The peptide is composed of 12 to 40 amino acids, according to any of the methods of the present invention from 1001 to 1067. [The present invention 1069] The method according to any one of 1001 to 1068 of the present invention, wherein the antigen of the predetermined type is derived from a tumor. [The present invention 1070] The method according to any one of 1001 to 1069 of the present invention, wherein the antigen of the predetermined type is presented on a tumor. [The present invention 1071] The method according to any one of 1001 to 1070 of the present invention, wherein the antigen of the predetermined type is a neoantigen derived from a tumor. [The present invention 1072] The method according to any one of 1001 to 1071 of the present invention, wherein the antigen of the predetermined type is an individualized antigen. [The present invention 1073] The method according to any one of 1001 to 1072 of the present invention, wherein the individualized antigen is an individualized neoantigen selected based on a model. [The present invention 1074] The method according to any one of 1001 to 1073 of the present invention, wherein the antigen of the predetermined type is a shared tumor antigen. [The present invention 1075] The method according to any one of 1001 to 1074 of the present invention, wherein the shared tumor antigen is a cancer / testis antigen. [The present invention 1076] The method according to any one of 1001 to 1075 of the present invention, wherein the shared tumor antigen is a cancer / testis-like antigen. [The present invention 1077] The method according to any one of 1001 to 1076 of the present invention, wherein the shared tumor antigen is a tumor-associated peptide antigen. [The present invention 1078] The method according to any one of 1001 to 1077 of the present invention, wherein the antigen of the predetermined type is specific to a particular type of tumor. [The present invention 1079] The method according to any one of 1001 to 1078 of the present invention, wherein the antigen of the predetermined type is a tumor-associated peptide antigen. [The present invention 1080] The method according to any one of 1001 to 1079 of the present invention, wherein at least a part of the at least one receptor sequence comprises at least one receptor clonotype. [The present invention 1081] Any method according to any one of the present inventions 1001 to 1080, wherein at least a part of the at least one receptor sequence comprises at least one receptor alpha chain, at least one receptor beta chain, or at least one pair of receptor alpha and receptor beta chains. [Present Invention 1082] Any method according to any one of the present inventions 1001 to 1081, wherein the step of identifying comprises sequencing at least one of the stimulated lymphocytes at the single cell level. [Present Invention 1083] Any method according to any one of the present inventions 1001 to 1082, wherein the step of identifying comprises sequencing at least one of the co-stimulated lymphocytes at the single cell level. [Present Invention 1084] Any method according to any one of the present inventions 1001 to 1083, wherein at least a part of the at least one receptor sequence comprises at least one antigen recognition sequence. [Present Invention 1085] Any method according to any one of the present inventions 1001 to 1084, wherein the at least one of the stimulated lymphocytes and the at least one of the co-stimulated lymphocytes together are less than 1000 T cells per 1,000,000 T cells present in the mixture of lymphocytes. [Present Invention 1086] The step of preparing the mixture of lymphocytes, i) isolating at least one lymphocyte that binds to the predetermined type of antigen from a population of PBMCs, ii) proliferating the at least one isolated lymphocyte comprising the steps Any method according to any one of the present inventions 1001 to 1085, further comprising the step. [Present Invention 1087] Any method according to any one of the present inventions 1001 to 1086, wherein the at least two lymphocytes bind to the predetermined type of antigen, and the proliferating comprises polyclonally proliferating the at least two lymphocytes. [Present Invention 1088] Any method according to any one of inventions 1001 to 1087, wherein the at least one of the stimulated lymphocytes and the at least one of the costimulated lymphocytes are, in total, less than 1000 T cells per 10,000,000 lymphocytes present in the population of PBMCs. [Invention 1089] Any method according to any one of inventions 1001 to 1088, wherein the mixture of lymphocytes is a product primed using professional antigen-presenting cells. [Invention 1090] Any method according to any one of inventions 1001 to 1089, wherein the plurality of cells presenting at least one of the second predetermined types of antigen present the plurality of predetermined types of antigen within a predetermined concentration range. [Invention 1091] Any method according to any one of inventions 1001 to 1090, wherein the plurality of cells presenting at least one of the second predetermined types of antigen are prepared by pulsing the plurality of cells with an amount of the predetermined type of antigen. [Invention 1092] Any method according to any one of inventions 1001 to 1091, wherein the predetermined concentration range is based on the predicted concentration of the predetermined type of antigen in the tumor. [Invention 1093] Any method according to any one of inventions 1001 to 1092, wherein the step of binding comprises binding the at least first binding T cell to a P-loaded MHC protein, where P is the predetermined type of antigen. [Invention 1094] Any method according to any one of inventions 1001 to 1093, wherein the MHC protein is an MHC class I protein. [Invention 1095] Any method according to any one of inventions 1001 to 1094, wherein the P-loaded MHC protein is present in a P-loaded MHC protein multimer. [Invention 1096] Any method according to any one of inventions 1001 to 1095, wherein the first plurality of T cells and the second plurality of T cells are derived from a common population of PBMCs. [Invention 1097] The method according to any one of the present inventions 1001 to 1096, wherein the first plurality of T cells and the second plurality of T cells are derived from one or more healthy donors. [The present invention 1098] The method according to any one of the present inventions 1001 to 1097, wherein the one or more healthy donors are at least partially human leukocyte antigen (HLA) compatible with the subject. [The present invention 1099] The method according to any one of the present inventions 1001 to 1098, wherein the one or more healthy donors are at least partially HLA compatible with the subject with respect to the presentation of the predetermined type of antigen. [The present invention 1100] The method according to any one of the present inventions 1001 to 1099, wherein the one or more healthy donors are completely HLA compatible with the subject. [The present invention 1101] The method according to any one of the present inventions 1001 to 1100, wherein the one or more healthy donors are selectively HLA compatible with the subject. [The present invention 1102] The method according to any one of the present inventions 1001 to 1101, wherein the one or more healthy donors are compatible with the subject with respect to HLA-A. [The present invention 1103] The method according to any one of the present inventions 1001 to 1102, wherein the one or more healthy donors are compatible with the subject with respect to HLA-B. [The present invention 1104] The method according to any one of the present inventions 1001 to 1103, wherein the one or more healthy donors are compatible with the subject with respect to HLA-C. [The present invention 1105] The one or more healthy donors are compatible with the subject with respect to HLA-DP in the method according to any one of the present inventions 1001 to 1104. [The present invention 1106] The method according to any one of the present inventions 1001 to 1105, wherein the one or more healthy donors are compatible with the subject with respect to HLA-DQ. [The present invention 1107] Any one of the methods of the present invention 1001 to 1106, wherein the one or more healthy donors are compatible with the subject with respect to HLA-DR. [The present invention 1108] Any one of the methods of the present invention 1001 to 1107, wherein the one or more healthy donors are at least partially HLA-incompatible with the subject. [The present invention 1109] Any one of the methods of the present invention 1001 to 1108, wherein the one or more healthy donors are completely HLA-incompatible with the subject. [The present invention 1110] Any one of the methods of the present invention 1001 to 1109, wherein the one or more healthy donors are selectively HLA-incompatible with the subject. [The present invention 1111] Any one of the methods of the present invention 1001 to 1110, wherein the one or more healthy donors are incompatible with the subject with respect to HLA-A. [The present invention 1112] Any one of the methods of the present invention 1001 to 1111, wherein the one or more healthy donors are incompatible with the subject with respect to HLA-B. [The present invention 1113] Any one of the methods of the present invention 1001 to 1112, wherein the one or more healthy donors are incompatible with the subject with respect to HLA-C. [The present invention 1114] Any one of the methods of the present invention 1001 to 1113, wherein the one or more healthy donors are incompatible with the subject with respect to HLA-DP. [The present invention 1115] Any one of the methods of the present invention 1001 to 1114, wherein the one or more healthy donors are incompatible with the subject with respect to HLA-DQ. [The present invention 1116] Any one of the methods of the present invention 1001 to 1115, wherein the one or more healthy donors are incompatible with the subject with respect to HLA-DR. [The present invention 1117] Any of the methods of the present invention 1001-1116, wherein the one or more healthy donors are incompatible with the subject with respect to HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, or a combination of two or more of the foregoing. [The present invention 1118] Any of the methods of the present invention 1001-1117, wherein the first plurality of T cells and the second plurality of T cells comprise naive CD8 + T cells. [The present invention 1119] Any of the methods of the present invention 1001-1118, wherein the first plurality of T cells and the second plurality of T cells comprise naive T cells. [The present invention 1120] Any of the methods of the present invention 1001-1119, wherein the first plurality of T cells and the second plurality of T cells comprise memory T cells. [The present invention 1121] Any of the methods of the present invention 1001-1120, wherein the first plurality of T cells and the second plurality of T cells comprise CD8 + T cells. [The present invention 1122] Any of the methods of the present invention 1001-1121, wherein the first plurality of T cells and the second plurality of T cells comprise CD4 + T cells. [The present invention 1123] Any of the methods of the present invention 1001-1122, wherein the first plurality of T cells and the second plurality of T cells comprise CD4 + CD8 + T cells. [The present invention 1124] Any of the methods of the present invention 1001-1123, wherein the first plurality of T cells and the second plurality of T cells comprise CD4 - CD8 + T cells. [The present invention 1125] Any of the methods of the present invention 1001-1124, wherein the first plurality of T cells and the second plurality of T cells comprise CD4 + CD8 - T cells. [The present invention 1126] Any method according to any one of inventions 1001 to 1125, wherein the plurality of cells presenting at least one of the second predetermined types of antigens includes one or more tumor cells. [The present invention 1127] Any method according to any one of inventions 1001 to 1126, wherein the plurality of cells presenting at least one of the second predetermined types of antigens includes one or more dendritic cells. [The present invention 1128] Any method according to any one of inventions 1001 to 1127, wherein the plurality of cells presenting at least one of the second predetermined types of antigens includes one or more macrophages. [The present invention 1129] Any method according to any one of inventions 1001 to 1128, wherein the plurality of cells presenting at least one of the second predetermined types of antigens includes one or more monocytes. [The present invention 1130] Any method according to any one of inventions 1001 to 1129, wherein the plurality of cells presenting at least one of the second predetermined types of antigens includes one or more B cells. [The present invention 1131] Any method according to any one of inventions 1001 to 1130, wherein the plurality of cells presenting at least one of the second predetermined types of antigens includes one or more cells presenting at least one of the second predetermined types of antigens, and expresses the predetermined type of antigen. [The present invention 1132] Any method according to any one of inventions 1001 to 1131, further comprising the step of detecting the binding by flow cytometry. [The present invention 1133] Any method according to any one of inventions 1001 to 1132, wherein one of the first predetermined types of antigens is bound to magnetic beads, and the method further comprises the step of detecting the at least first antigen-binding T cells by magnetic separation. [The present invention 1134] The method according to any one of the present inventions 1001 to 1133, further comprising the step of detecting said activation by flow cytometry. [The present invention 1135] The method according to any one of the present inventions 1001 to 1134, further comprising the step of detecting said at least first functional T cells by magnetic separation. [The present invention 1136] The method according to any one of the present inventions 1001 to 1135, further comprising the step of detecting said activation, which comprises detecting one or more biomarkers. [The present invention 1137] The method according to any one of the present inventions 1001 to 1136, wherein said one or more biomarkers comprise CD137. [The present invention 1138] The method according to any one of the present inventions 1001 to 1137, further comprising the step of detecting said activation, which comprises detecting the presence of one or more molecules suggesting T cell activation. [The present invention 1139] The method according to any one of the present inventions 1001 to 1138, wherein said one or more molecules comprise interferon gamma. [The present invention 1140] The method according to any one of the present inventions 1001 to 1139, further comprising the step of detecting said activation, which comprises detecting T cell proliferation. [The present invention 1141] The method according to any one of the present inventions 1001 to 1140, further comprising the step of inducing said plurality of T cells from at least two T cells each individually bound to at least two P-loaded MHC proteins. [The present invention 1142] The method according to any one of the present inventions 1001 to 1141, wherein said step of inducing comprises growing said at least first T cell and said at least second T cell. [The present invention 1143] The method according to any one of the present inventions 1001 to 1142, wherein said step of growing comprises growing said at least first T cell and said at least second T cell polyclonally. [The present invention 1144] Any method according to any one of the present inventions 1001 to 1143, wherein the at least first T cell and the at least second T cell are in a mixture during the proliferation. [The present invention 1145] Any method according to any one of the present inventions 1001 to 1144, wherein the at least first T cell and the at least second T cell are separated from each other before the proliferation. [The present invention 1146] Any method according to any one of the present inventions 1001 to 1145, wherein the forming step includes indirect T cell receptor crosslinking. [The present invention 1147] Any method according to any one of the present inventions 1001 to 1146, wherein the forming step is limited to a single polyclonal proliferation. [The present invention 1148] Any method according to any one of the present inventions 1001 to 1147, wherein the forming step includes multiple polyclonal proliferations. [The present invention 1149] Any method according to any one of the present inventions 1001 to 1148, wherein isolating at least one additional T cell that binds to the predetermined type of antigen follows at least one of the multiple polyclonal proliferations. [The present invention 1150] Any method according to any one of the present inventions 1001 to 1149, wherein the at least first functional T cell has a dissociation constant of less than 50 μM for the P-loaded MHC protein. [The present invention 1151] Any method according to any one of the present inventions 1001 to 1150, wherein the at least first functional T cell has a half-life of 2 to 10 seconds for the P-loaded MHC protein. [The present invention 1152] The predetermined type of antigen is a tumor-associated peptide antigen, and the at least first functional T cell has i) a dissociation constant of less than 50 μM for the P-loaded MHC protein; and ii) a half-life in the range of 2 to 10 seconds for the P-loaded MHC protein. Any method according to any one of the present inventions 1001 to 1151. [The present invention 1153] Any method of the present invention from 1001 to 1152, wherein at least one of the plurality of activators is antigenic with respect to the predetermined type of antigen. [The present invention 1154] Any method of the present invention from 1001 to 1153, wherein the cell presenting the predetermined type of antigen is an antigen-presenting cell. [The present invention 1155] Any method of the present invention from 1001 to 1154, wherein the antigen-presenting cell is a professional antigen-presenting cell. [The present invention 1156] Any method of the present invention from 1001 to 1155, wherein at least a portion of the at least one T cell receptor sequence is present in at least 0.005% of the combination of at least a portion of the first plurality of T cells and at least a portion of the second plurality of T cells. [The present invention 1157] Any method of the present invention from 1001 to 1156, wherein at least one of the plurality of activators is antigenic with respect to the predetermined type of antigen. [The present invention 1158] Any method of the present invention from 1001 to 1157, wherein at least one of the plurality of first activators is immunogenic with respect to the predetermined type of antigen and / or at least one of the plurality of second activators is immunogenic with respect to the predetermined type of antigen. [The present invention 1159] Any method of the present invention from 1001 to 1158, wherein at least one of the plurality of first activators is antigenic with respect to the predetermined type of antigen and / or at least one of the plurality of second activators is antigenic with respect to the predetermined type of antigen. [The present invention 1160] Any method of the present invention from 1001 to 1159, wherein at least one of the plurality of first activators contains the predetermined type of antigen and / or at least one of the plurality of second activators contains the predetermined type of antigen. [The present invention 1161] Any method of the present invention from 1001 to 1160, wherein at least one of the plurality of first activators is a cell presenting the predetermined type of antigen and / or at least one of the plurality of second activators is a cell presenting the predetermined type of antigen. [The present invention 1162] Any method of the present invention from 1001 to 1161, wherein at least one of the plurality of first activators contains a P-loaded MHC protein and / or at least one of the plurality of second activators contains a P-loaded MHC protein. [The present invention 1163] Any method of the present invention from 1001 to 1162, wherein at least one of the plurality of first activators is a cell that endogenously expresses the predetermined type of antigen and / or at least one of the plurality of second activators is a cell that endogenously expresses the predetermined type of antigen. [The present invention 1164] Any method of the present invention from 1001 to 1163, wherein at least one of the plurality of first activators contains a P-loaded MHC protein and / or at least one of the plurality of second activators is a cell that endogenously expresses the predetermined type of antigen. [The present invention 1165] Any method of the present invention from 1001 to 1164, wherein the dissociation constant corresponds to the binding between at least a part of at least one T cell receptor sequence and the P-loaded MHC protein. [The present invention 1166] Any method of the present invention from 1001 to 1165, wherein the threshold value is less than 1000 μM. [The present invention 1167] Any method of the present invention from 1001 to 1166, wherein the predetermined first type of antigen is a first peptide and the predetermined second type of antigen is a second peptide. [The present invention 1168] Any method of the present invention from 1001 to 1167, wherein the first peptide is expressed by a variant of the gene that expresses the second peptide. [The present invention 1169] Any method of the present invention from 1001 to 1168, wherein the first peptide is expressed by an allele of the gene expressing the second peptide. [The present invention 1170] Any method of the present invention from 1001 to 1169, wherein the second peptide is expressed by a wild-type gene. [The present invention 1171] Any method of the present invention from 1001 to 1170, wherein the first peptide is a neoantigen and the second peptide is expressed by a related wild-type gene. [The present invention 1172] Any method of the present invention from 1001 to 1171, wherein the first peptide and the second peptide differ by at least 5 amino acids. [The present invention 1173] Any method of the present invention from 1001 to 1172, wherein the first peptide and the second peptide differ by 5 to 15 amino acids. [The present invention 1174] Any method of the present invention from 1001 to 1173, wherein the first peptide and the second peptide have a sequence identity of less than 75%. [The present invention 1175] Any method of the present invention from 1001 to 1174, wherein the first peptide and the second peptide have a sequence identity of 55% to 80%. [The present invention 1176] Any method of the present invention from 1001 to 1175, wherein the step of identifying the first antigen-activated T cells includes contacting a portion of the mixture of T cells with cells that endogenously present the predetermined first type of antigen. [The present invention 1177] Any method of the present invention from 1001 to 1176, wherein the step of identifying the second antigen-activated T cells includes contacting a portion of the mixture of T cells with cells that endogenously present the predetermined second type of antigen. [The present invention 1178] The method according to any one of 1001 to 1177 of the present invention, wherein the step of identifying the first antigen-activated T cells includes contacting a portion of the mixture of T cells with cells loaded with the predetermined first type of antigen. [The present invention 1179] The method according to any one of 1001 to 1178 of the present invention, wherein the step of identifying the second antigen-activated T cells includes contacting a portion of the mixture of T cells with cells loaded with the predetermined second type of antigen. [The present invention 1180] The method according to any one of 1001 to 1179 of the present invention, wherein the P-binding T cells are identified using a bioinformatics filter that compares at least a plurality of portions of the T cell receptor sequences of at least a portion of the plurality of contacted P-binding T cells with at least a plurality of portions of a predetermined T cell receptor sequence. [The present invention 1181] The method according to any one of 1001 to 1180 of the present invention, wherein the step of dividing includes dividing the plurality of contacted P-binding T cells into a plurality of groups, and at least one of the groups consists of T cells having at least a plurality of portions of a common T cell receptor sequence. [The present invention 1182] The method according to any one of 1001 to 1181 of the present invention, wherein the step of dividing includes dividing the plurality of contacted P-binding T cells into a plurality of groups, and at least one of the groups consists of T cells having at least a plurality of portions of a T cell receptor sequence characterized by at least 70% sequence identity to each other. [The present invention 1183] The method according to any one of 1001 to 1182 of the present invention, wherein the step of dividing includes dividing the plurality of contacted P-binding T cells into a plurality of groups, and at least one of the groups consists of T cells having at least a plurality of portions of a T cell receptor sequence in which at most one amino acid differs from each other. [The present invention 1184] The step of dividing includes dividing the plurality of P-binding T cells brought into contact into a plurality of groups, and at least one of the groups consists of T cells having at least a plurality of portions of a T cell receptor sequence that differ only by conservative substitutions, according to any of the methods of the present invention from 1001 to 1183. [The present invention 1185] The step of dividing includes grouping of lymphocyte interactions by paratope hotspots (GLIPH), according to any of the methods of the present invention from 1001 to 1184. [The present invention 1186] At least a plurality of portions of the common T cell receptor sequence are at least a plurality of portions of the CDR3 region, according to any of the methods of the present invention from 1001 to 1185. [The present invention 1187] At least a plurality of portions of the CDR3 region include a linear amino acid sequence having a length of 6 to 35 amino acids, according to any of the methods of the present invention from 1001 to 1186. [The present invention 1188] At least a plurality of portions of the CDR3 region do not include a stem region, according to any of the methods of the present invention from 1001 to 1187. [The present invention 1189] At least a plurality of portions of the CDR3 region include a CDR3 beta chain portion, according to any of the methods of the present invention from 1001 to 1188. [The present invention 1190] The step of dividing is performed using an algorithm, according to any of the methods of the present invention from 1001 to 1189. [The present invention 1191] The algorithm includes an analysis of the similarity of the plurality of expression rate profiles, according to any of the methods of the present invention from 1001 to 1190. [The present invention 1192] The plurality of expression rate profiles include expression rates regarding one or more activation markers suggesting a functional response to P, according to any of the methods of the present invention from 1001 to 1191. [The present invention 1193] Any method of the present invention from 1001 to 1192, wherein the one or more activation markers include CD137, CD69, CD25, Ki67, CD107, CD122, CD27, CD28, CD95, CD134, killer cell lectin-like receptor G1 (KLRG1), CD38, or CD154. [The present invention 1194] Any method of the present invention from 1001 to 1193, wherein the one or more activation markers are selected from the group consisting of CD137, CD69, CD25, Ki67, and CD107, or a combination thereof. [The present invention 1195] Any method of the present invention from 1001 to 1194, wherein the algorithm is a cluster analysis algorithm. [The present invention 1196] Any method of the present invention from 1001 to 1195, wherein the algorithm includes the t-distributed stochastic neighbor embedding method. [The present invention 1197] Any method of the present invention from 1001 to 1196, wherein the functional response to the measured P includes the detection of one or more activation markers and / or one or more secreted molecules. [The present invention 1198] Any method of the present invention from 1001 to 1197, wherein the one or more activation markers include CD137, CD69, CD25, Ki67, CD107, CD122, CD27, CD28, CD95, CD134, killer cell lectin-like receptor G1 (KLRG1), CD38, or CD154. [The present invention 1199] Any method of the present invention from 1001 to 1198, wherein the one or more activation markers are selected from the group consisting of CD137, CD69, CD25, Ki67, and CD107, or a combination thereof. [The present invention 1200] Any method of the present invention from 1001 to 1199, wherein the one or more secreted molecules include one or more cytokines. [The present invention 1201] Any of the methods of the present invention 1001 to 1200, wherein the one or more cytokines are interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin-2 (IL-2), or a combination of two or more of the foregoing. [The present invention 1202] Any of the methods of the present invention 1001 to 1201, wherein the one or more secreted molecules include granzyme. [The present invention 1203] Any of the methods of the present invention 1001 to 1202, wherein the one or more secreted molecules include perforin. [The present invention 1204] Any of the methods of the present invention 1001 to 1203, wherein the measured functional response to the P includes detection of T cell proliferation. [The present invention 1205] Any of the methods of the present invention 1001 to 1204, wherein the first plurality of T cells and the second plurality of T cells are derived from a common starting population of PBMCs. [The present invention 1206] Any of the methods of the present invention 1001 to 1205, wherein the plurality of expression rate profiles are obtained from a series of single cell transcriptome analyses. [The present invention 1207] Any of the methods of the present invention 1001 to 1206, wherein the T cells included in the one of the plurality of T cell clusters express the predetermined first activation marker at an average of a second expression rate that exceeds a first expression rate threshold. [The present invention 1208] Any of the methods of the present invention 1001 to 1207, wherein the T cells included in the one of the plurality of T cell clusters express the second activation marker at an average of a second expression rate that exceeds a second expression rate threshold. [The present invention 1209] Any of the methods of the present invention 1001 to 1208, further comprising the step of identifying the plurality of P-binding T cells by matching the T cell receptor sequences of the plurality of P-binding T cells to a predetermined T cell receptor sequence. [The present invention 1210] Any method of the present invention from 1001 to 1209, wherein the predetermined T cell receptor sequence is determined by sequencing a second plurality of T cells bound to a P-loaded MHC protein. [The present invention 1211] Any method of the present invention from 1001 to 1210, wherein the activation marker is not expressed or is downregulated in at least two other T cells present in another one of the plurality of T cell clusters, and the at least two other T cells do not show a functional response when measured. [The present invention 1212] Any method of the present invention from 1001 to 1211, wherein the candidate antigen is a neoantigen. [The present invention 1213] Any method of the present invention from 1001 to 1212, wherein the antigen-specific vaccine is for the treatment of cancer. [The present invention 1214] Any method of the present invention from 1001 to 1213, wherein the predetermined type of antigen is a neoantigen. [The present invention 1215] Any method of the present invention from 1001 to 1214, wherein the neoantigen is a peptide. [The present invention 1216] Any method of the present invention from 1001 to 1215, wherein the peptide is composed of 8 to 15 amino acids. [The present invention 1217] Any method of the present invention from 1001 to 1216, wherein the peptide is composed of 12 to 40 amino acids. [The present invention 1218] Any method of the present invention from 1001 to 1217, wherein the neoantigen is derived from a tumor. [The present invention 1219] Any method of the present invention from 1001 to 1218, wherein the tumor is a solid tumor. [The present invention 1220] Any method of the present invention from 1001 to 1219, wherein the neoantigen is presented on the tumor. [The present invention 1221] The method according to any one of the methods 1001 to 1220 of the present invention, wherein the neoantigen is an individualized neoantigen. [The present invention 1222] The method according to any one of the methods 1001 to 1221 of the present invention, wherein the neoantigen is a shared tumor neoantigen. [The present invention 1223] The method according to any one of the methods 1001 to 1222 of the present invention, wherein the shared tumor neoantigen is a tumor-associated peptide neoantigen. [The present invention 1224] The method according to any one of the methods 1001 to 1223 of the present invention, wherein the neoantigen is specific to a particular type of tumor. [The present invention 1225] The method according to any one of the methods 1001 to 1224 of the present invention, wherein the neoantigen is a tumor-associated peptide neoantigen. [The present invention 1226] The method according to any one of the methods 1001 to 1225 of the present invention, wherein the neoantigen is selected from one or more neoantigens identified by a model. [The present invention 1227] The method according to any one of the methods 1001 to 1226 of the present invention, wherein the model is calibrated by machine learning. [The present invention 1228] The method according to any one of the methods 1001 to 1227 of the present invention, wherein the one or more neoantigens are individualized neoantigens. [The present invention 1229] The method according to any one of the methods 1001 to 1228 of the present invention, wherein the one or more neoantigens are present in a list of shared neoantigens. [The present invention 1230] The method according to any one of the methods 1001 to 1229 of the present invention, wherein the neoantigen is selected from one or more neoantigens identified by an artificial intelligence model. [The present invention 1231] The method according to any one of the methods 1001 to 1230 of the present invention, wherein the artificial intelligence model includes a neural network. [The present invention 1232] The method according to any one of the methods 1001 to 1231 of the present invention, wherein the neoantigen is selected from a set of presentation likelihoods. [The present invention 1233] Any method of the present invention 1001-1232, wherein the individualized neoantigen is selected based on one or more of the machine learning methods, software, and / or systems disclosed in the cited references. [The present invention 1234] Any method of the present invention 1001-1233, wherein the predetermined type of antigen is a viral antigen. [The present invention 1235] Any method of the present invention 1001-1234, wherein in vitro priming is excluded. [The present invention 1236] A composition obtained by any one of the methods of the present invention 1001-1235. [The present invention 1237] A composition comprising an artificial T cell receptor selective for a predetermined type of antigen, i) a) Analyzing a mixture of natural T cells to identify antigen-binding T cells and antigen-activated T cells for the predetermined type of antigen; b) Identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the antigen-activated T cells, wherein the at least a portion of the at least one T cell receptor sequence contains the at least a portion of the CDR3 region; At least a portion of the CDR3 region selected thereby, ii) A T cell receptor fragment And a composition comprising [The present invention 1238] A T cell comprising an artificial T cell receptor or a fragment thereof obtained by any one of the methods of the present invention 1001-1235. [The present invention 1239] The T cell of the present invention 1238 for use in the treatment of cancer. [The present invention 1240] A kit comprising the composition of the present invention 1236 or 1237. [The present invention 1241] A kit for use in any one of the methods 1001 - 1235 of the present invention.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0109] The present disclosure generally relates to a discovery that a mixture of T cells (e.g., a mixture of naive T cells derived from a starting PBMC sample) can be more rapidly and with fewer steps identified for T cell receptor development suitable for immunotherapy by dividing the mixture into a plurality of portions that are separately assayed for antigen binding and functionality. T cell receptors from each portion can be sequenced, and based on both antigen binding and functional T cells, overlapping T cell receptors are identified for further development. The present disclosure more specifically, in part, is based on the discovery that this approach does not necessarily require in vitro priming of the starting sample and thus can reduce deleterious effects caused by downregulation of T cell receptors and / or exposure to high concentrations of antigen. Further, functional assays can be performed by exposing the T cells to activators (e.g., antigen presenting cells or tumor cells) that present antigen at physiological concentrations, and thus there is a higher likelihood of identifying T cell receptors that actually give rise to a functional T cell line.

