Polypeptide constructs with novel binding affinities and uses thereof - Patents.com

Abstract

The present disclosure relates generally to polypeptide constructs, and in particular to T cell receptor (TCR) constructs that have binding affinity for a specific cognate antigen. The disclosure further provides compositions and methods useful for producing the constructs, as well as methods for diagnosing, preventing, and / or treating diseases associated with cells expressing the cognate antigen recognized by the polypeptide construct.

Description

[Technical field]

[0001] Related Patent Cross-References This application claims priority to U.S. Provisional Patent Application No. 63 / 156,026, filed March 3, 2021, the disclosure of which is incorporated by reference in its entirety, including any drawings.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant No. U54 CA232568-01 awarded by the National Cancer Institute. The government has certain rights in this invention.

[0003] The present disclosure relates generally to the field of immunology, and in particular to polypeptide constructs that have binding affinity for specific antigens. The present disclosure also provides compositions and methods useful for generating such constructs, as well as methods for the diagnosis, prevention, and / or treatment of health disorders associated with cells expressing the cognate antigen that the polypeptide construct recognizes. [Background technology]

[0004] In recent years, the widespread use of immune checkpoint blockade and T cell-based immunotherapy to treat patients with solid tumors requires a better understanding of T cell specificity within cancer. For example, T cell receptors (TCRs) have recently emerged as a promising approach for immunotherapy and have been the subject of clinical trials conducted by several pharmaceutical and biotechnology companies. TCRs have emerged as having therapeutic and diagnostic capabilities and can be modified similarly to antibody molecules. The affinity of TCRs, especially for specific antigens, can enhance the value of various therapeutic approaches, such as adoptive immunotherapy.

[0005] However, despite the widespread use of immunotherapy to treat cancer, there is limited overall understanding of the specificity of T cells in cancer. For example, although antigen specificity is a key determinant of T cell function, the challenges posed by TCR diversity and polymorphism of human leukocyte antigen (HLA) alleles pose a major obstacle to understanding the full range of antigens recognized by tumor-infiltrating T cells. Furthermore, despite the availability of advanced techniques using single-cell sequencing to analyze T cell morphology and repertoires, the specificity of the majority of tumor-infiltrating T cells remains unknown in all solid tumors. This is mainly due to the lack of tools to analyze the different TCR repertoires in the context of highly polymorphic human leukocyte antigen (HLA) alleles. For example, although next-generation sequencing techniques have made it relatively easy and inexpensive to sequence large numbers of TCRs, a major question that remains at the center of debate is how these very large repertoires can be analyzed. This problem is based on the fact that there may be hundreds or even thousands of potential TCR sequences for the same peptide-MHC (major histocompatibility complex) specificity.

[0006] Elucidating the specificity of tumor-infiltrating T cells is therefore important for understanding how T cell-intrinsic factors shape their interactions with the tumor-immune system and influence therapies aimed at harnessing T cell responses against cancer. Summary of the Invention

[0007] The present disclosure generally relates to the field of immunology. More specifically, novel polypeptide constructs are provided herein that have binding affinity for specific antigens. The present disclosure also provides compositions and methods useful for producing such polypeptide constructs, as well as methods for diagnosing, preventing, and / or treating diseases associated with cells expressing the cognate antigen that the polypeptide construct recognizes. In particular, the present disclosure further provides recombinant cells, such as lymphocytic T cells, that are genetically engineered to express the polypeptide constructs disclosed herein and directed against cells of interest, such as cancer cells.

[0008] In one aspect, provided herein are various constructs comprising at least one complementarity determining region (CDR) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106.

[0009] Non-limiting exemplary embodiments of constructs of the present disclosure may include one or more of the following features: In some embodiments, at least one CDR has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-56. In some embodiments, at least one CDR has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, at least one CDR has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 57-106. In some embodiments, at least one CDR has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:64. In some embodiments, the construct is a single chain construct or a double chain construct. In some embodiments, the construct is selected from the group consisting of: (a) a T cell receptor (TCR); (b) an antibody; and (c) a functional derivative or fragment of (a) or (b). In some embodiments, the construct is a TCR construct comprising a TCR alpha chain and a TCR beta chain operably linked to each other. In some embodiments, the construct is a TCR construct comprising a CDR3 beta in its beta chain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs:1-106. In some embodiments, the CDR3β has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1 to 56. In some embodiments, the CDR3β has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18.In some embodiments, the CDR3β has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 57-106. In some embodiments, the CDR3β has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 64. In some embodiments, the construct further comprises a CDR3α sequence within its α chain.

[0010] In some embodiments, the constructs disclosed herein are antigen-binding fragments (Fab), single chain variable fragments (scFv), single chain antibodies, single domain antibodies (sdAbs), V H Domain, V L Domain, V H The antibody construct is selected from the group consisting of an H domain, a bispecific antibody, or a functional fragment of either thereof.

[0011] In another aspect, provided herein is a recombinant nucleic acid, which is a nucleic acid comprising a nucleic acid sequence encoding a construct of the invention.

[0012] Non-limiting exemplary embodiments of the nucleic acids of the present disclosure may include one or more of the following features: In some embodiments, the nucleic acid sequence is operably linked to a heterologous nucleic acid sequence. In some embodiments, the nucleic acid molecule is further configured as an expression cassette or expression vector. In some embodiments, the vector is a plasmid vector or a viral vector. In some embodiments, the viral vector is derived from a lentivirus, adenovirus, adeno-associated virus, baculovirus, or retrovirus.

[0013] In another aspect, some embodiments of the present disclosure relate to a genetically engineered cell comprising one or more of (a) the constructs of the present disclosure and / or (b) the recombinant nucleic acids of the present disclosure. Non-limiting example embodiments of the cells of the present disclosure may include one or more of the following features: In some embodiments, the genetically engineered cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (T H ), cytotoxic T cells (T CTL ), memory T cells, γδ T cells, other T cells, hematopoietic stem cells, or hematopoietic stem cell precursors.

[0014] In some embodiments, the immune cells are lymphocytes. In some embodiments, the lymphocytes are T lymphocytes or T lymphocyte precursors. In some embodiments, the T lymphocytes are CD4+ T cells or CD8+ T cells. In some embodiments, the T lymphocytes are CD8+ T cytotoxic lymphocyte cells selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, the T lymphocytes are CD4+ T helper lymphocyte cells selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

[0015] In a related aspect, some embodiments of the present disclosure relate to a cell culture comprising at least one genetically engineered cell of the present disclosure and a medium.

[0016] In another aspect, some embodiments disclosed herein relate to a method of producing a genetically engineered cell, the method comprising: (a) providing a host cell capable of protein expression; and (b) transducing the provided host cell with a recombinant nucleic acid of the present disclosure to produce the genetically engineered cell. Thus, in a related aspect, genetically engineered cells produced by the methods of the present disclosure are also provided herein. In a further related aspect, some embodiments of the present disclosure relate to a cell culture comprising at least one genetically engineered cell of the present disclosure and a medium.

[0017] In one aspect, provided herein are various pharmaceutical compositions comprising a pharma- ceutically acceptable carrier and one or more of: (a) a construct of the present disclosure; (b) a recombinant nucleic acid of the present disclosure; and / or (c) a genetically engineered cell of the present disclosure.

[0018] Non-limiting exemplary embodiments of pharmaceutical compositions of the present disclosure may include one or more of the following features: In some embodiments, the composition comprises a recombinant nucleic acid of the present disclosure and a pharma- ceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or lipid nanoparticle. In some embodiments, the composition comprises a genetically engineered cell of the present disclosure and a pharma- ceutically acceptable carrier.

[0019] In another aspect, some embodiments of the present disclosure relate to a method of preventing and / or treating a disease in a subject in need thereof, comprising administering to the subject a composition comprising one or more of: (a) a construct of the present disclosure; (b) a recombinant nucleic acid of the present disclosure; (C) a genetically engineered cell of the present disclosure; and (d) a pharmaceutical composition of the present disclosure.

[0020] Non-limiting exemplary embodiments of the disclosed methods of preventing and / or treating a disease in a subject in need thereof may include one or more of the following features: In some embodiments, the disease is associated with immune checkpoint inhibition. In some embodiments, the method is a checkpoint inhibition immunotherapy. In some embodiments, the checkpoint inhibition immunotherapy is an anti-PD1 checkpoint therapy or an anti-PD1-L1 checkpoint therapy. In some embodiments, the disease is associated with lung cancer and is selected from adenocarcinoma, squamous cell carcinoma, small cell carcinoma, non-small cell carcinoma, adenosquamous carcinoma, small cell lung carcinoma, large cell carcinoma, neuroendocrine carcinoma of the lung, non-small cell lung carcinoma (NSCLC), undifferentiated non-small cell carcinoma, other non-small cell carcinoma not specified, lung squamous cell carcinoma, bronchioloalveolar carcinoma, sarcomatoid carcinoma, pleomorphic carcinoma, carcinosarcoma, pulmonary blastoma, metastatic carcinoma of unknown primary, primary lung lymphoepithelioma-like carcinoma, and benign neoplasms of the lung. In some embodiments, the lung cancer is an NSCLC selected from the group consisting of squamous cell carcinoma, adenocarcinoma, large cell carcinoma, carcinoid tumor, pleomorphic carcinoma, salivary gland carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, and unclassified carcinoma. In some embodiments, the NSCLC includes stage I NSCLC or stage II NSCLC. In some embodiments, the cancer is non-metastatic cancer, metastatic cancer, multidrug resistant cancer, or recurrent cancer. In some embodiments, the administered composition inhibits tumor growth or cancer metastasis in the subject.

[0021] In some embodiments, provided herein are methods of preventing and / or treating a disease that is a malignancy associated with a viral infection in a subject in need thereof. In some embodiments, the disease is a malignancy associated with infection by Epstein-Barr Virus (EBV). In some embodiments, the malignancy is associated with EBV infection and is selected from the group consisting of Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphoma, nasopharyngeal carcinoma, gastric cancer, post-transplant lymphoproliferative disorders, B-lymphoproliferative disorders, T / NK lymphoproliferative disorders, T / NK lymphoma / leukemia, leiomyosarcoma, and lymphoepithelioma-like carcinoma.

[0022] In some embodiments of the method of preventing and / or treating a disease in a subject in need thereof, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the composition is administered to the subject individually as a first therapy (monotherapy) or in combination with at least one additional therapy. In some embodiments, the at least one additional therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, or surgery. In some embodiments, the at least one additional therapy is selected from the group consisting of anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single dose, fractionated, focal, whole organ radiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptively transferred T cells, anticancer vaccine, and oncolytic virus. In some embodiments, the first therapy and the at least one additional therapy are administered concomitantly. In some embodiments, the first therapy and the at least one additional therapy are administered simultaneously. In some embodiments, the first therapy and the at least one additional therapy are administered sequentially. In some embodiments, the first treatment is applied before the at least one additional treatment. In some embodiments, the first treatment is applied after the at least one additional treatment. In some embodiments, the first treatment is applied before and / or after the at least one additional treatment. In some embodiments, the first treatment and the at least one additional treatment are applied in alternation. In some embodiments, the first treatment and the at least one additional treatment are applied together in a single formulation.

[0023] In another aspect, some embodiments of the present disclosure relate to kits for carrying out the methods disclosed herein. Some embodiments relate to kits for methods of diagnosing, preventing, and / or treating a disease in a subject in need thereof, the kits comprising one or more of a construct of the present disclosure; a recombinant nucleic acid of the present disclosure; a genetically engineered cell of the present disclosure; and a pharmaceutical composition of the present disclosure.

[0024] In another aspect, the present specification provides the use of one or more of the construct of the present disclosure; the recombinant nucleic acid of the present disclosure; the genetically engineered cell of the present disclosure; and the pharmaceutical composition for the prevention and / or treatment of disease. In some embodiments, the disease is a proliferative disorder. In some embodiments, the proliferative disorder is cancer. In some embodiments, the disease is a malignant tumor associated with an infectious disease. In some embodiments, the infectious disease is a bacterial or viral infection.