[0110] The T cell receptor is a highly diverse heterodimer consisting of a combination of alpha ("α") and beta ("β") chains (αβ TCR) or gamma delta ("γδ") chains (γδ TCR). The T cell receptor chains consist of a variable region important for antigen recognition and a constant region. The variable regions of the T cell receptor α and δ chains are encoded by several variable (V) and joining (J) genes, while the T cell receptor β and γ chains are additionally encoded by diversity (D) genes. Each TCR chain contains three hypervariable loops named complementarity-determining regions (CDR1-3) in its structure. CDR1 and 2 are encoded by the V gene and are required for the interaction between the MHC complex and the TCR. However, CDR3 is encoded by the junction region between the V and J or D and J genes and is therefore highly variable. Since CDR3 is the region of the TCR that directly contacts the peptide antigen, it plays an essential role in the interaction between the peptide-MHC complex and the TCR. For this reason, CDR3 is often used as the region of interest for determining the T cell receptor clonotype, as it is very unlikely that two T cells will express the same CDR3 nucleotide sequence unless they are derived from T cells that have proliferated clonally.

[0111] Accordingly, certain embodiments can provide, for example, methods, compositions, assays, systems, devices, and / or kits for identifying at least one T cell receptor component having antigen-binding and antigen-activating T cells against a given type of antigen. For example, in some embodiments, the present disclosure provides methods for selecting T cell receptor clonotypes.

[0112] In certain embodiments, a method for selecting a T cell receptor clonotype includes analyzing a mixture of T cells to identify antigen-binding T cells and antigen-activated T cells for a given type of antigen, and identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the antigen-activated T cells. In some embodiments, a method for selecting a T cell receptor clonotype includes analyzing a mixture of naive T cells to identify antigen-binding T cells and functional T cells for a given type of antigen, and identifying at least a portion of at least one T cell receptor sequence shared by at least one of the antigen-binding T cells and at least one of the functional T cells.