[0025] In another aspect, the present disclosure provides the use of one or more of the constructs, recombinant nucleic acids, engineered cells, or pharmaceutical compositions of the present disclosure in the manufacture of a medicament for treating a health disorder. In some embodiments, the disorder is a proliferative disorder. In some embodiments, the proliferative disorder is cancer. In some embodiments, the disorder is a malignant tumor associated with an infectious disease. In some embodiments, the infectious disease is a bacterial or viral infection.

[0026] In yet another aspect, various methods for obtaining the constructs disclosed herein are provided herein, comprising the steps of: (a) identifying a plurality of T cell receptors (TCRs) associated with a health disorder; (b) determining the sequence of CDR3β present in each of the identified TCRs; (c) generating a construct comprising the CDR3β sequence determined in (b), the construct being capable of binding to one or more cognate antigens. In some embodiments, the disorder is a proliferative disease. In some embodiments, the method further comprises identifying one or more antigens commonly recognized by the CDR3β sequences.

[0027] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings, detailed description, and claims. [Brief description of the drawings]

[0028] [Figure 1A] Figures 1A-1C summarize the results of the experiments carried out to validate specificity groups with TCR CDR3β sequences from lung cancer patients. Figure 1A shows a schematic diagram of the four steps involved in inferring TCR specificity by the GLIPH2 (Grouping of Lymphocyte Interactions by Paratope Hotspots) algorithm. Step 1 involves obtaining the CDR3β sequences of T cell receptors. In this step, 778,938 CDR3β sequences from the University of Texas MD Anderson Cancer Center (MDACC) population were used as input for the GLIPH2 analysis. Step 2 involves the detection of short sequence motifs within the CDR3β sequences from multiple patients. These common motifs are predicted to be involved in direct association with antigen peptides loaded on HLA molecules. In this step, 66,094 specificity groups were validated by multiple evaluation criteria (see also Figure 3A). Step 3 involves the validation of 4,226 clonally amplified specificity groups. Several limiting conditions are used to infer specific specificity groups, including a) enrichment of Vβ genes, b) minimum number of distinct CDR3β sequences=3, c) minimum number of patients=3, d) enrichment of clonally amplified CDR3β clonotypes, and e) enrichment of HLA alleles. Step 4 involves the establishment of 435 clonally amplified tumor-enriched specificity groups. [Figure 1B]Figure 1B shows the association of tumor-enriched specificity groups in lung cancer. The most amplified CDR3β sequences from tumors belonged to 435 tumor-enriched specificity groups, whereas CDR3β sequences from lung tissues of healthy donors and COPD patients did not. This trend was validated by tumors from a second NSCLC population (TRACERx (TRAcking Cancer Evolution through therapy (Rx)) collective, n=202, validation check). In paired t-tests, *** indicates p<0.001; * indicates p<0.05, and NS indicates no significant difference. [Figure 1C] Figure 1C shows a network analysis of 396 specificity groups annotated with CDR3β sequences from HLA tetramers containing influenza virus (Flu, red), Epstein-Barr virus (EBV, green), and cytomegalovirus (CMV, blue) antigens. Each point represents a specificity group, and an edge connecting two specificity groups indicates the presence of identical CDR3β sequences. [Figure 2A] Figures 2A-2C summarize the results of experiments performed to show the clones of virus-specific CD8+ T cells expanded in patients responding to anti-PD1 treatment. Figure 2A shows a comparison of the CDR3β clone frequencies pre- and post-treatment in the peripheral blood of patients M1 (left) and M2 (right). The CDR3β clones predicted to recognize the viral antigen are highlighted. [Figure 2B] Figure 2B shows specificity groups containing post-treatment amplified CDR3β clones (column 5, CDR3β sequences) from patients M1 or M2 (column 6, patient ID) annotated by viral tetramer CDR3β sequences (columns 2-4, antigen and HLA alleles of the tetramer). Enrichment of A*02:01 or B*35:01 alleles is shown (last two columns, p-values ​​from hypergeometric distribution test). CDR3α / β sequences of two EBV-associated amplified clones from patient M2 are shown at the bottom. [Figure 2C]FIG. 2C shows that TCR27-Jurkat cell lines (CDR3β: CASSTGDSNQPQHF; SEQ ID NO: 64, top diagram) and TCR28-Jurkat cell lines (CDR3β: CASSARTGELFF; SEQ ID NO: 18, bottom diagram) were generated and their reactivity against predicted EBV antigens in the context of B*35 as shown in FIG. 2B was tested. TCR27- and TCR28-Jurkat cells were co-cultured with T2-B*35 cells stimulated with the indicated peptides (indicated at the top of each diagram). The level of activation was quantified by the expression of CD69. The sequence: LPFDFTPGY (SEQ ID NO: 107) was used as a control peptide in these experiments. [Figure 3A] FIG. 3A shows the pathway of specificity inference, which summarizes the available data on 178 HLA-typed NSCLC patients from MD Anderson Cancer Center (MDACC). [Figure 3B] Figure 3B shows that a low proportion of TCR clonotypes from adjacent lung tissues fall into the tumor-enriched specificity group. The proportions of the top 20 most amplified CDR3β clonotypes from adjacent lung tissues of patients from the MDACC NSCLC population (n=178 samples, left) and the TRACERx population (n=63 samples, right) were quantified for 435 tumor-enriched specificity groups as in Figure 1B (%, log10 transformed). The same analysis was performed for the remaining CDR3β clonotypes (Non-exp; no amplification). ND indicates no statistically significant difference. [Figure 3C] FIG. 3C displays the network of 71 clonally amplified specificities, color-coded and annotated by the 10 designated tetramers. [Figure 3D]Figure 3D provides a schematic summary of the computational validation of TCR specificity groups using HLA tetramer sequences. From left to right, network analysis of 71 clonally amplified specificity groups, color-coded as in Figure 1C; two large influenza (Flu)-related communities (red) are circled, and the CDR3β members of this specificity group are highlighted by the previously reported short motifs "RS" and "GxY" highlighted in red font; a heat map shows the individual CDR3β members (columns) of the Flu-related specificity group (with the "RS" motif) (rows), and the level of common CDR3β members between specificity groups within the circled communities; a table shows an example of the "SIRSS%E" specificity group containing the short "RS" motif (bold) annotated with the tetramer sequences of five Flu specificities (bottom), and the number of different CDR3β members from tumors and Vβ gene usage (top). [Figure 3E] Figure 3E shows that the 394 specificity groups annotated with the designated tetramers (keys) were organized into distinct communities through common CDR3β sequences, similar to Figure 1 C. Edge density indicates the number of common CDR3β sequences between any two connected nodes. [Figure 3F] Figure 3F shows a community plot similar to Figure 3E. The edge colors represent the common CDR3β sequences among specificity groups with the same specificity (red) or different specificity (blue) defined by the tetramer-derived sequences (labeled in different colors, E). 588 (edges) (92.74%) of all linkages (n=634) are labeled in red. [Figure 3G] Figure 3G shows 71 clonally amplified specificity groups similar to Figure 3C. Edge colors represent common CDR3β sequences among specificity groups (Figure 3C) with identical (red) or different (blue) specificities as defined by tetramer-derived sequences. 92 (edges) (100%) of all linkages (n=92) are labeled in red. [Figure 4A]Figures 4A-4B summarize the results of CDR3β sequences predicted to recognize CMV, Flu, and EBV, showing no difference in their distribution between tumor and non-diseased lungs. Figure 4A is a volcano plot showing the relative distribution of CDR3β sequences with predicted specificity for CMV (blue), Flu (red), or EBV (green) across tumor and non-diseased lungs by comparing multiple patients with a Poisson test. The y-axis shows the negative log10 transformed p-value of the Poisson test, and the x-axis shows the log2 transformed fold difference (T / N) between tumor and (adjacent) non-diseased lungs. [Figure 4B] Figure 4B shows the overall frequency of clonal types in tumor lung (right) or non-diseased lung (left) predicted to recognize antigens from EBV, CMV, or Flu by GLIPH2. Each point is a patient and the overall frequency is shown as a log10 transformed value (1st and 3rd quartiles represent the 25th and 75th percentiles, respectively). [Diagram 5] Figure 5 is a schematic diagram of the combined single-cell TCR-Seq and single-cell RNA-Seq (scRNA-seq) procedures. CD45+CD3+ T cells were sorted from single-cell suspensions of lung tumor samples from NSCLC patients at Stanford University. Single-cell TCR-Seq was performed using nested multiplex PCR as previously described (Han et al., 2014. Nat. Biotechnol. 32, 684). Single-cell RNseq was performed according to previous methods (Picelli et al., 2014. Nat. Protoc. 9, 171) with modifications, as detailed in the method. TCR repertoires were reconstructed from the single-cell TCR-Seq pathway and integrated from the scRNA-seq data using the Tracer for GLIPH2 Analysis (TraCeR) algorithm (Stubbington et al., 2016. Nat. Methods 13, 329). [Figure 6A] Figure 6A shows pre- and post-treatment CT scan images from NSCLC patients M1 (top panel) and M2 (bottom panel) treated with anti-PD1 therapy. Tumors are highlighted with red arrows. [Figure 6B-6C] Figure 6B shows that T2 (174 CEM.T2) cells were transduced with a lentiviral vector encoding the full-length coding sequence of WT human HLA-B*35:01. Cells were selected with puromycin and surface B*35 expression was quantified by FACS in the presence or absence of the control peptide "LPFDFTPGY" (SEQ ID NO: 107) as previously reported (Takamiya et al., 1994. Int Immunol. Vol. 6, 255). Figure 6C is a volcano plot showing the comparison of 66,094 common specificity groups between tumor lung (T) and adjacent lung (N) by Poisson test. The y-axis represents the negative log10 transformed p-value of the Poisson test and the x-axis represents the log2 transformed fold difference between tumor lung and adjacent lung (T / N). The size of the dots represents the level of clonal amplification. Specificity groups (n = 11) annotated with CDR3β sequences, pathogen-associated tetramers as in Figure 2B, are highlighted according to the respective CDR3β sequences of the amplified clones (column 5 of Figure 2B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Detailed Description of the Invention The present disclosure relates generally to compositions and methods, particularly for the diagnosis, prevention, and / or treatment of health disorders. More specifically, novel polypeptide constructs are provided herein that have binding affinity for a particular cognate antigen. The present disclosure also provides compositions and methods useful for generating such polypeptide constructs, as well as methods for diagnosing, preventing, and / or treating diseases associated with cells expressing the cognate antigen recognized by the polypeptide constructs. In particular, recombinant cells, such as lymphocytic T cells, engineered to express the polypeptide constructs disclosed herein and directed against cells of interest, such as cancer cells. As discussed in more detail below, the present disclosure describes a combined bioinformatics and antigen screening approach to identify novel common tumor antigens in lung cancer. In some embodiments, the disclosed approach implements an improved version of the GLIPH algorithm (grouping of lymphocyte interactions with paratope hotspots), i.e., GLIPH2 (references

[23] and

[24] ), to infer T cell specificity for common antigens at a global level. Using TCR repertoires from 178 HLA-typed lung cancer patients, GLIPH2 identified over 400 specificity groups predicted to recognize common tumor antigens in a defined HLA environment. Subsequent analysis was then performed by predicted HLA-B*35 constraint analysis, which led to the prioritization of two specific specificity groups, namely TCR27 and TCR28. As described in more detail below, additional analysis revealed that specificity group TCR27 possesses the following motifs for antigen recognition: "STGD%NQP", "%TGDSNQP", "ST%DSNQP", "STG%SNQP", and "S%GDSNQP", where "%" indicates various amino acids (Gee et al., 2018). Non-limiting examples of CDR3β sequences of TCR27 specificity groups include, for example, the sequences provided in the sequence listing as SEQ ID NOs: 57-106. The TCR28 specificity group possesses the motifs "SARTG%, " "S%RTGE", "SAR%GE", "SA%TGE", and "SART%E".Non-limiting examples of CDR3β sequences of the TCR28 specificity group include the sequences provided in the sequence listing as SEQ ID NOs: 1 to 56.