[0113] Also provided herein is a method for selecting a shared receptor sequence within lymphocytes, the method including analyzing a mixture of lymphocytes to identify stimulated lymphocytes and co-stimulated lymphocytes for a given type of antigen, and identifying at least a portion of at least one receptor sequence shared by at least one of the stimulated lymphocytes and at least one of the co-stimulated lymphocytes.

[0114] The disclosure also provides a method for selecting a T cell receptor. In some embodiments, the method for selecting a T cell receptor includes binding at least a first antigen-binding T cell to at least one of at least a first given type of antigen, the step including contacting a first plurality of T cells with at least one of at least a first given type of antigen, activating at least a first functional T cell, the step including contacting a second plurality of T cells with a plurality of cells presenting at least one of at least a second given type of antigen, and identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0115] In other embodiments, a method for selecting a T cell receptor comprises binding at least a first antigen-binding T cell present in a first plurality of T cells to at least one of at least a first class I P-MHC protein multimer, wherein P is an antigen of a predetermined type, the step of contacting the first plurality of T cells with at least one of the first class I P-MHC protein multimers; activating at least a first functional T cell present in a second plurality of T cells, the step of contacting the second plurality of T cells with a plurality of cells presenting at least one of at least a first class II P-MHC protein multimer; and identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell. In yet other embodiments, a method for selecting a T cell receptor comprises binding at least a first antigen-binding T cell present in a first plurality of T cells to at least one of at least a first class II P-MHC protein multimer, wherein P is an antigen of a predetermined type, the step of contacting the first plurality of T cells with at least one of the first class II P-MHC protein multimers; activating at least a first functional T cell present in a second plurality of T cells, the step of contacting the second plurality of T cells with a plurality of cells presenting at least a first class I P-MHC protein; and identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0116] In some embodiments, a method for selecting a T cell receptor comprises binding at least a first antigen-binding T cell present in a first plurality of T cells to one of at least a first class I P-MHC protein multimer, wherein P is an antigen of a predetermined type, and contacting the first plurality of T cells with one of the first class I P-MHC protein multimers; activating at least a first functional T cell present in a second plurality of T cells, comprising contacting the second plurality of T cells with a plurality of cells presenting at least the first class I P-MHC protein; and identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell. In other embodiments, a method for selecting a T cell receptor comprises binding at least a first antigen-binding T cell present in a first plurality of T cells to one of at least a first class II P-MHC protein multimer, wherein P is an antigen of a predetermined type, and contacting the first plurality of T cells with one of the first class II P-MHC protein multimers; activating at least a first functional T cell present in a second plurality of T cells, comprising contacting the second plurality of T cells with a plurality of cells presenting at least the first class II P-MHC protein multimer; and identifying at least a portion of at least one T cell receptor sequence common to at least one antigen-binding T cell and at least one functional T cell.

[0117] In yet another embodiment, a method for selecting a T cell receptor comprises isolating a first T cell from a plurality of T cells, wherein the first T cell is bound to a P-loaded MHC protein and P is an antigen of a predetermined type; further isolating a second T cell from the plurality of T cells, wherein the second T cell expresses at least one biomarker indicative of activation by an antigen of a predetermined type; and matching at least a portion of the T cell receptor sequence of the first T cell with at least a portion of the T cell receptor sequence of the second T cell.

[0118] Methods for detecting antigen-binding T cells are also provided herein. In some embodiments, a method for detecting antigen-binding T cells comprises isolating at least one T cell that binds to an antigen of a predetermined type from a population of PBMCs; forming a plurality of cognate T cells, comprising expanding the at least one isolated T cell; binding at least a first binding T cell to at least a first binder, comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of binders, wherein at least one of the plurality of binders comprises an antigen of a predetermined type; and confirming that the at least a first binding T cell is configured to be activated by a cell presenting an antigen of a predetermined type. Antigen-binding T cells can be detected by an assay using a binder disclosed herein or in one of the incorporated references.

[0119] The present disclosure also provides a method for detecting a functional T cell receptor clonotype, comprising the steps of isolating at least one T cell that binds to a predetermined type of antigen from a population of PBMCs, forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell, activating at least a first functional T cell, the step comprising contacting a T cell derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic for the predetermined type of antigen, and confirming that at least the first functional T cell is configured to bind to a P-loaded MHC protein, wherein P is the predetermined type of antigen.

[0120] It is understood that the present disclosure also provides a method of combining one or more of the above methods. For example, a method for detecting a functional T cell receptor clonotype can be combined with a method for detecting antigen-binding T cells to facilitate the selection of T cell receptors that bind to a specific antigen and are functional. Thus, in some embodiments, a method for selecting a T cell receptor specific for a predetermined type of antigen, comprising isolating a first plurality of T cells, wherein at least a portion of the first plurality of T cells binds to a plurality of P-loaded MHC proteins, wherein P is the predetermined type of antigen, further isolating a second plurality of T cells, wherein in the presence of a plurality of activators, at least a portion of the second plurality of T cells upregulates one or more activation signaling molecules and / or expresses one or more activation markers, wherein at least one of the plurality of activators is immunogenic for the predetermined type of antigen, and identifying at least a portion of at least one T cell receptor sequence common to both at least a portion of the first plurality of T cells and at least a portion of the second plurality of T cells.

[0121] In other embodiments, a method for selecting a T cell receptor specific for a predetermined type of antigen comprises the steps of isolating a first plurality of T cells, wherein at least a portion of the first plurality of T cells express one or more first activation markers in the presence of a plurality of first activators; further isolating a second plurality of T cells, wherein at least a portion of the second plurality of T cells upregulate one or more second activation markers and / or express one or more activation signaling molecules in the presence of a plurality of second activators; and identifying at least one of at least a portion of the first plurality of T cells and at least a portion of the second plurality of T cells that have a dissociation constant less than a threshold for a P-loaded MHC protein, wherein at least a portion of a common T cell receptor sequence is present and P is the predetermined type of antigen.

[0122] Thus, in certain embodiments, for example, at least one T cell receptor component can comprise at least one T cell receptor component. In certain embodiments, for example, at least one T cell receptor component can comprise at least one T cell receptor clonotype. In certain embodiments, for example, at least one T cell receptor component can comprise at least one T cell receptor alpha chain. In certain embodiments, for example, at least one T cell receptor component can comprise at least one T cell receptor beta chain. In certain embodiments, for example, at least one T cell receptor component can comprise at least a pair of T cell receptor alpha chain and T cell receptor beta chain. In certain embodiments, for example, the step of identifying can comprise sequencing at least one binding T cell at the single cell level. In certain embodiments, for example, the step of identifying can comprise sequencing at least one functional T cell at the single cell level. In certain embodiments, for example, at least one T cell receptor component can comprise at least one CDR3 sequence. In certain embodiments, for example, at least one CDR3 sequence can comprise an amino acid sequence (e.g., a linear sequence) consisting of 16 to 106 amino acids. In some embodiments, at least one CDR3 sequence can comprise an amino acid sequence (e.g., a linear sequence) consisting of 6 to 35 amino acids. In yet further embodiments, at least one CDR3 sequence can comprise an amino acid sequence (e.g., a linear sequence) consisting of 6 to 12 amino acids. In certain embodiments, for example, at least one CDR3 sequence can exclude the stem region. In certain embodiments, for example, at least one CDR3 sequence can comprise the CDR3 beta chain moiety(ies).

[0123] Sequencing of the T cell receptor can be performed using methods known in the art, such as the methods disclosed in the incorporated references. For example, by way of example and without limitation, sequencing can be performed by restriction enzyme digestion of the query DNA followed by gel electrophoresis and Southern blotting using probes for known T cell receptor genes; next-generation sequencing (NGS) (e.g., Illumina sequencing platform, Ion Torrent or Pacific Biosciences); or PCR-based assays. There are several approaches for extracting CDR data from sequencing reads and determining the clonotype. For example, one exemplary strategy for characterizing the CDR3 sequence is T cell receptor profiling, which involves amplifying cDNA or genomic DNA from the T cell receptor beta chain CDR3 (β-CDR3) locus using pre-designed PCR primers followed by deep sequencing. Another exemplary approach involves T cell receptor profiling based on RNA sequencing (RNA-seq), which provides data from all transcribed genes present in the sample and enables simultaneous analysis of TCRα, TCRβ, TCRγ, and TCRδ. However, it is understood that the above methods are merely exemplary and any sequencing method known in the art may be used to determine the T cell receptor sequence.

[0124] In certain embodiments, for example, the step of identifying at least one T cell receptor component of antigen-binding and antigen-activated T cells can include comparing the T cell receptor from a first sample containing antigen-binding T cells (some of which may or may not be antigen-activated) to the T cell receptor from a second sample containing antigen-activated T cells (some of which may or may not be antigen-binding). In certain embodiments, for example, comparing can include matching any of the T cell receptor components between the T cells from the first sample and the T cells from the second sample.

[0125] In certain embodiments, for example, a given type of antigen may be a peptide. In certain embodiments, for example, the peptide can consist of at least 8 amino acids. In some embodiments, the peptide consists of 9 amino acids. In some embodiments, the peptide consists of 10 amino acids. In some embodiments, the peptide consists of 11 amino acids. In some embodiments, the peptide consists of 12 amino acids. In some embodiments, the peptide consists of 13 amino acids. In some embodiments, the peptide consists of 14 amino acids. In some embodiments, the peptide consists of 15 amino acids. However, the peptide can also consist of more than 15 amino acids, and it is understood that the lengths of the peptides above are merely exemplary.

[0126] In certain embodiments, for example, a given type of antigen may be a peptide that is 8 - 20 amino acids. For example, in some embodiments, the peptide can consist of 8 - 15 amino acids. In some embodiments, the peptide can consist of 8 - 12 amino acids. In certain embodiments, for example, the peptide can consist of at least 12 amino acids, such as 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 35 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids or 40 amino acids.

[0127] In certain embodiments, for example, a given type of antigen may be a peptide that is 12 - 40 amino acids. In certain embodiments, the peptide can consist of 12 - 30 amino acids. In some embodiments, the peptide can consist of 12 - 20 amino acids.

[0128] Major histocompatibility complex (MHC) class I and class II proteins share the task of presenting peptides on the cell surface for recognition by T cells. Immunogenic peptide-MHC class I (pMHCI) complexes are presented on nucleated cells and recognized by cytotoxic CD8+ T cells. Presentation of pMHCII by antigen-presenting cells [e.g., dendritic cells (DCs), macrophages or B cells], on the other hand, can activate CD4+ T cells and lead to the modulation and regulation of effector cells. In all cases, it is the clonotype T cell receptor that interacts with a given pMHC complex and potentially results in sustained cell:cell contact formation and T cell activation.

[0129] Thus, in certain embodiments, for example, a given type of antigen (e.g., a peptide) can have a binding affinity for an MHC protein (e.g., an MHC class I protein or an MHC class II protein). In certain embodiments, for example, a given type of antigen can have a binding affinity for an MHC class I protein of less than 1000 μM. In some embodiments, a given type of antigen can have a binding affinity for an MHC class I protein of less than 100 μM. In some embodiments, a given type of antigen can have a binding affinity for an MHC class I protein of less than 50 μM. In some embodiments, a given type of antigen can have a binding affinity for an MHC class I protein of less than 10 μM. In some embodiments, a given type of antigen can have a binding affinity for an MHC class I protein of less than 1 μM. In some embodiments, a given type of antigen can have a binding affinity for an MHC class I protein of less than 0.1 μM.

[0130] In certain embodiments, for example, a given type of antigen may be a neoantigen (e.g., an antigen having at least one modification distinct from the corresponding wild-type parental antigen). In certain embodiments, for example, a neoantigen can include modifications to a parental antigen due to mutations in tumor cells. In certain embodiments, for example, the mutations can include frameshift or non-frameshift indels. In certain embodiments, for example, the mutations can include missense substitutions. In certain embodiments, for example, the mutations can include nonsense substitutions. In certain embodiments, for example, the mutations can include splice site alterations. In certain embodiments, for example, the mutations can include genomic rearrangements. In certain embodiments, for example, the mutations can include gene fusions. In certain embodiments, for example, the mutations can include genomic rearrangements and / or expression alterations that give rise to neoORFs (neoopen reading frames). In certain embodiments, for example, the genomic rearrangements can include one or more insertions. In certain embodiments, for example, the genomic rearrangements can include one or more deletions. In certain embodiments, for example, the mutations can include splice variants. In certain embodiments, for example, a neoantigen can include modifications to a parental antigen due to post-translational modifications specific to tumor cells. In certain embodiments, for example, the post-translational modifications can include abnormal phosphorylation. In certain embodiments, for example, the post-translational modifications can include splice antigens generated by the proteasome. In certain embodiments, for example, a neoantigen can be derived from a tumor. In certain embodiments, for example, the tumor can be a solid tumor. In certain embodiments, for example, a neoantigen can be presented on the surface of a tumor. In certain embodiments, for example, a neoantigen can be a tumor neoantigen. In certain embodiments, for example, a tumor neoantigen can be present in a subject's tumor cells or tissue but not in the subject's corresponding normal cells or tissue.In certain embodiments, for example, tumor neoantigens can be overexpressed in a subject's tumor cells or tissue as compared to expression in the subject's corresponding normal cells or tissue. In certain embodiments, for example, the neoantigen can be an individualized neoantigen. In certain embodiments, for example, the neoantigen can be a shared tumor neoantigen. In certain embodiments, for example, the shared tumor neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be specific to a particular type of tumor. In certain embodiments, for example, the neoantigen can be a tumor-associated peptide neoantigen. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by a model. In certain embodiments, for example, one or more neoantigens can be individualized neoantigens. In certain embodiments, for example, one or more neoantigens can be present in a list of shared neoantigens. In certain embodiments, for example, the neoantigen can be selected from one or more neoantigens identified by an artificial intelligence model. In certain embodiments, for example, the model can be calibrated using machine learning. In certain embodiments, for example, the artificial intelligence model can include a neural network. In certain embodiments, for example, the neoantigen can be selected from a set of presentation likelihoods. In certain embodiments, for example, the neoantigen can be determinable using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0131] In certain embodiments, for example, the neoantigen can be a tumor neoantigen. In certain embodiments, for example, the tumor neoantigen can be present in the subject's tumor cells or tissue, but not in the subject's corresponding normal cells or tissue. In certain embodiments, for example, the tumor neoantigen can be overexpressed in the subject's tumor cells or tissue as compared to expression in the subject's corresponding normal cells or tissue. In certain embodiments, for example, the tumor neoantigen can be determined using one or more of the machine learning methods, software, and / or systems disclosed in the incorporated references.

[0132] In certain embodiments, for example, neoantigens can be associated with certain types of cancer. In certain embodiments, for example, the cancer can be lung cancer, bladder cancer, stomach cancer, rectal cancer, endometrial cancer, thyroid cancer, renal papillary cells, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer (e.g., lower-grade glioma, glioblastoma), B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia, non-small cell lung cancer (e.g., squamous cell carcinoma (SCC)) and small cell lung cancer, and can be selected from the group consisting of combinations of two or more of the foregoing cancers. In certain embodiments, for example, the cancer can be selected from a subgroup of the foregoing group. In certain embodiments, for example, the cancer can be an epithelial cancer. In certain embodiments, for example, the cancer can be a blood cancer.

[0133] Accordingly, provided herein is also a method for screening candidate neoantigens for immunogenicity, the method comprising: isolating from a population of PBMCs at least one T cell that binds to a candidate neoantigen; forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell; and activating at least a first functional T cell, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic for the candidate neoantigen.