[0030] As discussed above, widespread use of immune checkpoint inhibition and T cell-based immunotherapy to treat patients with solid tumors requires a deeper understanding of T cell specificity within cancer. However, despite the availability of advanced techniques to analyze T cell morphology and repertoire using single cell sequencing techniques, the specificity of the majority of tumor-infiltrating T cells remains unclear in all solid tumors. In recent years, the specificity of the few tumor-infiltrating T cells reported thus far includes T cells that recognize mutated antigens, non-mutated (common) antigens, and viral antigens. In the era of immune checkpoint inhibition, attention has recently been focused on mutated antigens (e.g., neoantigens). As neoantigens refer to a kind of "modified self" antigen, T cells that recognize this class of antigens have been shown to display an active phenotype and respond vigorously within tumors. Non-mutated tumor antigens include differentiation antigens expressed within their normal tissue counterparts (e.g., melanoma-associated antigens) or self antigens whose expression is restricted to immune privileged sites, germline tissues, or embryos. There are numerous examples of targeting these forms of tumor antigens with adoptive T cell therapy. In addition, T cells with specificity for viruses (HPV, EBV, Merkel cell polyomavirus, etc.) are also the focus of research into virus-associated cancers.

[0031] In contrast, the role of other forms of T cell specificity in solid tumors remains to be elucidated. For example, numerous reports have described the presence of virus-specific T cells in tumors, such as influenza virus (Flu)- and cytomegalovirus (CMV)-specific T cells in lung cancer. However, in the absence of direct evidence that such viruses play a role in the oncogenesis of lung cancer and other solid tumors, these viruses are often assumed to be largely independent of the tumor immune response and are referred to as "bystander cells." As described in more detail below, the experimental data presented herein identify specific CD8+ T cell classes from cancer cells and pathogens and their cross-reactive antigens. This finding is consistent with the hypothesis that maintaining a broad T cell repertoire to protect against viruses and other pathogens may depend on cross-reactivity. T cells specific for self-antigens have been detected in the peripheral blood of healthy individuals and, although excised in the thymus, have not been clonally excised, may be able to circumvent immunological "blind spots" for viruses and other pathogens. Because cancer cells are histologically similar to their tissue of origin and can express self-antigens, experiments are planned to investigate the possibility that some tumor-infiltrating T cells are indeed specific for ubiquitously expressed, non-mutated self-antigens. Comprehensive analysis and detailed characterization of T cell specificity within the tumor microenvironment can provide a fundamental understanding of T cell responses beyond phenotypic characterization and provide important insights into how the immune system recognizes tumors, normal tissues, and pathogens.

[0032] Tumor-infiltrating T cells remain largely unknown, mainly due to the lack of methods to analyze the diverse TCR repertoires in the context of highly polymorphic human leukocyte antigen (HLA) alleles. For example, next-generation sequencing technologies have made it relatively easy and cheap to sequence large numbers of TCRs, but a major central challenge remains how to be able to analyze these very large repertoires.

[0033] This is because there can be hundreds or thousands of possible TCR sequences for the same peptide-MHC specificity. The GLIPH algorithm (Glanville et al., 2018), and more recently its improved version (GLIPH2; Huang et al., 2020), have been developed to analyze the antigen specificity of T cells across the whole body and infer the specificity of T cells solely based on their CDR3β sequences. These algorithms can rapidly analyze a large number of sequences, parse them into TCR specificity groups (also simply called specificity groups), and predict possible MHC allele restrictions. As described in more detail below, we used GLIPH2 to analyze 778,938 different TCRβ CDR3 sequences (also called CDR3β sequences) from 178 HLA-typed non-small cell lung cancer (NSCLC) patients with surgically resectable disease. In total, we derived 4,300 high-confidence specificity groups. Of these, 449 were found to be tumor-enriched compared to non-diseased lung tissue. It was also found that up to 35% of all tumor-infiltrating T cell repertoires in a patient were predicted to share common antigen specificities. Selected specificity groups were then validated by identifying novel clonotypes predicted to recognize known viral antigens in the context of specific HLA, and these predictions were experimentally confirmed. Tumors were then preferentially enriched, prioritizing two specificity groups predicted to recognize antigens in the context of HLA-B*35. Phenotypically, these cross-reactive CD8+ T cells adopted an effector cell morphology, thereby expressing several genes found in activated NK cells, and did not express the exhaustion markers PD-1 or CD39. In summary, the experimental data described herein provide direct evidence that tumor-infiltrating T cells may cross-react to recognize tumor antigens and pathogen-derived antigens.

[0034] As described in more detail below, the experimental data disclosed herein establish a novel approach to find common tumor antigens and the T cells that recognize them. In particular, some experimental data presented herein show clonally amplified EBV-specific CDR3β sequences in patients with clinical response to anti-PD-1 treatment, suggesting that pathogen cross-reactivity may be an important feature in the interplay between tumorigenesis and T cell immunity. Overall, the data disclosed herein provide a versatile approach to comprehensively analyze common T cell specificities in human cancers and to identify specific antigens using yeast display libraries. This data not only serves as a resource for further T cell research in lung cancer, but also explains why a portion of seemingly "random" virus-specific T cells assemble in the tumor microenvironment and suggests a way that this assembly may contribute to tumorigenesis.

[0035] A non-limiting workflow of the method for finding novel common tumor antigens in a target cancer, such as lung cancer, generally begins with a comprehensive analysis of the T cell specificity landscape in human lung cancer. Using the bioinformatics method GLIPH2, 778,938 CDR3β sequences from 178 patients were analyzed and a specificity group enriched for 449 tumors was constructed. Two such TCRs with inferred specificity associated with HLA-B*35 were identified. The platform for T cell antigen identification as disclosed herein combines two technologies. As the first technology, the GLIPH2 algorithm performs universal inference of global T cell specificity with accurate prediction of HLA restriction. Further information regarding the GLIPH2 algorithm can be found in Huang et al., Nat Biotechnol, 2020, the contents of which are incorporated by reference. Inference of common specificity and HLA landscape is used to prioritize candidate TCRs associated with the disease for downstream antigen detection. Second, the rich diversity of yeast display libraries greatly facilitates antigen identification and allows for the detection of cross-reactive antigens. Unlike other MHC / peptide libraries constructed in mammalian cells, the yeast display library used in the experiments described below contains 10 8 The yeast display library incorporates over 100 randomly permuted peptide sequences. In the past, the success of antigen identification using yeast display libraries has been limited due to the uncertainty of HLA restrictions. The work described herein overcomes this limitation by using the GLIPH2 algorithm to infer the exact HLA context of candidate TCRs before screening the yeast library against the antigen.

[0036] As discussed above, clarifying the specificity of tumor-infiltrating T cells is important to understand how T cell intrinsic factors shape tumor-immune system interactions and influence therapies aimed at harnessing T cell responses against cancer. Complementing the current understanding of T cell exhaustion as a mechanism of tumor immune evasion, the studies described herein demonstrate that T cell specificity for self-antigens also plays a role. Without being bound by any particular theory, it is believed that T cell specificity for self-antigens partially explains why previous studies have observed low reactivity of tumor-infiltrating T cells against autologous tumors.

[0037] Furthermore, the concept that immunological exposure to environmental pathogens may affect immune responses to tumors has been theorized in the past, but the mechanisms are not well understood. In the late 19th century and early 19th century, William Coley developed a mixed bacterial vaccine called Coley toxin to treat cancer patients with some success. In modern times, Bacillus Calmette-Guerin (BCG) is routinely used as an immunotherapy for early-stage bladder cancer. More recently, the gut microbiota has been shown to be an important determinant of immunotherapy responsiveness in cancer. In pancreatic cancer, a unique microbiota has been observed in patients with the longest postoperative survival. Although the mechanism of action in these various cases may involve cell types of the innate immune system, cross-reactive T cells that recognize both tumor and pathogens appear to play a key role. Furthermore, because the lung is exposed to respiratory pathogens, the interactions between these antigens and tumor antigens may be particularly important in understanding the adaptive immune response to lung cancer.

[0038] The experimental results described herein demonstrate that categorizing intratumoral T cell specificity as tumor-specific bystander cells or pathogen-specific bystander cells does not fully capture all the possibilities of T cell antigen recognition. As described in more detail below, intratumoral T cells may also be cross-reactive to both tumor antigens and pathogen-derived antigens, thus aiding in a more detailed understanding of intratumoral T cell specificity. The disclosed approach to discovering this particular class of TCRs also provides a novel method for detecting additional tumor antigens. This is because a deeper understanding of how cross-reactive T cells recognize tumor antigens and pathogen-derived antigens may help advance cell therapy, checkpoint therapy, and vaccine strategies against cancer. The experimental data disclosed herein demonstrate that an individual's encounter with environmental pathogens can shape an adaptive immune response against cancer, which leads to ideas that can be used to improve immunotherapy in patients.

[0039] definition Unless otherwise defined, all technical terms, notations, and other scientific or technical terms used herein are intended to have the meanings commonly understood by those skilled in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as indicating a substantial difference to what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well known and commonly used by those skilled in the art using conventional methods.

[0040] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes one or more cells, as well as mixtures thereof. "A and / or B" is used herein to include all of "A," "B," "A or B," and "A and B."

[0041] The term "about" as used herein has its ordinary meaning of "approximately." If the degree of approximation is not clear from the context, "about" means within ±10% of the stated numerical value in all cases including the stated numerical value, or rounded to the nearest significant figure. When ranges are specified, the bounding numerical values ​​are included.

[0042] As used herein, the terms "administration" and "administering" refer to the delivery of a bioactive composition or formulation by a route of administration, including, but not limited to, oral, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical, or a combination thereof. The terms include, but are not limited to, administration by a medical professional and self-administration.

[0043] The terms "cell", "cell culture", and "cell line" refer not only to a particular cell, cell culture, or cell line, but also to the progeny or potential progeny of that cell, cell culture, or cell line, regardless of the number of transfers or passages in culture. Of course, not all progeny are exactly the same as the parent cell. This is because certain modifications have occurred in subsequent generations, either due to mutations (e.g., deliberate or accidental mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that the progeny may not actually be identical to the parent cell, and this particular modification is still included within the scope of the term as used herein, so long as the progeny retains the same function as the original cell, cell culture, or cell line.

[0044] The term "effective," "therapeutically effective," or "pharmacologically effective" amount or number of a construct, nucleic acid, cell, or composition of the subject of the present disclosure generally refers to an amount or number of the construct, nucleic acid, cell, or composition sufficient to achieve a specified purpose (e.g., achieve the effect for which it is administered, prevent or treat a disease, inhibit a microbial infection, or alleviate one or more symptoms of a health disorder) relative to the absence of the composition, etc. An example of an effective amount or number is an amount or number sufficient to contribute to the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a therapeutically effective amount. "Alleviation" of one or more symptoms generally refers to a reduction in the severity or frequency of the symptoms, or the elimination of one or more symptoms. The exact amount or number of constructs, nucleic acids, cells, or compositions will depend on the purpose of the treatment and can be determined by one of skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0045] The term "operably linked" as used herein refers to a physical or functional link between two or more elements, such as a polypeptide sequence or a polynucleotide sequence, which allows the elements to operate in the intended manner.For example, the term "operably linked" when used in the context of an orthogonal DNA target sequence or a promoter sequence in a nucleic acid construct or engineered response element described herein means that the orthogonal DNA target sequence and the promoter are in frame with the polynucleotide of interest encoding a protein or RNA at a suitable space and distance so that the binding of each by a transcription factor or RNA polymerase can affect transcription.Of course, operably linked elements can be contiguous or non-contiguous.