[0134] The present disclosure also provides a method for screening candidate antigens for antigen-specific vaccines. In some embodiments, the method comprises isolating at least one T cell that binds to a candidate antigen from a population of PBMCs, forming a plurality of cognate T cells, the step comprising expanding the at least one isolated T cell, and activating at least a first functional T cell, the step comprising contacting T cells derived from the plurality of cognate T cells with at least one of a plurality of activators that are immunogenic with respect to the candidate antigen.

[0135] In certain embodiments, a given type of antigen may be an antigen selected from a publicly available database containing selected T cell receptor (TCR) sequences with known antigen specificities, such as the VDJdb database (vdjdb.cdr3.net). In some embodiments, a given type of antigen is a predicted antigen. In other embodiments, a given type of antigen is an experimentally verified antigen.

[0136] In certain embodiments, for example, source T cells for the systems and methods can be provided. In certain embodiments, for example, the source T cells can be derived from PBMC. In certain embodiments, for example, the source T cells can be derived from bone marrow. In certain embodiments, for example, the source T cells can be derived from the thymus. In certain embodiments, for example, the source T cells can be derived from a tissue biopsy. In certain embodiments, for example, the source T cells can be derived from a tumor. In certain embodiments, for example, the source T cells can be derived from lymph node tissue. In certain embodiments, for example, the source T cells can be derived from gut-associated lymphoid tissue. In certain embodiments, for example, the source T cells can be derived from mucosa-associated lymphoid tissue. In certain embodiments, for example, the source T cells can be derived from spleen tissue. In certain embodiments, for example, the source T cells can be derived from lymphoid tissue. In certain embodiments, for example, the source T cells can be derived from a tumor (e.g., one of the tumors disclosed herein). In certain embodiments, for example, the source T cells can be obtained from a T cell line. In certain embodiments, for example, the source T cells can be obtained from an autologous source. In certain embodiments, for example, the source T cells can be obtained from an allogeneic source. In certain embodiments, for example, the source T cells can be obtained from a single individual. In certain embodiments, for example, the single individual can be healthy (e.g., free of one or more preselected diseases). In certain embodiments, for example, the single individual may have one or more preselected diseases (e.g., a preselected cancer). In certain embodiments, for example, the source T cells can be obtained from a population of individuals. In certain embodiments, for example, the population of individuals can be healthy (e.g., free of one or more preselected diseases). In certain embodiments, for example, the population of individuals may have one or more preselected diseases (e.g., a preselected cancer).

[0137] In certain embodiments, for example, the source T cells can be derived from cells obtained by leukapheresis of the individual's circulating blood. In certain embodiments, for example, the source T cells can be derived from cells obtained by apheresis of the individual's circulating blood. In certain embodiments, for example, the obtained cells can include lymphocytes. In certain embodiments, for example, the obtained lymphocytes can include T cells and, optionally, one or more of monocytes, granulocytes, B cells, other nucleated white blood (while blood) cells, erythrocyte cells, and platelets.

[0138] In certain embodiments, for example, the obtained cells can be washed to remove plasma and placed in an appropriate buffer or medium for subsequent processing to obtain source T cells. In certain embodiments, for example, the cells can be washed with phosphate-buffered saline (PBS). In certain embodiments, for example, the washing solution may not contain calcium cations, magnesium cations, or any divalent cations. In certain embodiments, for example, the washing can be performed using a semi-automatic flow-through centrifuge. In certain embodiments, for example, following the washing, the cells can be resuspended in a liquid. In certain embodiments, for example, the liquid can include a biocompatible buffer. In certain embodiments, for example, the biocompatible buffer can include calcium cation-free and magnesium cation-free PBS. In certain embodiments, for example, undesirable components of the cells obtained by apheresis can be removed and the cells can be directly resuspended in a culture medium.

[0139] In certain embodiments, for example, at least one T cell receptor component can be present in less than one T cell per 1,000,000 source T cells. For example, in some embodiments, at least one T cell receptor component can be present in less than one T cell per 2,000,000 source T cells. In some embodiments, at least one T cell receptor component can be present in less than one T cell per 5,000,000 source T cells. In some embodiments, at least one T cell receptor component can be present in less than one T cell per 10,000,000 source T cells.

[0140] In certain embodiments, for example, the methods disclosed herein can exclude T cell priming. In certain embodiments, for example, the methods disclosed herein can obviate the need to isolate antigen-presenting cells from a blood sample for the purpose of priming T cells. In certain embodiments, for example, the obviation of the need to isolate antigen-presenting cells from a blood sample for the purpose of priming T cells can reduce the total volume of blood required by at least 25% (e.g., at least 50%, or 30% - 70%) compared to methods that utilize T cell priming.

[0141] A variety of methods for isolating T cells are known in the art, and it is understood that any of the isolation methods can be used in combination with the present invention. In certain embodiments, for example, source T cells can be isolated from peripheral blood lymphocytes by lysing red blood cells and centrifuging through a PERCOLL™ gradient. In certain embodiments, for example, source T cells can be isolated from peripheral blood lymphocytes by Ficoll-Paque separation. In certain embodiments, for example, source T cells can be isolated from peripheral blood lymphocytes using a microfluidic device. In certain embodiments, for example, positive or negative selection can be used to obtain a mixture of T cells from source T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) CD28 + T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) naive CD8 + T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) naive T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) memory T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) CD8 + T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) CD4 + T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) CD4 + CD8 + T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) CD4 - CD8 + T cells. In certain embodiments, for example, the mixture of T cells can contain (or can be enriched for) CD4 + CD8 -It can contain (or can be enriched with) T cells. In certain embodiments, for example, a mixture of T cells is CD45RA + It can contain (or can be enriched with). In certain embodiments, for example, a mixture of T cells is CD45RO + It can contain (or can be enriched with) T cells. In certain embodiments, for example, a mixture of T cells is CD3 + / CD28 + It can contain (or can be enriched with) T cells.

[0142] As described above, a desired population of T cells can be obtained from source T cells using positive selection, negative selection, or a combination of both. In certain embodiments, for example, an anti-marker agent (e.g., an antibody) can be conjugated to magnetic beads and magnetic separation can be performed to positively select T cells. In other embodiments, for example, an antibody can be conjugated to a surface marker specific to undesired cells of a T cell subset to negatively select the T cell subset. In certain embodiments, for example, negative selection can include cell sorting. In certain embodiments, for example, negative selection can include selection by negative magnetic immunoadhesion. In certain embodiments, for example, negative selection can include selection by flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. In certain embodiments, for example, a mixture of T cells enriched in CD4 + can be obtained by exposing source T cells to monoclonal antibodies (e.g., biotinylated monoclonal antibodies that can be bound to anti-biotin magnetic beads) against one or more (e.g., all) of CD14, CD20, CD11b, CD16, HLA-DR, and CD8, followed by enrichment (e.g., including magnetic separation) and characterization by flow cytometry. In certain embodiments, for example, CD8 +A mixture of T cells enriched in T cells can be obtained by exposing the source T cells to monoclonal antibodies (e.g., biotinylated monoclonal antibodies that can be conjugated to anti-biotin magnetic beads) against one or more (e.g., all) of CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, anti-HLA-DR, CD235a (glycophorin A), CD244, and CD4, followed by enrichment (e.g., including magnetic separation) and characterization by flow cytometry. The above examples regarding positive selection, negative selection, or a combination of both are non-limiting, and it is understood that any marker specific to the desired cell population may be used for positive selection, or any marker specific to the undesired cell population may be used for negative selection.

[0143] Accordingly, methods for negatively selecting T cell receptor clonotypes are also provided herein. In some embodiments, the method includes analyzing a mixture of T cells to identify a first antigen-binding T cell and a first antigen-activated T cell against a predetermined first type of antigen, and a second antigen-activated T cell against a predetermined second type of antigen, and identifying at least a portion of at least one T cell receptor sequence that is shared by at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells and is not shared with any of the second antigen-activated T cells. In other embodiments, a method for negatively selecting T cell receptor clonotypes includes analyzing a mixture of T cells to identify a first antigen-activated T cell and a first antigen-binding T cell against a predetermined first type of antigen, and a second antigen-binding T cell against a predetermined second type of antigen, and identifying at least a portion of at least one T cell receptor sequence that is shared by at least one of the first antigen-binding T cells and at least one of the first antigen-activated T cells and is not shared with any of the second antigen-binding T cells.

[0144] In certain embodiments, for example, a mixture of T cells can be prepared by freezing a source of T cells washed in a freezing solution (e.g., washed as described herein). In certain embodiments, for example, the freezing solution can contain PBS. In certain embodiments, for example, the PBS can contain dimethyl sulfoxide (DMSO) (e.g., 20% DMSO, etc., 5 - 40% DMSO). In certain embodiments, for example, the PBS can contain human serum albumin (HSA) (e.g., 8% HSA, etc., 1 - 30% HSA). In certain embodiments, for example, the freezing solution can contain other suitable cell freezing components. In certain embodiments, for example, the freezing solution can be an undiluted freezing solution. In certain embodiments, for example, the freezing solution can be diluted. In certain embodiments, for example, the freezing solution can contain PBS diluted 1:1 with a medium and containing 20% DMSO and 8% human serum albumin (HSA) (or other suitable cell freezing components). In certain embodiments, for example, the mixture of T cells can be frozen at -80°C and stored in the vapor phase of a liquid nitrogen storage tank.

[0145] In certain embodiments, for example, at least one T cell receptor component can be present in less than 1 T cell per 1,000 T cells in the T cell mixture. For example, in some embodiments, at least one T cell receptor component can be present in less than 1 T cell per 10,000 T cells in the T cell mixture. In some embodiments, at least one T cell receptor component can be present in less than 1 T cell per 100,000 T cells in the T cell mixture. In some embodiments, at least one T cell receptor component can be present in less than 1 T cell per 1,000,000 T cells in the T cell mixture. In some embodiments, at least one T cell receptor component can be present in less than 1 T cell per 10,000,000 T cells in the T cell mixture.

[0146] In certain embodiments, for example, at least one T cell receptor component can be present in at least 0.0005% of the T cells in a T cell mixture. For example, in some embodiments, at least one T cell receptor component can be present in at least 0.005% of the T cells in a T cell mixture. In some embodiments, at least one T cell receptor component can be present in at least 0.05% of the T cells in a T cell mixture. In some embodiments, at least one T cell receptor component can be present in at least 0.5% of the T cells in a T cell mixture. In some embodiments, at least one T cell receptor component can be present in at least 5% of the T cells in a T cell mixture. In some embodiments, at least one T cell receptor component can be present in at least 10% of the T cells in a T cell mixture. In some embodiments, at least one T cell receptor component can be present in at least 15% of the T cells in the T cell mixture (the at least one T cell receptor component can be present in at least one T cell receptor component can be present in at least 15% of the T cells in the T cell mixture).

[0147] In certain embodiments, for example, at least one T cell receptor component can be present in less than 5% of the T cells in the T cell mixture. In some embodiments, at least one T cell receptor component can be present in less than 0.5% of the T cells in the T cell mixture. In some embodiments, at least one T cell receptor component can be present in less than 0.005% of the T cells in the T cell mixture. In some embodiments, at least one T cell receptor component can be present in less than 0.0005% of the T cells in the T cell mixture (the at least one T cell receptor component can be present in at least one T cell receptor component can be present in less than 0.0005% of the T cells in the T cell mixture).

[0148] Certain embodiments can include, for example, enriching a subpopulation of T cells from a population of T cells (e.g., using one or more of the methods, assays, or systems disclosed in the incorporated references). In certain embodiments, for example, the enriching step can include enriching a subpopulation of T cells that exhibit antigen binding or antigen activation to a given type of antigen. In certain embodiments, for example, the enriching step can include contacting the population of T cells with a binding agent and subsequently separating members of the subpopulation that bind to the binding agent from the population of T cells.

[0149] In certain embodiments, for example, the binder can comprise at least a portion (or a part) of a given type of antigen. In certain embodiments, for example, the binder can comprise at least a portion of a given type of antigen that is bound to at least a portion of an MHC protein. In certain embodiments, for example, the MHC protein may be an MHC class I protein. In certain embodiments, for example, the MHC protein may be an MHC class II protein. In certain embodiments, for example, the binder can comprise at least a portion of a given type of antigen that is bound to an aptamer. In certain embodiments, for example, the binder can comprise at least a portion of a given type of antigen that is bound to an affimer. In certain embodiments, for example, the binder can comprise at least a portion of a given type of antigen that is bound to an antibody. In certain embodiments, for example, the binder can comprise a multimer (e.g., a tetramer comprising at least a portion of a given type of antigen that is bound to at least a portion of an MHC protein). In certain embodiments, for example, the binder may be linked to magnetic beads to facilitate magnetic separation or to a fluorophore to facilitate isolation by fluorescence flow cytometry.

[0150] In certain embodiments, for example, the member can bind to the binder with a dissociation constant of from 0.01 μM to 1000 μM. For example, in some embodiments, the member can bind to the binder with a dissociation constant of from 0.1 μM to 100 μM. In some embodiments, the member can bind to the binder with a dissociation constant of from 0.5 μM to 50 μM. In some embodiments, the member can bind to the binder with a dissociation constant of from 1 μM to 50 μM. In some embodiments, the member can bind to the binder with a dissociation constant of from 1 μM to 25 μM. In some embodiments, the member can bind to the binder with a dissociation constant of from 25 μM to 75 μM. In some embodiments, the member can bind to the binder with a dissociation constant of from 10 μM to 50 μM.

[0151] In further embodiments, for example, the member can bind to the binder with a dissociation constant of less than 1000 μM. In some embodiments, the member can bind to the binder with a dissociation constant of less than 100 μM. In some embodiments, the member can bind to the binder with a dissociation constant of less than 75 μM. In some embodiments, the member can bind to the binder with a dissociation constant of less than 50 μM. In some embodiments, the member can bind to the binder with a dissociation constant of less than 40 μM. In some embodiments, the member can bind to the binder with a dissociation constant of less than 30 μM. In some embodiments, the member can bind to the binder with a dissociation constant of less than 20 μM. In some embodiments, the member can bind to the binder with a dissociation constant of less than 10 μM.

[0152] In certain embodiments, for example, the member can bind to the binder with a half-life of from 0.1 second to 100 seconds. For example, in some embodiments, the member can bind to the binder with a half-life of from 1 second to 50 seconds. In some embodiments, the member can bind to the binder with a half-life of from 1 second to 25 seconds. In some embodiments, the member can bind to the binder with a half-life of from 1 second to 10 seconds. In some embodiments, the member can bind to the binder with a half-life of from 2 seconds to 10 seconds. In some embodiments, the member can bind to the binder with a half-life of from 2 seconds to 7 seconds. In some embodiments, the member can bind to the binder with a half-life of from 2 seconds to 5 seconds.

[0153] In certain embodiments, for example, the member can bind to the binder with a half-life of at least 0.1 seconds. For example, in some embodiments, the member can bind to the binder with a half-life of at least 0.5 seconds. In some embodiments, the member can bind to the binder with a half-life of at least 1 second. In some embodiments, the member can bind to the binder with a half-life of at least 2 seconds. In some embodiments, the member can bind to the binder with a half-life of at least 5 seconds. In some embodiments, the member can bind to the binder with a half-life of at least 10 seconds.

[0154] In certain embodiments, for example, the member can bind to the binder with a dissociation constant of less than 50 μM and a half-life of between 2 and 10 seconds.

[0155] Certain embodiments can include, for example, the step of expanding one or more T cells (e.g., using one or more of the methods, assays, or systems disclosed in the incorporated references). In certain embodiments, for example, the expanding step can include expanding a plurality of T cells that are positively selected for antigen binding or antigen activation against a predetermined type of antigen. In certain embodiments, for example, the expanding step can include polyclonal expansion.