[0046] "Operably linked" in the context of a polypeptide construct refers to a physical connection (e.g., direct or indirect connection) between amino acid sequences (e.g., different sections, moieties, or domains) to provide a designated activity of the construct. In the present disclosure, regions or domains of the constructs of the present disclosure may be operably linked to retain proper folding, processing, targeting, expression, binding, and other functional properties of the construct in a cell. Unless otherwise stated, the sections, moieties, and domains of the constructs of the present disclosure are operably linked to each other. The operably linked sections, moieties, and domains of the constructs disclosed herein may be contiguous or non-contiguous (e.g., linked to each other via a linker).

[0047] The term "percentage of identity" as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are identical, or have a specified percentage of identical nucleotides or amino acids (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection. See, e.g., the NCBI website (ncbi.nlm.nih.gov / BLAST). Such sequences are said to be "substantially identical". This definition also refers to or may be applied to the complement of a sequence. This definition also includes sequences with deletions and / or additions, and includes sequences with substitutions.Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990).Sequence identity can be measured using sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with its default parameters.

[0048] The term "pharmaceutically acceptable excipient" as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of the compound of interest to a subject. Thus, "pharmaceutically acceptable excipient" may include substances that are called pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" as used herein includes, but is not limited to, physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) may also be incorporated into the composition.

[0049] As used herein, a "subject" or "individual" includes animals, such as humans (e.g., human individuals) and non-human animals. In some embodiments, a "subject" or "individual" is a patient under the care of a physician. Thus, a subject may be a human patient or individual who has, is at risk of, or is suspected of having a disease (e.g., cancer) and / or one or more symptoms of the disease in question. A subject may also be an individual who has been diagnosed at or after diagnosis as being at risk for the disease in question. The term "non-human animals" includes all vertebrates, including mammals, e.g., rodents such as mice, non-human primates, and other mammals such as sheep, dogs, cows, chickens, and non-mammals such as amphibians, reptiles, etc.

[0050] The term "vector" as used herein refers to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid molecule is generally linked to, e.g., inserted into, a vector nucleic acid molecule. Generally, a vector is capable of replicating when associated with appropriate control elements. The term "vector" includes cloning and expression vectors, as well as viral and integrating vectors. An "expression vector" is a vector that includes a regulatory region, which allows DNA sequences and fragments to be expressed in vitro and / or in vivo. A vector may include a sequence that induces autonomous replication in a cell, or may include a sequence sufficient to allow integration into a host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses. In some embodiments, the vector is a gene delivery vector. In some embodiments, the vector is used as a gene delivery vehicle to transfer genes into cells.

[0051] It should be understood that aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended, and does not exclude additional elements, methods, or steps not recited. As used herein, "consisting" excludes elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation of the term "comprising" herein, particularly in describing components of a composition or steps of a method, is understood to encompass those compositions and methods that consist essentially of and consist of the recited components or steps.

[0052] Those skilled in the art will appreciate that when a range of values ​​is presented, all ranges disclosed herein encompass any and all possible subranges and combinations of subranges. It will be readily recognized that any range recited is fully indicated and capable of dividing the same range into at least two, three, four, five, ten, etc. As a non-limiting example, each of the ranges described herein can be readily divided into a bottom third, a middle third, a top third, etc. Those skilled in the art will also appreciate that all phrases such as "up to," "at least," "greater than," "less than," etc. refer to ranges that include the recited numbers and can then be divided into subranges as described above. Finally, those skilled in the art will appreciate that each range includes its individual components. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1, 2, 3, 4, or 5 items, and so forth. In this specification, a particular range is indicated by the term "about" before the numerical value. In this specification, the term "about" is used to provide literal support for the exact number preceded by the term, as well as a number close to or approximately the number preceded by the term. In determining whether a number is close to or approximately a specifically stated number, in the context in which an unstated number that is close to or approximately the number is presented, the unstated number may be considered to provide a substantial equivalence to the specifically stated number.

[0053] Headings such as (a), (b), (i), etc. are provided merely to facilitate reading of the specification and claims. The use of headings in the specification or claims does not require that the steps or elements be performed in alphabetical or numerical order, or that they be performed in the order in which they are presented.

[0054] For clarity, it is understood that certain features of the present disclosure described in the context of another embodiment may be provided in combination in a single embodiment. Conversely, for brevity, various features of the present disclosure described in the context of a single embodiment may also be provided individually or in any suitable subcombination. All combinations of the embodiments related to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and expressly disclosed. Moreover, all subcombinations of the various embodiments and their elements are also specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0055] T cell receptor TCR is a heterodimeric cell surface protein of the immunoglobulin superfamily, associated with the invariant protein of the CD3 complex, involved in mediating signal transduction. TCR and antibodies are molecules evolved to recognize different classes of antigens (ligands). TCR is an antigen-specific molecule responsible for the recognition of antigenic peptides presented in the context of products of the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APCs) or any nucleated cell (e.g., all human cells in the body except red blood cells). In contrast, antibodies generally recognize soluble or cell surface antigens and do not require antigen presentation by MHC. This system endows T cells, via the TCR, with the potential to recognize the entire array of intracellular antigens expressed by cells (including viral and bacterial proteins) that are processed intracellularly into short peptides, bound to intracellular MHC molecules, and delivered to the surface as peptide-MHC complexes (pepMHC). This system allows virtually any foreign protein (such as mutated cancer antigens or viral proteins) or aberrantly expressed proteins to serve as targets for T cells.

[0056] TCRs generally exist in αβ and γδ forms, which are structurally similar but have quite different anatomical locations and functions. The extracellular portion of the naturally occurring heterodimeric αβ TCR generally consists of two polypeptides, the α and β chains, each of which has a membrane-proximal constant domain and a membrane-distal variable domain. The constant and variable domains each contain intrachain disulfide bonds. The variable domains contain highly polymorphic loops similar to the complementarity determining regions (CDRs) of antibodies, embedded within framework sequences, one of which is a hypervariable region called CDR3. There are several types of α chain variable (Vα) regions and several types of β chain variable (Vβ) regions, which are separated by frameworks, the CDR1 and CDR2 sequences, and a partially defined CDR3 sequence. The use of TCR gene therapy overcomes several current obstacles, for example, to confer the desired specificity to the patient's own T cells and generate sufficient numbers of T cells in a short period of time to avoid T cell exhaustion. Furthermore, TCRs can be transduced into T cells with central memory or stem cell characteristics, ensuring better persistence and function during metastasis.Furthermore, TCR-engineered T cells can be infused into chemotherapy- or radiation-induced lymphopenic cancer patients, allowing efficient engraftment and preventing immunosuppression.