[0156] Certain embodiments can include, for example, progressively enriching a population of antigen - binding T cells for a given type of antigen by continuous enrichment, followed by growing the starting mixture of T cells multiple times, e.g., 2 times (i.e., enrichment→growth→enrichment or enrichment→growth→enrichment→growth). In some embodiments, the starting mixture of T cells can be grown at least 3 times. In some embodiments, the starting mixture of T cells can be grown at least 4 times. In some embodiments, the starting mixture of T cells can be grown at least 5 times. In some embodiments, the starting mixture of T cells can be grown more than 5 times. In certain embodiments, for example, a portion of the progressively enriched population (i.e., the population of T cells resulting from continuous enrichment) can be further selected for antigen activation (e.g., by exposing the progressively enriched population of antigen - binding T cells to cells presenting a given type of antigen and further enriching based on the presence of one or more activation markers).

[0157] In certain embodiments, for example, antigen - activated T cells can be formed by contacting T cells with an activator, and the resulting antigen - activated T cells can be detected by detecting the expression of one or more activation markers and / or secreted molecules. In certain embodiments, for example, antigen - activated T cells can be formed by contacting T cells with an activator, and the resulting antigen - activated T cells can be detected by detecting the expression of one or more activation markers disclosed in one of this specification or the incorporated references.

[0158] In certain embodiments, for example, one or more activation markers can include cell surface markers. In certain embodiments, for example, one or more activation markers can include signaling molecules (e.g., molecules that are upregulated in response to T cell activation). In certain embodiments, for example, one or more activation markers and / or secreted molecules can include CD137 (also known as 4-1BB or Tnsfr9), interferon gamma (IFN-γ), tumor necrosis factor alpha (TNFα), interleukin-2 (IL-2), CD69, upregulation of MHC class I protein, upregulation of MHC class II protein, Ki67, perforin, granzyme, CD122, CD27, CD28, CD95, CD134, killer cell lectin-like receptor G1 (KLRG1), CD38, CD154, or combinations of two or more of the foregoing. In certain embodiments, the activation marker may be CD137. In other embodiments, the activation marker may be IFN-γ. In some embodiments, the activation marker may be TNFα. In some embodiments, the activation marker may be IL-2. In some embodiments, the activation marker may be CD69. In some embodiments, the activation marker may be upregulation of MHC class I protein. In some embodiments, the activation marker may be upregulation of MHC class II protein. In some embodiments, the activation marker may be Ki67. In some embodiments, the activation marker may be CD137 and IFN-γ. In some embodiments, the activation marker may be CD137, IFN-γ, and IL-2. It is understood that the exemplary activation markers above are non-limiting and any T cell activation marker known in the art can be used in conjunction with the disclosure provided herein.

[0159] The present disclosure also provides a method for identifying T cell activation markers. In some embodiments, the method comprises contacting a first plurality of T cells with a plurality of antigen-presenting cells, wherein the first plurality of T cells comprises a plurality of antigen-binding T cells and the antigen is a predetermined type of antigen; measuring a plurality of expression rate profiles for at least a portion of the contacted plurality of antigen-binding T cells; dividing at least a portion of the contacted plurality of antigen-binding T cells into a plurality of T cell clusters; measuring a functional response to the antigen in at least two T cells present in at least a portion of the contacted plurality of antigen-binding T cells; mapping the expression rate profiles to the plurality of T cell clusters to identify one of the plurality of T cell clusters comprising the at least two T cells; and identifying activation markers expressed by the at least two T cells.

[0160] In certain embodiments, for example, two or more of the activation markers and / or secreted molecules can be detected in separate portions of the T cell and in individual T cells between two sub-portions that share the same at least one identified T cell component. In certain embodiments, for example, two or more of the activation markers and / or secreted molecules can be detected together in a single portion of the T cell.

[0161] In certain embodiments, for example, the activator can include a predetermined type of antigen. In certain embodiments, for example, the activator can further include a co-stimulatory ligand. In certain embodiments, for example, the co-stimulatory ligand can be one or more of the ligands selected from the group consisting of an antibody or an antigen-binding fragment thereof that specifically binds to CD28, CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BBL, 4-1BB, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), CD40, LIGHT, an antibody or an antigen-binding fragment thereof that specifically binds to HVEM, an antibody or an antigen-binding fragment thereof that specifically binds to CD40L, an antibody or an antigen-binding fragment thereof that specifically binds to OX40, and an antibody or an antigen-binding fragment thereof that specifically binds to 4-1BB. In certain embodiments, for example, the co-stimulatory ligand can be selected from the group consisting of monoclonal antibodies, F(ab’)2, Fab, scFv, and single-chain antibodies. In certain embodiments, for example, the co-stimulatory ligand can be a humanized monoclonal antibody or fragment. In certain embodiments, for example, the co-stimulatory ligand can be a humanized mouse monoclonal antibody or a fully human antibody against CD28. In certain embodiments, for example, the co-stimulatory ligand can be a humanized monoclonal antibody or an antigen-binding fragment thereof. In certain embodiments, for example, a predetermined type of antigen (e.g., a predetermined type of antigen complexed with an MHC protein) and the co-stimulatory ligand covalently bind to the surface of the paramagnetic particles.

[0162] In certain embodiments, for example, the activator can comprise a predetermined type of antigen on the surface of the cell. In certain embodiments, for example, the activator can comprise one or more dendritic cells. In certain embodiments, for example, the activator can comprise one or more antigen-presenting cells (e.g., one or more professional antigen-presenting cells). In certain embodiments, for example, the activator can comprise one or more artificial antigen-presenting cells. In certain embodiments, for example, the activator can comprise one or more macrophages. In certain embodiments, for example, the activator can comprise one or more B cells.

[0163] In certain embodiments, for example, the activator can comprise one or more cancer cells. For example, in some embodiments, the cancer cells are derived from solid tumors. In some embodiments, the cancer cells are derived from hematological malignancies. In yet further embodiments, the cancer cells are circulating tumor cells. In certain embodiments, for example, the cancer can be selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia, non-small cell lung cancer and small cell lung cancer, and combinations of two or more of the foregoing cancers. In certain embodiments, for example, the cancer can be selected from a subgroup of the foregoing group.

[0164] In certain embodiments, for example, the activator can comprise one or more cells related to a cancerous tumor. In certain embodiments, for example, the tumor can be selected from the group consisting of lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia, non-small cell lung cancer and small cell lung cancer, and tumors related to two or more combinations of the aforementioned cancers. In certain embodiments, for example, the tumor can be selected from a subgroup of the aforementioned group.

[0165] In certain embodiments, any of the aforementioned activators can be formed by antigen loading with a certain content of a predetermined type of antigen. In certain embodiments, for example, the antigen loading can be configured to present a predetermined concentration of a predetermined type of antigen on the activator surface (e.g., each of the activators can have the same concentration of a predetermined type of antigen). In certain embodiments, for example, the activator can be formed excluding antigen loading.

[0166] In certain embodiments, for example, the activator can present a predetermined type of antigen at a physiologically appropriate concentration. In certain embodiments, for example, the activator can present a predetermined type of antigen at a concentration determined by pulsing a plurality of cells with a solution containing a predetermined type of antigen for a predetermined period, and the solution containing the predetermined type of antigen has a concentration of 0.000001 μM to 100 μM. For example, in some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.000001 μM to 0.00001 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.00001 μM to 0.0001 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.0001 μM to 0.001 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.001 to 0.01 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.01 to 0.1 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.0001 μM to 100 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.001 μM to 100 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.01 μM to 10 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 0.1 μM to 10 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 1 μM to 100 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 1 μM to 50 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 1 μM to 25 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 5 μM to 25 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 10 μM to 100 μM. In some embodiments, the solution containing a predetermined type of antigen has a concentration of 10 μM to 30 μM.

[0167] In certain embodiments, for example, the solution can contain a predetermined type of antigen at a concentration of less than 100 μM. For example, in some embodiments, the solution can contain a predetermined type of antigen at a concentration of less than 75 μM. In some embodiments, the solution can contain a predetermined type of antigen at a concentration of less than 50 μM. In some embodiments, the solution can contain a predetermined type of antigen at a concentration of less than 25 μM. In some embodiments, the solution can contain a predetermined type of antigen at a concentration of less than 10 μM. In some embodiments, the solution can contain a predetermined type of antigen at a concentration of less than 1 μM.

[0168] In any of the above embodiments, for example, the predetermined period may be from 1 hour to 36 hours. For example, in some embodiments, the predetermined period may be from 6 hours to 24 hours. In some embodiments, the predetermined period may be from 6 hours to 12 hours. In some embodiments, the predetermined period may be from 12 hours to 24 hours. In some embodiments, the predetermined period may be from 9 hours to 18 hours.

[0169] In any of the above embodiments, for example, the predetermined period may be at least 1 hour. In some embodiments, the predetermined period may be at least 4 hours. In some embodiments, the predetermined period may be at least 8 hours. In some embodiments, the predetermined period may be at least 12 hours. In some embodiments, the predetermined period may be at least 18 hours. In some embodiments, the predetermined period may be at least 24 hours.

[0170] In any of the above embodiments, for example, the predetermined period may be less than 168 hours. In some embodiments, the predetermined period may be less than 72 hours. In some embodiments, the predetermined period may be less than 36 hours. In some embodiments, the predetermined period may be less than 24 hours. In some embodiments, the predetermined period may be less than 12 hours.

[0171] In any of the foregoing embodiments, for example, the predetermined period can be repeated one or more times. For example, in some embodiments, the activator pulses a plurality of cells with a solution containing a predetermined type of antigen over any of the predetermined periods described herein, and can present the predetermined type of antigen at any of the concentrations described herein, and then can be re-challenged one more time. In some embodiments, the antigen is re-challenged two more times. In further embodiments, the antigen is re-challenged three more times. In still further embodiments, the antigen is re-challenged four more times. In yet further embodiments, the antigen is re-challenged five more times. In other embodiments, the antigen is re-challenged more than five times. In some embodiments, the antigen is re-challenged more than ten times.

[0172] In certain embodiments, for example, a particular T cell can bind to an activator with a dissociation constant of 0.01 μM to 1000 μM to form an activated T cell. For example, in some embodiments, a particular T cell can bind to an activator with a dissociation constant of 0.1 μM to 100 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of 0.5 μM to 50 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of 1 μM to 50 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of 1 μM to 25 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of 25 μM to 75 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of 10 μM to 50 μM.

[0173] In certain embodiments, for example, a particular T cell can bind to an activator with a dissociation constant of less than 1000 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of less than 100 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of less than 75 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of less than 50 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of less than 40 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of less than 30 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of less than 20 μM. In some embodiments, a particular T cell can bind to an activator with a dissociation constant of less than 10 μM.

[0174] In certain embodiments, for example, a particular T cell can bind to an activator with a half-life of from 0.1 second to 100 seconds to form an antigen-activated T cell. For example, in some embodiments, a particular T cell can bind to an activator with a half-life of from 1 second to 50 seconds. In some embodiments, a particular T cell can bind to an activator with a half-life of from 1 second to 25 seconds. In some embodiments, a particular T cell can bind to an activator with a half-life of from 1 second to 10 seconds. In some embodiments, a particular T cell can bind to an activator with a half-life of from 2 seconds to 10 seconds. In some embodiments, a particular T cell can bind to an activator with a half-life of from 2 seconds to 7 seconds. In some embodiments, a particular T cell can bind to an activator with a half-life of from 2 seconds to 5 seconds.

[0175] In certain embodiments, for example, a particular T cell can bind to an activator with a half-life of at least 0.1 second. For example, in some embodiments, a particular T cell can bind to an activator with a half-life of at least 0.5 second. In some embodiments, a particular T cell can bind to an activator with a half-life of at least 1 second. In some embodiments, a particular T cell can bind to an activator with a half-life of at least 2 seconds. In some embodiments, a particular T cell can bind to an activator with a half-life of at least 5 seconds. In some embodiments, a particular T cell can bind to an activator with a half-life of at least 10 seconds.

[0176] In certain embodiments, for example, a particular T cell can bind to an activator with a dissociation constant of less than 50 μM and a half-life of 2 to 10 seconds to form an antigen-activated T cell.

[0177] FIG. 1 shows a schematic diagram of a method for identifying a T cell receptor having at least a portion shared by antigen-binding T cells and antigen-activated T cells against a predetermined type of antigen (e.g., neoantigens such as individualized neoantigens or shared neoantigens including neoantigens selected by a model whose parameters are adjusted using machine learning). A sample is processed (100) to obtain a mixture containing a plurality of different T cells. The mixture can include, for example, PBMCs such as PBMCs obtained by processing a leukapheresis sample from a healthy donor to remove cells that are positive for one or more of CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, anti-HLA-DR, CD235a (glycophorin A), CD244, and CD4. The plurality of different T cells can include, for example, naive CD8 + T cells. The mixture is incubated with a P-loaded MHC protein (P is a predetermined type of antigen), and then the T cells bound thereto are isolated, whereby naive CD8 +Enrich T cells (102). The P-loaded MHC proteins can be provided, for example, in the form of magnetically labeled multimers (facilitating isolation by magnetic separation) or fluorescently labeled multimers (facilitating isolation by fluorescence flow cytometry). The isolated T cells are expanded, for example, by polyclonal proliferation (104) and divided into a first, a second, and optionally a third T cell population. The first T cell population is incubated with the P-loaded MHC protein and subsequently the P-binding T cells bound thereto are isolated to assess for P-binding T cells (106). The second T cell population is exposed to cells presenting a predetermined type of antigen, an activation marker or a secreted molecule suggesting T cell activation is detected, and the activated T cells are isolated to assess for P-activated T cells (108). The cells presenting a predetermined type of antigen can, for example, present a predetermined type of antigen at a physiologically appropriate content. The cells presenting a predetermined type of antigen can, for example, be professional antigen-presenting cells. The cells presenting a predetermined type of antigen can, for example, be tumor cells derived from the subject. The activation marker can, for example, include CD137. The P-activated T cells can be isolated by contacting with a magnetically labeled anti-activation marker antibody and subsequently magnetic separation. The optional third T cell population, if present, is similarly exposed to yet another cell presenting a predetermined type of antigen, yet another activation marker or yet another secreted molecule suggesting T cell activation is detected, and yet another P-activated T cell is isolated to assess for P-activated T cells (110). The T cell receptors derived from the isolated P-binding T cells and P-activated T cells are sequenced at the single cell level (112, 114, 116), the resulting sequences are compared (118), and a T cell receptor having at least a portion of a sequence common among the P-binding T cells, P-activated T cells, and optionally yet another P-activated T cell is identified.

[0178] Figure 1 illustrates one particular exemplary embodiment, but other variations also fall within the scope of the present disclosure. In certain embodiments, for example, PBMCs can be obtained from whole blood samples. In certain embodiments, for example, a plurality of different T cells can include CD-4 + T cells, and the mixture can be enriched for CD-4 + T cells. In certain embodiments, for example, a plurality of different T cells can include memory T cells, and the mixture can be enriched for memory T cells. In certain embodiments, for example, the process can include removing a biomarker panel different from the panel shown depending on the T cell composition desired for enrichment. In certain embodiments, for example, the expanded T cells can be further divided into third and fourth T cell populations to assay for second and third activation markers and / or secreted molecules. In certain embodiments, for example, P-activated T cells can be isolated by contacting them with a fluorescently labeled anti-activation marker antibody and passing them through a fluorescence flow cytometer.

[0179] A schematic diagram of a method including a negative selection step for identifying a T cell receptor having at least a portion shared by antigen-binding T cells and antigen-activated T cells against a predetermined type of antigen (e.g., neoantigens such as personalized neoantigens or shared neoantigens including neoantigens selected by a model whose parameters are adjusted using machine learning) is shown in FIG. 2. A sample is processed (200) to obtain a mixture containing a plurality of different T cells. The mixture can include, for example, PBMCs such as PBMCs obtained by processing a leukapheresis sample from a healthy donor to remove cells that are positive for one or more of CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, anti-HLA-DR, CD235a (glycophorin A), CD244, and CD4. The plurality of different T cells can include, for example, naive CD8 from a healthy donor +It can contain T cells. The mixture is incubated with a P-loaded MHC protein (P is a predetermined type of antigen), and then the T cells bound thereto are isolated, whereby naive CD8 + T cells are enriched (202). The P-loaded MHC protein can be provided, for example, in the form of a magnetically labeled multimer (facilitating isolation by magnetic separation) or a fluorescently labeled multimer (facilitating isolation by fluorescence flow cytometry). The isolated T cells are expanded, for example, by polyclonal proliferation (204) and divided into a first, a second, and a third T cell population. The first T cell population is incubated with the P-loaded MHC protein, and then the P-binding T cells bound thereto are isolated to evaluate for P-binding T cells (206). The second T cell population is exposed to cells presenting a predetermined type of antigen, detects an activation marker or a secreted molecule suggesting T cell activation, and isolates the activated T cells to evaluate for P-activated T cells (208). The cells presenting a predetermined type of antigen can present, for example, a physiologically appropriate content of the predetermined type of antigen. The cells presenting a predetermined type of antigen can be, for example, professional antigen-presenting cells. The cells presenting a predetermined type of antigen can be, for example, tumor cells derived from a subject. The activation marker can include, for example, CD137. The P-activated T cells can be isolated by contacting with a magnetically labeled anti-activation marker antibody and then magnetic separation. The third T cell population is exposed to yet another cell presenting a different type of antigen Q (Q is different from P), detects yet another activation marker or yet another secreted molecule suggesting T cell activation, and isolates the Q-activated T cells to evaluate for Q-activated T cells (210). The T cell receptors derived from the isolated P-binding T cells, P-activated T cells, and Q-activated T cells are sequenced at the single cell level (212, 214, 216), and the resulting sequences are compared (218) to identify a T cell receptor having at least a portion of a sequence that is common between the P-binding T cells and the P-activated T cells but not present in the Q-activated T cells.

[0180] Figure 2 illustrates certain exemplary embodiments, and other variations are within the scope of the present disclosure. In certain embodiments, for example, PBMCs can be obtained from whole blood samples. In certain embodiments, for example, a plurality of different T cells can include CD-4 + T cells, and the mixture can be enriched for CD-4 + T cells. In certain embodiments, for example, a plurality of different T cells can include memory T cells, and the mixture can be enriched for memory T cells. In certain embodiments, for example, the treatment can include removal of a biomarker panel different from the panel shown depending on the T cell composition desired for enrichment. In certain embodiments, for example, the expanded T cells can be further divided into third and fourth T cell populations (or additional T cell populations) and evaluated for P-activated and / or Q-activated T cells using additional activation markers and / or secreted molecules. In certain embodiments, for example, the expanded T cells can be negatively selected for T cells activated by yet another predetermined antigen (i.e., in addition to Q). In certain embodiments, for example, T cells can be negatively selected based on binding to a predetermined antigen rather than activation. In certain embodiments, for example, P-activated T cells can be isolated by contacting them with a fluorescently labeled anti-activation marker antibody and passing them through a fluorescence flow cytometer. In certain embodiments, for example, the third T cell population can be examined in a medium free of Q to detect and isolate T cells that give false positive results for activation in the absence of antigen.

[0181] A schematic diagram of a method for identifying activation markers suggesting activation of T cell receptors against antigens of a specified type (e.g., neoantigens such as individualized neoantigens or shared neoantigens including neoantigens selected by a model whose parameters are adjusted using machine learning) is shown in FIG. 3. A sample is processed (300) to obtain a mixture containing a plurality of different T cells. The mixture can include, for example, PBMC such as PBMC obtained by processing a leukapheresis sample from a healthy donor to remove cells that are positive for one or more of CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, anti-HLA-DR, CD235a (glycophorin A), CD244, and CD4. The plurality of different T cells can include, for example, naive CD8 + T cells from a healthy donor. The mixture is incubated with a P-loaded MHC protein (where this P is an antigen of a specified type), and subsequently the T cells bound thereto are isolated, whereby naive CD8 +Enrich T cells (302). The P-loaded MHC protein can be provided, for example, in the form of a magnetically labeled multimer (facilitating isolation by magnetic separation) or a fluorescently labeled multimer (facilitating isolation by fluorescence flow cytometry). The isolated T cells are expanded, for example, by polyclonal proliferation (304) and divided into a first and a second T cell population. The first T cell population is incubated with the P-loaded MHC protein, and subsequently the P-binding T cells bound thereto are isolated, and the T cell receptor for the isolated P-binding T cells is sequenced at the single cell level (308) to assess the P-binding T cells (306). The second T cell population is incubated with cells presenting a predetermined type of antigen (310), and at least a first activation marker (e.g., CD137 and / or a secreted molecule suggesting activation such as those disclosed herein, e.g., interferon gamma) is measured to determine which members of the second T cell population are activated, and subsequently, the genetic expression profile (e.g., by transcriptome analysis) and sequence of the T cell receptor for the second T cell population are determined at the single cell level (312). (a) Divide the second T cell population into a plurality of T cell clusters (figuratively); (b) identify P-binding clusters within the plurality of T cell clusters by comparing the T cell receptor sequences of the P-binding T cells for the first T cell population with the sequences of the T cell receptors for the second T cell population; and (c) analyze the second T cell population by detecting which of the identified P-binding clusters contain at least a threshold number of cells presenting at least the first activation marker (314). Evaluate the genetic expression profile of the detected P-binding clusters (316) to identify yet another activation marker characteristic of P-activation of T cells.

[0182] Figure 3 illustrates one particular exemplary embodiment, but other variants are also within the scope of the present disclosure. In one particular embodiment, for example, PBMCs can be obtained from a whole blood sample. In one particular embodiment, for example, a plurality of different T cells can + include CD-4+ The T cells may be enriched. In certain embodiments, for example, a plurality of different T cells can include memory T cells, and the mixture may be enriched in memory T cells. In certain embodiments, for example, the process can include removal of a biomarker panel different from the panel shown depending on the T cell composition desired for enrichment. In certain embodiments, for example, rather than forming clusters based on detection of at least a first activation marker, the clusters can be formed based on similarity of the T cell receptor sequences (e.g., a cluster of T cells having at least a portion of a T cell receptor sequence characterized by sequence identity above a predetermined threshold). In certain embodiments, for example, the selected clustering method may not depend on at least a first activation marker, and measurement of at least a first activation marker is omitted.

[0183] The present disclosure also provides a composition comprising one or more of the components of the methods described herein. For example, in one embodiment, a composition is provided herein that includes an artificial T cell receptor selective for a predetermined type of antigen, at least a portion of the CDR3 region, which is at least a portion of the CDR3 region shared by at least one antigen-binding T cell and at least one antigen-activated T cell identified by analyzing a mixture of natural T cells to identify antigen-binding T cells and antigen-activated T cells for the predetermined type of antigen, and a T cell receptor fragment. In another embodiment, a composition is provided herein that includes P-binding T cells having at least one activation marker. In yet another embodiment, a composition is provided herein that includes a T cell receptor identified using the methods provided herein. In a further embodiment, a composition is provided herein that includes a T cell receptor clonotype identified using the methods provided herein.

[0184] Kits are also provided herein that include one or more of the components of the methods and compositions described herein. For example, in one embodiment, the kit includes a predetermined type of antigen. In another embodiment, the kit includes an assay for identifying activation markers. In a further embodiment, the kit includes an artificial T cell receptor selective for a predetermined type of antigen. In some embodiments, the kit includes a T cell receptor clonotype. It is further understood that the kits encompassed herein can be used in any of the methods disclosed herein. Incorporation by reference

[0185] Without limitation, the following documents are hereby incorporated by reference in their entirety: U.S. Patent Application Publication No. 2017 / 0212984; No. 2017 / 0192011; No. 2017 / 0003288; U.S. Patent No. 10,055,540; No. 10,066,265; International Patent Application Publication No. WO2018 / 175585; WO2018 / 165475; WO2018 / 085453; WO2017 / 075141; WO2015 / 106151; European Patent No. EP2327763; European Patent Application No. EP2327763; Alanio, C. et al., "Enumeration of human-antigen-specific CD8+ T cells reveals conserved precursor frequencies," Blood 115:18 (2010) 3718-3725; Moon, J. J. et al., "Naive CD4+ T cell frequencies varies for different epitopes and predicts repertoire diversity and response magnitude," Immunity 27:2 (August 2007) 203-213; Rius, C. et al., "Peptide-MHC Class I Tetramers Can Fail to Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations," J. Immunology 200 (2018) 2263-2279; Aleksic, M. et al., "Different affinity windows for virus and cancer-specific T-cell receptors - implications for therapeutic strategies," European J. Immunology 42:12 (December 2012) 3174-3179; Dimopoulos, N. et al., "Combining MHC tetramer and intracellular cytokine staining for CD8+ T cells to reveal antigenic epitopes naturally presented on tumor cells," J. Immunological Methods 340 (2009) 90-94; Kao H. et al., "A New Strategy for Tumor Antigen Discovery Based on in Vitro Priming of Naive T Cells with Dendritic Cells," Clinical Cancer Research 7 (2001) 773s-780s; Glanville, J. et al. "Identifying specificity groups in the T cell receptor repertoire," Nature 547:7661 (2017) 94-98; Bulik-Sullivan, B. et al., "Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification," Nature Biotechnology, AOP (December 11, 2018) 1-14; De Simone, D., "Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges," Frontiers In Immunology 9 (2018) Article 1638, 7 pages; Bossi, G., et al., "Examining the presentation of tumor-associated antigens on peptide-pulsed T2 cells" Oncolmmunology 2:11 (2013) e26840-1 to e26840-6; Purbhoo, M.A., et al., "Quantifying and Imaging NY-ESO-1 / LAGE-1-Derived Epitopes on Tumor Cells Using High Affinity T Cell Receptors" J Immunology 176 (2006) 7308-7316, Rosati et al., "Overview of methodologies for T-cell receptor repertoire analysis" BMC Biotechnology (2017) 17(1):61; Mahe, E. et al., "T cell clonality assessment: past, present and future" J Clin Pathol. (2018) Mar;71(3):195-200; and Bagaev DV, et al. "VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium," Nucleic Acids Res. 2020 Jan 8;48(D1):D1057-D1062 (collectively, the "incorporated references").

Example

[0186] (Example 1) Identification of Antigen-Binding Properties of ASSLPTTMNY (SEQ ID NO:1)-Specific T Cells and Antigen-Activated T Cell Receptors This example demonstrates the successful identification and selection of T cell receptor clonotypes present in antigen-specific and functional T cells using ASSLPTTMNY (SEQ ID NO:1) as an exemplary antigen and HLA-A*0101 as an exemplary gene encoding a class I MHC molecule. Although the examples provided herein are described using ASSLPTTMNY (SEQ ID NO:1) as an exemplary antigen, it is understood that the method can be performed using any antigen less than 50 amino acids in length.

[0187] Peripheral blood mononuclear cells (PBMCs) were obtained from leukapheresis samples from healthy donors who were HLA-A*0101 compatible. The PBMCs were exposed to biotinylated antibodies, magnetically labeled with streptavidin-coated microbeads, and subsequently magnetically sorted to exclude cells that were positive for CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, anti-HLA-DR, CD235a (glycophorin A), CD244, and CD4. Naïve CD8 + T cells were stained with a live / dead marker and lineage markers and isolated by passing them through a fluorescence-activated cell sorting machine. Naïve CD8 + T cells were polyclonally expanded to obtain a T cell sample. Next, the T cell sample was divided into three parts for the identification of antigen-binding and antigen-activated T cell receptors, and the assay was repeated twice.

[0188] To determine the T cell receptor sequences that emerged in antigen-binding T cells, an antigen-MHC assay (preformed) was performed using ASSLPTTMNY (SEQ ID NO: 1) as an exemplary antigen. The first part of the T cell sample was stained with a fluorescent reporter-labeled antigen-MHC protein tetramer and passed through a fluorescence-activated cell sorting machine. T cells that were positively stained with the fluorescent reporter suggested antigen binding.

[0189] In parallel, T cell activation after exposure to the exemplary antigen ASSLPTTMNY (SEQ ID NO: 1) was determined by separately measuring CD137 expression in one part of the cells and IFN-γ secretion in another part of the cells. CD137, a member of the tumor necrosis factor receptor (TNFR) family, has been successfully used to identify antigen-reactive cells in both the CD4+ and CD8+ T cell compartments. IFN-γ secretion was used as an exemplary cytokine suggesting a T cell functional response.

[0190] To detect CD137 expression, a portion of the T cell sample was stimulated overnight with autologous PBMC pulsed with 10 μM of the exemplary ASSLPTTMNY (SEQ ID NO: 1) antigen, stained with a magnetic-labeled CD137 antibody, and isolated by magnetic separation. Cells that were positively stained for CD137 suggested T cell activation in response to the specific antigen.

[0191] To detect IFN-γ secretion, another portion of the T cell sample was stimulated overnight with autologous PBMC pulsed with 10 μM of the antigen, assayed using the Miltenyi IFN-γ secretion assay to isolate cells expressing IFN-γ. Cells that secreted IFN-γ suggested T cell activation in response to the specific antigen.

[0192] Following each of the different assays, positive hits were sequenced to determine the T cell receptor sequences. T cells were sequenced at the single cell level using 10× Genomics single cell resolution paired immune TCR profiling. Chromium cell barcodes and unique molecular identifiers were tagged to the sequencing reads to determine the frequency of the complete T cell receptor sequences.

[0193] CD137 + Comparisons between the T cell receptor sequences of T cells and T cells bound to the antigen (Figure 4) and between the T cell receptor sequences of IFN-γ secreting T cells and T cells bound to the antigen (Figure 5) demonstrated that T cell receptor sequences that exhibit high frequencies of both antigen binding and T cell activation can be identified. Additionally, this result suggested that T cell receptor sequences (reference "A") could be identified using both the CD137 assay and the IFN-γ secretion assay (Table 2).

[0194] The sequence of reference "A" is

Chemical Formula

[0195] The TCR alpha and beta chains possess three hypervariable regions named complementarity-determining regions (CDR1, 2, and 3). CDR3 is involved in the recognition and binding to the processed antigen peptide, leading to the clonal expansion of T cells. Sequencing of the T cell receptor alpha chain VJ region and the T cell receptor beta chain VJ region also revealed the CDR3 of each of the TCR alpha and beta chains (underlined peptides). Specifically, the CDR3 region of the alpha variable region of reference "A" had a peptide sequence of CASPVDRGSTLGRLYF (SEQ ID NO: 21), and the CDR3 region of the beta variable region of reference "A" had a peptide sequence of CASSQVGTGSYEQYF (SEQ ID NO: 22).

[0196] Similarly, in a second independent experiment using the same exemplary antigen, ASSLPTTMNY (SEQ ID NO: 1), CD137 + Comparison between the T cell receptor sequences of T cells and T cells bound to the exemplary antigen (Figure 6) and comparison between the T cell receptor sequences of IFN-γ secreting T cells and T cells bound to the exemplary antigen (Figure 7) suggested that the T cell receptor sequences for T cells with the highest frequency for both antigen binding and CD137 expression or both antigen binding and IFN-γ secretion shared a common T cell receptor sequence (reference "B") (Table 2).

[0197] Sequencing of the T cell receptor sequence (reference "B") [Chemical formula] The T cell receptor alpha chain VJ region having a peptide sequence of and [Chemical formula] [Chemical formula] The VJ region of the T cell receptor beta chain having the peptide sequence of

[0198] The CDR3 region of the alpha variable region of reference "B" has the peptide sequence of CALSEARQYSGAGSYQLTF (SEQ ID NO: 23), and the CDR3 region of the beta variable region of reference "B" has the peptide sequence of CASSLEWGPYEQYF (SEQ ID NO: 24).

[0199] Overall, these results demonstrate that it is possible to succeed in the selection of T cell receptor clonotypes present in antigen-specific and functional T cells, which represents a novel approach towards the development of a T cell line that is therapeutically effective with a physiologically appropriate concentration of antigen. (Example 2) Identification of the antigen-binding properties of HSEVGLPVY (SEQ ID NO: 2)-specific T cells and the antigen-activated T cell receptor

[0200] This example demonstrates that, in combination with HLA-A*0101 as an exemplary MHC class I-encoding gene and using HSEVGLPVY (SEQ ID NO: 2) as another exemplary antigen, it was possible to succeed in the identification and selection of T cell receptor clonotypes present in antigen-specific and functional T cells.

[0201] T cell samples were aliquoted and separately tested for antigen binding and T cell activation using the CD137 expression assay as described above in Example I. Positive hits were sequenced to determine the T cell receptor sequences. The T cell receptor sequences that emerged in both antigen-binding T cells and antigen-activated T cells were noted (Table 3). Figures 8 and 9 show the results of the first and second of three replicates of the CD137 assay, respectively.

[0202] As shown in Table 3, for each of the T cell receptor sequence references (“I” to “I”), the peptide sequences of the T cell receptor alpha chain VJ region (SEQ ID NOs: 7 to 13) and the peptide sequences of the T cell receptor beta chain VJ region (SEQ ID NOs: 14 to 20) were determined. Notably, references “H”, “I”, “J” and “L” were among the T cell receptor sequences that appeared in both antigen-binding T cells and antigen-activated T cells in at least two repetitions of the CD137 test (Figures 8 and 9).

[0203] In addition, the CDR3 region of each of the identified T cell receptor sequences was determined (see the text in Table 3, shown in bold and underlined). Specifically, the CDR3 region of the alpha variable region of reference "I" had a peptide sequence of CAENSGGYQKVTF (SEQ ID NO: 25), and the CDR3 region of the beta variable region of reference "I" had a peptide sequence of CASSVGDHTIYF (SEQ ID NO: 26). The CDR3 region of the alpha variable region of reference "J" had a peptide sequence of CAMREGYRDDKIIF (SEQ ID NO: 27), and the CDR3 region of the beta variable region of reference "J" had a peptide sequence of CASSFSSGGAHEQFF (SEQ ID NO: 28). The CDR3 region of the alpha variable region of reference "K" had a peptide sequence of CAVNDYKLSF (SEQ ID NO: 29), and the CDR3 region of the beta variable region of reference "K" had a peptide sequence of CASSIGWNYEQYF (SEQ ID NO: 30). The CDR3 region of the alpha variable region of reference "L" had a peptide sequence of CILPNAGNMLTF (SEQ ID NO: 31), and the CDR3 region of the beta variable region of reference "L" had a peptide sequence of CATRGTGTQPQHF (SEQ ID NO: 32). The CDR3 region of the alpha variable region of reference "M" had a peptide sequence of CAGPREYGNKLVF (SEQ ID NO: 33), and the CDR3 region of the beta variable region of reference "M" had a peptide sequence of CASSVGGQGEVVQYF (SEQ ID NO: 34). The CDR3 region of the alpha variable region of reference "N" had a peptide sequence of CATDGKRVTGGGNKLTF (SEQ ID NO: 35), and the CDR3 region of the beta variable region of reference "N" had a peptide sequence of CASSLWRTGELFF (SEQ ID NO: 36). The CDR3 region of the alpha variable region of reference "O" had a peptide sequence of CADAPGSSYKLIF (SEQ ID NO: 37), and the CDR3 region of the beta variable region of reference "O" had a peptide sequence of CASSQVPHEQYF (SEQ ID NO: 38).

[0204] Collectively, these results further illustrate that the methods provided herein can successfully select T cell receptor clonotypes present in antigen-specific and functional T cells. Additionally, this result demonstrates the reproducibility of the T cell receptor sequences identified by this method. (Example 3) Identification of Antigen-Binding and Antigen-Activating T Cell Receptors

[0205] This example demonstrates that the method provided herein can also identify T cell receptors capable of sensing antigens presented by class II molecules of the major histocompatibility complex (MHC).

[0206] Peripheral blood mononuclear cells (PBMCs) can be obtained from leukapheresis samples of class II HLA-derived white blood cells, such as healthy donors who are HLA-DRB*101:01 compatible. PBMCs are exposed to biotinylated antibodies, magnetically labeled with streptavidin-coated microbeads, and subsequently magnetically sorted to exclude cells that are positive for CD45RO, CD14, CD15, CD16, CD19, CD25, CD34, CD36, CD57, CD123, CD235a (glycophorin A), CD244, and CD8. Naïve CD4 + T cells can be isolated by staining with live / dead markers and lineage markers and passing them through a fluorescence-activated cell sorting machine. Naïve CD4 + T cells can be polyclonally expanded to obtain a T cell sample.

[0207] The T cell samples can be aliquoted and separately tested for antigen binding and T cell activation using the CD137 expression assay as described above in Example I. The positive hits can be sequenced to determine the T cell receptor sequences. T cell receptor sequences that occur in both antigen-binding T cells and antigen-activated T cells can be determined, and for each of the T cell receptor sequence references, a specific peptide sequence for the T cell receptor alpha chain VJ region and a peptide sequence for the T cell receptor beta chain VJ region can be determined. In addition, the CDR3 region of each of the identified T cell receptor sequences can be determined.

[0208] This example demonstrates T cell receptor sequences that can interact with antigens presented by class II MHC molecules.

Table 1

Table 2-1

Table 2-2

Table 2-3

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 3-9

[0209] All publications and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0210] Preferred embodiments of the invention have been shown and described herein, it being apparent to those skilled in the art that such embodiments are provided by way of example only. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of such claims and their equivalents be covered thereby.

[0211] Array information SEQUENCE LISTING <110> GRITSTONE BIO, INC. <120> SELECTION OF T CELL RECEPTORS <150> US 62 / 812,572 <151> 2019-03-01 <160> 142 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <220> <223> Antigen <400> 1 Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr 1 5 10 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <220> <223> Antigen <400> 2 His Ser Glu Val Gly Leu Pro Val Tyr 1 5 <210> 3 <211> 136 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference A <400> 3 Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Ser Pro Val Asp Arg Gly Ser Thr Leu Gly Arg Leu Tyr Phe Gly 115 120 125 Arg Gly Thr Gln Leu Thr Val Trp 130 135 <210> 4 <211> 139 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference B <400> 4 Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Glu Ala Arg Gln Tyr Ser Gly Ala Gly Ser Tyr Gln Leu 115 120 125 Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ile 130 135 <210> 5 <211> 133 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference A <400> 5 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His Leu Val Met 20 25 30 Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Leu Gly His 35 40 45 Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro Leu Glu Leu 50 55 60 Met Phe Val Tyr Asn Phe Lys Glu Gln Thr Glu Asn Asn Ser Val Pro 65 70 75 80 Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu Phe Leu His 85 90 95 Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Gln Val Gly Thr Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr 115 120 125 Arg Leu Thr Val Thr 130 <210> 6 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference B <400> 6 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Glu Trp Gly Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr 130 <210> 7 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference I <400> 7 Met Ala Gly Ile Arg Ala Leu Phe Met Tyr Leu Trp Leu Gln Leu Asp 1 5 10 15 Trp Val Ser Arg Gly Glu Ser Val Gly Leu His Leu Pro Thr Leu Ser 20 25 30 Val Gln Glu Gly Asp Asn Ser Ile Ile Asn Cys Ala Tyr Ser Asn Ser 35 40 45 Ala Ser Asp Tyr Phe Ile Trp Tyr Lys Gln Glu Ser Gly Lys Gly Pro 50 55 60 Gln Phe Ile Ile Asp Ile Arg Ser Asn Met Asp Lys Arg Gln Gly Gln 65 70 75 80 Arg Val Thr Val Leu Leu Asn Lys Thr Val Lys His Leu Ser Leu Gln 85 90 95 Ile Ala Ala Thr Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys Ala Glu 100 105 110 Asn Ser Gly Gly Tyr Gln Lys Val Thr Phe Gly Thr Gly Thr Lys Leu 115 120 125 Gln Val Ile 130 <210> 8 <211> 134 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference J <400> 8 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Arg Glu Gly Tyr Arg Asp Asp Lys Ile Ile Phe Gly Lys Gly 115 120 125 Thr Arg Leu His Ile Leu 130 <210> 9 <211> 125 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference K <400> 9 Met Val Leu Lys Phe Ser Val Ser Ile Leu Trp Ile Gln Leu Ala Trp 1 5 10 15 Val Ser Thr Gln Leu Leu Glu Gln Ser Pro Gln Phe Leu Ser Ile Gln 20 25 30 Glu Gly Glu Asn Leu Thr Val Tyr Cys Asn Ser Ser Ser Val Phe Ser 35 40 45 Ser Leu Gln Trp Tyr Arg Gln Glu Pro Gly Glu Gly Pro Val Leu Leu 50 55 60 Val Thr Val Val Thr Gly Gly Glu Val Lys Lys Leu Lys Arg Leu Thr 65 70 75 80 Phe Gln Phe Gly Asp Ala Arg Lys Asp Ser Ser Leu His Ile Thr Ala 85 90 95 Ala Gln Pro Gly Asp Thr Gly Leu Tyr Leu Cys Ala Val Asn Asp Tyr 100 105 110 Lys Leu Ser Phe Gly Ala Gly Thr Thr Val Thr Val Arg 115 120 125 <210> 10 <211> 125 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference L <400> 10 Met Lys Leu Val Thr Ser Ile Thr Val Leu Leu Ser Leu Gly Ile Met 1 5 10 15 Gly Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu Glu 20 25 30 Glu Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr Asp 35 40 45 Tyr Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr Val 50 55 60 Ile His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu Ala 65 70 75 80 Ile Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala Thr 85 90 95 Leu Arg Asp Ala Ala Val Tyr Tyr Cys Ile Leu Pro Asn Ala Gly Asn 100 105 110 Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys 115 120 125 <210> 11 <211> 128 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference M <400> 11 Met Leu Leu Ile Thr Ser Met Leu Val Leu Trp Met Gln Leu Ser Gln 1 5 10 15 Val Asn Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val Gln 20 25 30 Glu Gly Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser 35 40 45 Asn Ile Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu 50 55 60 Ile Gln Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Thr 65 70 75 80 Phe Gln Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala 85 90 95 Thr Gln Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Gly Pro Arg Glu 100 105 110 Tyr Gly Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys 115 120 125 <210> 12 <211> 134 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference N <400> 12 Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu 1 5 10 15 Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser 20 25 30 Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser 35 40 45 Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val 50 55 60 His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg 65 70 75 80 Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile 85 90 95 Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Asp 100 105 110 Gly Lys Arg Val Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr Gly 115 120 125 Thr Gln Leu Lys Val Glu 130 <210> 13 <211> 126 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference O <400> 13 Met Trp Gly Ala Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr 1 5 10 15 Ala Gly Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly 20 25 30 Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly 35 40 45 Leu Ser Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser 50 55 60 Tyr Asn Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe 65 70 75 80 Leu Ser Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln 85 90 95 Met Lys Asp Ser Ala Ser Tyr Phe Cys Ala Asp Ala Pro Gly Ser Ser 100 105 110 Tyr Lys Leu Ile Phe Gly Ser Gly Thr Arg Leu Leu Val Arg 115 120 125 <210> 14 <211> 130 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference I <400> 14 Met Gly Phe Arg Leu Leu Cys Cys Val Ala Phe Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr 20 25 30 Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp 35 40 45 Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe 50 55 60 Leu Ile Gln Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu 65 70 75 80 Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn 85 90 95 Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Val Gly Asp His Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr 115 120 125 Val Val 130 <210> 15 <211> 133 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference J <400> 15 Met Ser Ile Ser Leu Leu Cys Cys Ala Ala Phe Pro Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Arg Ile Leu 20 25 30 Lys Ile Gly Gln Ser Met Thr Leu Gln Cys Thr Gln Asp Met Asn His 35 40 45 Asn Tyr Met Tyr Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Lys Leu 50 55 60 Ile Tyr Tyr Ser Val Gly Ala Gly Ile Thr Asp Lys Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Glu Leu Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Phe Ser Ser Gly Gly Ala His Glu Gln Phe Phe Gly Pro Gly Thr 115 120 125 Arg Leu Thr Val Leu 130 <210> 16 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference K <400> 16 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Ile Gly Trp Asn Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr 130 <210> 17 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference L <400> 17 Met Gly Pro Gly Leu Leu His Trp Met Ala Leu Cys Leu Leu Gly Thr 1 5 10 15 Gly His Gly Asp Ala Met Val Ile Gln Asn Pro Arg Tyr Gln Val Thr 20 25 30 Gln Phe Gly Lys Pro Val Thr Leu Ser Cys Ser Gln Thr Leu Asn His 35 40 45 Asn Val Met Tyr Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu 50 55 60 Leu Phe His Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro 65 70 75 80 Asp Asn Phe Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp 85 90 95 Ile Arg Ser Pro Gly Leu Gly Asp Ala Ala Met Tyr Leu Cys Ala Thr 100 105 110 Arg Gly Thr Gly Thr Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu 115 120 125 Ser Ile Leu 130 <210> 18 <211> 134 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference M <400> 18 Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala 1 5 10 15 Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr 20 25 30 Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His 35 40 45 Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe 50 55 60 Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe 65 70 75 80 Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu 85 90 95 Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala 100 105 110 Ser Ser Val Gly Gly Gln Gly Glu Val Val Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr 130 <210> 19 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference N <400> 19 Met Gly Thr Arg Leu Leu Cys Trp Ala Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Glu Leu Thr Glu Ala Gly Val Ala Gln Ser Pro Arg Tyr Lys Ile Ile 20 25 30 Glu Lys Arg Gln Ser Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His 35 40 45 Ala Thr Leu Tyr Trp Tyr Gln Gln Ile Leu Gly Gln Gly Pro Lys Leu 50 55 60 Leu Ile Gln Phe Gln Asn Asn Gly Val Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Lys Leu Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Trp Arg Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg 115 120 125 Leu Thr Val Leu 130 <210> 20 <211> 130 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference O <400> 20 Met Gly Cys Arg Leu Leu Cys Cys Val Val Phe Cys Leu Leu Gln Ala 1 5 10 15 Gly Pro Leu Asp Thr Ala Val Ser Gln Thr Pro Lys Tyr Leu Val Thr 20 25 30 Gln Met Gly Asn Asp Lys Ser Ile Lys Cys Glu Gln Asn Leu Gly His 35 40 45 Asp Thr Met Tyr Trp Tyr Lys Gln Asp Ser Lys Lys Phe Leu Lys Ile 50 55 60 Met Phe Ser Tyr Asn Asn Lys Glu Leu Ile Ile Asn Glu Thr Val Pro 65 70 75 80 Asn Arg Phe Ser Pro Lys Ser Pro Asp Lys Ala His Leu Asn Leu His 85 90 95 Ile Asn Ser Leu Glu Leu Gly Asp Ser Ala Val Tyr Phe Cys Ala Ser 100 105 110 Ser Gln Val Pro His Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr 115 120 125 Val Thr 130 <210> 21 <211> 16 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference A <400> 21 Cys Ala Ser Pro Val Asp Arg Gly Ser Thr Leu Gly Arg Leu Tyr Phe 1 5 10 15 <210> 22 <211> 15 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference A <400> 22 Cys Ala Ser Ser Gln Val Gly Thr Gly Ser Tyr Glu Gln Tyr Phe 1 5 10 15 <210> 23 <211> 19 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference B <400> 23 Cys Ala Leu Ser Glu Ala Arg Gln Tyr Ser Gly Ala Gly Ser Tyr Gln 1 5 10 15 Leu Thr Phe <210> 24 <211> 14 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference B <400> 24 Cys Ala Ser Ser Leu Glu Trp Gly Pro Tyr Glu Gln Tyr Phe 1 5 10 <210> 25 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference I <400> 25 Cys Ala Glu Asn Ser Gly Gly Tyr Gln Lys Val Thr Phe 1 5 10 <210> 26 <211> 12 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference I <400> 26 Cys Ala Ser Ser Val Gly Asp His Thr Ile Tyr Phe 1 5 10 <210> 27 <211> 14 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference J <400> 27 Cys Ala Met Arg Glu Gly Tyr Arg Asp Asp Lys Ile Ile Phe 1 5 10 <210> 28 <211> 15 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference J <400> 28 Cys Ala Ser Ser Phe Ser Ser Gly Gly Ala His Glu Gln Phe Phe 1 5 10 15 <210> 29 <211> 10 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference K <400> 29 Cys Ala Val Asn Asp Tyr Lys Leu Ser Phe 1 5 10 <210> 30 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference K <400> 30 Cys Ala Ser Ser Ile Gly Trp Asn Tyr Glu Gln Tyr Phe 1 5 10 <210> 31 <211> 12 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference L <400> 31 Cys Ile Leu Pro Asn Ala Gly Asn Met Leu Thr Phe 1 5 10 <210> 32 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference L <400> 32 Cys Ala Thr Arg Gly Thr Gly Thr Gln Pro Gln His Phe 1 5 10 <210> 33 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference M <400> 33 Cys Ala Gly Pro Arg Glu Tyr Gly Asn Lys Leu Val Phe 1 5 10 <210> 34 <211> 15 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference M <400> 34 Cys Ala Ser Ser Val Gly Gly Gln Gly Glu Val Val Gln Tyr Phe 1 5 10 15 <210> 35 <211> 17 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference N <400> 35 Cys Ala Thr Asp Gly Lys Arg Val Thr Gly Gly Gly Asn Lys Leu Thr 1 5 10 15 Phe <210> 36 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference N <400> 36 Cys Ala Ser Ser Leu Trp Arg Thr Gly Glu Leu Phe Phe 1 5 10 <210> 37 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference O <400> 37 Cys Ala Asp Ala Pro Gly Ser Ser Tyr Lys Leu Ile Phe 1 5 10 <210> 38 <211> 12 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference O <400> 38 Cys Ala Ser Ser Gln Val Pro His Glu Gln Tyr Phe 1 5 10 <210> 39 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference C <400> 39 Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe 20 25 30 Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn 35 40 45 Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu 50 55 60 Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg 65 70 75 80 Phe Thr Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile 85 90 95 Arg Asp Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Pro 100 105 110 Arg Met Asp Ser Ser Tyr Lys Leu Ile Phe Gly Ser Gly Thr Arg Leu 115 120 125 Leu Val Arg Pro 130 <210> 40 <211> 140 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference D <400> 40 Met Leu Thr Ala Ser Leu Leu Arg Ala Val Ile Ala Ser Ile Cys Val 1 5 10 15 Val Ser Ser Met Ala Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser 20 25 30 Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thr Arg 35 40 45 Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu 50 55 60 Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile 65 70 75 80 Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn 85 90 95 Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys 100 105 110 Ala Leu Ser Glu Ala Arg Gln Tyr Ser Gly Ala Gly Ser Tyr Gln Leu 115 120 125 Thr Phe Gly Lys Gly Thr Lys Leu Ser Val Ile Pro 130 135 140 <210> 41 <211> 129 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference E <400> 41 Met Leu Leu Ile Thr Ser Met Leu Val Leu Trp Met Gln Leu Ser Gln 1 5 10 15 Val Asn Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val Gln 20 25 30 Glu Gly Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser 35 40 45 Asn Ile Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu 50 55 60 Ile Gln Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Thr 65 70 75 80 Phe Gln Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala 85 90 95 Thr Gln Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Gly Gln Gly Asn 100 105 110 Arg Asp Asp Lys Ile Ile Phe Gly Lys Gly Thr Arg Leu His Ile Leu 115 120 125 Pro <210> 42 <211> 131 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference F <400> 42 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 60 Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80 Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95 Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val 100 105 110 Lys Asp Asn Asn Ala Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val 115 120 125 Val Lys Pro 130 <210> 43 <211> 133 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference G <400> 43 Met Glu Thr Leu Leu Gly Val Ser Leu Val Ile Leu Trp Leu Gln Leu 1 5 10 15 Ala Arg Val Asn Ser Gln Gln Gly Glu Glu Asp Pro Gln Ala Leu Ser 20 25 30 Ile Gln Glu Gly Glu Asn Ala Thr Met Asn Cys Ser Tyr Lys Thr Ser 35 40 45 Ile Asn Asn Leu Gln Trp Tyr Arg Gln Asn Ser Gly Arg Gly Leu Val 50 55 60 His Leu Ile Leu Ile Arg Ser Asn Glu Arg Glu Lys His Ser Gly Arg 65 70 75 80 Leu Arg Val Thr Leu Asp Thr Ser Lys Lys Ser Ser Ser Leu Leu Ile 85 90 95 Thr Ala Ser Arg Ala Ala Asp Thr Ala Ser Tyr Phe Cys Ala Thr Ala 100 105 110 Val Phe Asn Phe Gly Asn Glu Lys Leu Thr Phe Gly Thr Gly Thr Arg 115 120 125 Leu Thr Ile Ile Pro 130 <210> 44 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference H <400> 44 Met Ala Gly Ile Arg Ala Leu Phe Met Tyr Leu Trp Leu Gln Leu Asp 1 5 10 15 Trp Val Ser Arg Gly Glu Ser Val Gly Leu His Leu Pro Thr Leu Ser 20 25 30 Val Gln Glu Gly Asp Asn Ser Ile Ile Asn Cys Ala Tyr Ser Asn Ser 35 40 45 Ala Ser Asp Tyr Phe Ile Trp Tyr Lys Gln Glu Ser Gly Lys Gly Pro 50 55 60 Gln Phe Ile Ile Asp Ile Arg Ser Asn Met Asp Lys Arg Gln Gly Gln 65 70 75 80 Arg Val Thr Val Leu Leu Asn Lys Thr Val Lys His Leu Ser Leu Gln 85 90 95 Ile Ala Ala Thr Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys Ala Glu 100 105 110 Asn Met Gly Gly Ala Gly Lys Ser Thr Phe Gly Asp Gly Thr Thr Leu 115 120 125 Thr Val Lys Pro 130 <210> 45 <211> 134 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference C <400> 45 Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys 20 25 30 Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His 35 40 45 Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe 50 55 60 Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro 65 70 75 80 Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn 85 90 95 Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Ser Thr Gly Ala Arg Arg Ser Arg Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr 130 <210> 46 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference D <400> 46 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Glu Trp Gly Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr 130 <210> 47 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference E <400> 47 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Glu Trp Gly Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr 130 <210> 48 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference F <400> 48 Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys 20 25 30 Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His 35 40 45 Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe 50 55 60 Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro 65 70 75 80 Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn 85 90 95 Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Leu Ser Ser Gly Leu Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr 130 <210> 49 <211> 134 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference G <400> 49 Met Gly Ser Arg Leu Leu Cys Trp Val Leu Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Val Lys Ala Gly Val Thr Gln Thr Pro Arg Tyr Leu Ile Lys 20 25 30 Thr Arg Gly Gln Gln Val Thr Leu Ser Cys Ser Pro Ile Ser Gly His 35 40 45 Arg Ser Val Ser Trp Tyr Gln Gln Thr Pro Gly Gln Gly Leu Gln Phe 50 55 60 Leu Phe Glu Tyr Phe Ser Glu Thr Gln Arg Asn Lys Gly Asn Phe Pro 65 70 75 80 Gly Arg Phe Ser Gly Arg Gln Phe Ser Asn Ser Arg Ser Glu Met Asn 85 90 95 Val Ser Thr Leu Glu Leu Gly Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Ser Met Thr Ser Gly Gly Pro Trp Glu Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Thr Val Thr 130 <210> 50 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Beta VJ region for Reference H <400> 50 Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90 95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr 100 105 110 Ser Gly Gly Val Ala Gly Val Arg Gln Phe Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu 130 <210> 51 <211> 14 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference C <400> 51 Cys Val Val Pro Arg Met Asp Ser Ser Tyr Lys Leu Ile Phe 1 5 10 <210> 52 <211> 16 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference C <400> 52 Cys Ala Ser Ser Ser Thr Gly Ala Arg Arg Ser Arg Glu Gln Tyr Phe 1 5 10 15 <210> 53 <211> 19 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference D <400> 53 Cys Ala Leu Ser Glu Ala Arg Gln Tyr Ser Gly Ala Gly Ser Tyr Gln 1 5 10 15 Leu Thr Phe <210> 54 <211> 14 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference D <400> 54 Cys Ala Ser Ser Leu Glu Trp Gly Pro Tyr Glu Gln Tyr Phe 1 5 10 <210> 55 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference E <400> 55 Cys Ala Gly Gln Gly Asn Arg Asp Asp Lys Ile Ile Phe 1 5 10 <210> 56 <211> 14 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference E <400> 56 Cys Ala Ser Ser Leu Glu Trp Gly Pro Tyr Glu Gln Tyr Phe 1 5 10 <210> 57 <211> 12 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference F <400> 57 Cys Ala Val Lys Asp Asn Asn Ala Arg Leu Met Phe 1 5 10 <210> 58 <211> 14 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference F <400> 58 Cys Ala Ser Ser Leu Ser Ser Gly Leu Tyr Glu Gln Tyr Phe 1 5 10 <210> 59 <211> 15 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference G <400> 59 Cys Ala Thr Ala Val Phe Asn Phe Gly Asn Glu Lys Leu Thr Phe 1 5 10 15 <210> 60 <211> 16 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference G <400> 60 Cys Ala Ser Ser Ser Met Thr Ser Gly Gly Pro Trp Glu Gln Tyr Phe 1 5 10 15 <210> 61 <211> 13 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the alpha variable region for Reference H <400> 61 Cys Ala Glu Asn Met Gly Gly Ala Gly Lys Ser Thr Phe 1 5 10 <210> 62 <211> 14 <212> PRT <213> Homo sapiens <220> <223> CDR3 region of the beta variable region for Reference H <400> 62 Cys Ala Thr Ser Gly Gly Val Ala Gly Val Arg Gln Phe Phe 1 5 10 <210> 63 <211> 135 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference P <400> 63 Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15 Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe 20 25 30 Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser 35 40 45 Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 50 55 60 Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80 Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95 Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110 Ala Met Arg Glu Gly Tyr Arg Asp Asp Lys Ile Ile Phe Gly Lys Gly 115 120 125 Thr Arg Leu His Ile Leu Pro 130 135 <210> 64 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference Q <400> 64 Met Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe 20 25 30 Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser Asn 35 40 45 Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys Arg Lys Glu 50 55 60 Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly Asn Glu Asp Gly Arg 65 70 75 80 Phe Thr Ala Gln Leu Asn Arg Ala Ser Gln Tyr Ile Ser Leu Leu Ile 85 90 95 Arg Asp Ser Lys Leu Ser Asp Ser Ala Thr Tyr Leu Cys Val Val Asn 100 105 110 Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu 115 120 125 Ser Val Ile Pro 130 <210> 65 <211> 132 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference R <400> 65 Met Ala Gly Ile Arg Ala Leu Phe Met Tyr Leu Trp Leu Gln Leu Asp 1 5 10 15 Trp Val Ser Arg Gly Glu Ser Val Gly Leu His Leu Pro Thr Leu Ser 20 25 30 Val Gln Glu Gly Asp Asn Ser Ile Ile Asn Cys Ala Tyr Ser Asn Ser 35 40 45 Ala Ser Asp Tyr Phe Ile Trp Tyr Lys Gln Glu Ser Gly Lys Gly Pro 50 55 60 Gln Phe Ile Ile Asp Ile Arg Ser Asn Met Asp Lys Arg Gln Gly Gln 65 70 75 80 Arg Val Thr Val Leu Leu Asn Lys Thr Val Lys His Leu Ser Leu Gln 85 90 95 Ile Ala Ala Thr Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys Ala Glu 100 105 110 Asn Ser Gly Gly Tyr Gln Lys Val Thr Phe Gly Thr Gly Thr Lys Leu 115 120 125 Gln Val Ile Pro 130 <210> 66 <211> 129 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference S <400> 66 Met Leu Leu Ile Thr Ser Met Leu Val Leu Trp Met Gln Leu Ser Gln 1 5 10 15 Val Asn Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val Gln 20 25 30 Glu Gly Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser 35 40 45 Asn Ile Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu 50 55 60 Ile Gln Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Thr 65 70 75 80 Phe Gln Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala 85 90 95 Thr Gln Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Gly Pro Arg Glu 100 105 110 Tyr Gly Asn Lys Leu Val Phe Gly Ala Gly Thr Ile Leu Arg Val Lys 115 120 125 Ser <210> 67 <211> 127 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference T <400> 67 Met Trp Gly Ala Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr 1 5 10 15 Ala Gly Gln Ser Leu Glu Gln Pro Ser Glu Val Thr Ala Val Glu Gly 20 25 30 Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Tyr Gly 35 40 45 Leu Ser Trp Tyr Gln Gln His Asp Gly Gly Ala Pro Thr Phe Leu Ser 50 55 60 Tyr Asn Ala Leu Asp Gly Leu Glu Glu Thr Gly Arg Phe Ser Ser Phe 65 70 75 80 Leu Ser Arg Ser Asp Ser Tyr Gly Tyr Leu Leu Leu Gln Glu Leu Gln 85 90 95 Met Lys Asp Ser Ala Ser Tyr Phe Cys Ala Val Arg Ala Gln Gly Asn 100 105 110 Ala Arg Leu Met Phe Gly Asp Gly Thr Gln Leu Val Val Lys Pro 115 120 125 <210> 68 <211> 126 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference U <400> 68 Met Lys Leu Val Thr Ser Ile Thr Val Leu Leu Ser Leu Gly Ile Met 1 5 10 15 Gly Asp Ala Lys Thr Thr Gln Pro Asn Ser Met Glu Ser Asn Glu Glu 20 25 30 Glu Pro Val His Leu Pro Cys Asn His Ser Thr Ile Ser Gly Thr Asp 35 40 45 Tyr Ile His Trp Tyr Arg Gln Leu Pro Ser Gln Gly Pro Glu Tyr Val 50 55 60 Ile His Gly Leu Thr Ser Asn Val Asn Asn Arg Met Ala Ser Leu Ala 65 70 75 80 Ile Ala Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu His Arg Ala Thr 85 90 95 Leu Arg Asp Ala Ala Val Tyr Tyr Cys Ile Leu Pro Asn Ala Gly Asn 100 105 110 Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro 115 120 125 <210> 69 <211> 136 <212> PRT <213> Homo sapiens <220> <223> Alpha VJ region for Reference V <400> 69 Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala...

Claims

[Claim 1] The invention described herein.

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