[0057] Compositions of the Disclosure As described in more detail below, one aspect of the present disclosure relates to novel polypeptide constructs that have binding affinity for specific cognate antigens. Additionally, recombinant nucleic acids encoding such polypeptide constructs are provided, as well as recombinant cells that are engineered to express the polypeptide constructs disclosed herein and directed to cells of interest, such as cancer cells.

[0058] A. Constructs of the Disclosure In one aspect, provided herein are various constructs comprising at least one complementarity determining region (CDR) having at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106.

[0059] Non-limiting exemplary embodiments of constructs of the present disclosure may include one or more of the following features: In some embodiments, the construct comprises at least one, at least two, or at least three CDRs having at least 70% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106. In some embodiments, the construct comprises at least one, at least two, or at least three CDRs having at least 70% sequence identity to the sequence of SEQ ID NO: 6. In some embodiments, the construct comprises at least one CDR having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106. In some embodiments, the construct comprises at least one CDR having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:6.

[0060] The CDR sequences of the constructs disclosed herein may be modified, e.g., mutated. Non-limiting examples of modifications of CDR sequences include substitution, deletion, addition, or insertion of 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less amino acid residues. In some embodiments, at least one CDR of the constructs disclosed herein comprises a sequence having 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106, in which at least one, at least two, at least three, at least four, or at least five amino acid residues in the sequence are replaced by different amino acid residues. In some embodiments, at least one CDR of the constructs disclosed herein comprises a sequence having 100% identity to a sequence of SEQ ID NO: 6, in which at least one, at least two, at least three, at least four, or at least five amino acid residues in the sequence are replaced by different amino acid residues. In some embodiments, at least one CDR comprises a sequence having 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106, in which one, two, three, four, or five amino acid residues are replaced by different amino acid residues. In some embodiments, at least one CDR comprises a sequence having 100% identity to a sequence of SEQ ID NO: 18, in which one, two, three, four, or five amino acid residues are replaced by different amino acid residues.

[0061] Those skilled in the art know that binding affinity can generally be used as a measure of the strength of a non-covalent interaction between two molecules, such as an antibody or functional fragment thereof and an antigen. In some cases, binding affinity can be used to describe a monovalent interaction (intrinsic activity). The binding affinity between two molecules is expressed as the dissociation constant (K D ) can be quantified by measuring the K D can be determined by measuring the rates of complex formation and dissociation, for example using surface plasmon resonance (SPR) techniques (Biacore). The rate constants corresponding to the association and dissociation of the monovalent complex are designated as the association rate constant k a (or k on ) and the dissociation rate constant k d (or koff ) is called K D is the formula: D =k d / k a By k a and k d The value of the dissociation constant can be determined directly by well-known methods, and can be calculated even for complex mixtures, for example by the method described by Caceci et al. (1984, Byte 9: 340-362). For example, K D may be set up using a double filter nitrocellulose filter binding assay, such as the assay disclosed by Wong and Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard assays for evaluating the binding ability of the engineered antibodies of the present disclosure to the target antigen are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometric analysis, as well as other assays exemplified elsewhere herein. The binding kinetics and binding affinity of the antibodies can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR) using, for example, the Biacore® system or KinExA. In some embodiments, the binding affinity of the constructs disclosed herein to the target antigen can be evaluated by the Scatchard method described in Frankel et al. (Mol. Immunol, 16: 101-106, 1979).

[0062] In some embodiments, the construct of the present disclosure may be (a) a TCR; (b) an antibody; or (c) a functional derivative or fragment of (a) or (b). Those skilled in the art will readily appreciate that the term "functional fragment thereof" or "functional derivative thereof" refers to a molecule that has a quantitative and / or qualitative biological activity in common with the wild-type molecule from which the fragment or derivative is derived. For example, a functional fragment or derivative of an antibody is a fragment or derivative that retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment or derivative is derived. For example, an antibody capable of binding to an epitope may be truncated at the N-terminus and / or C-terminus, and the retention of epitope binding activity is assessed using measurements known to those skilled in the art.

[0063] In some embodiments, the construct is a TCR construct comprising a TCR alpha chain and a TCR beta chain operably linked to each other. In some embodiments, the TCR alpha chain and the TCR beta chain are covalently linked to each other. In some embodiments, the TCR alpha chain and the TCR beta chain are linked to each other in a non-covalent manner. In some embodiments, the TCR alpha chain and the TCR beta chain are covalently linked to each other via a polypeptide linker. In some embodiments, the polypeptide linker is a cleavable linker. In some embodiments, the polypeptide linker comprises an autoproteolytic peptide. In some embodiments, the autoproteolytic peptide comprises one or more autoproteolytic cleavage sites derived from calcium-dependent serine endoprotease (furin), porcine teschovirus-1 2A (P2A), foot and mouth disease virus (FMDV) 2A (F2A), equine rhinitis A virus (ERAV) 2A (E2A), zosea asignavirus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), flacherie disease virus 2A (BmIFV2A), or combinations thereof. In some embodiments, the TCR α and TCR β chains are covalently linked to each other via the P2A cleavage site. The present disclosure provides both single chain and multi-chain TCR constructs. In some embodiments, the TCR constructs of the present disclosure may be provided as single chain α or β molecules, or γ and δ molecules, or as two-chain constructs composed of both α and β chains, or γ and δ chains.

[0064] In some embodiments, the TCR constructs of the present disclosure may be provided as single chain TCRs (scTCRs). The scTCR may comprise a polypeptide of the variable region of a first TCR chain (e.g., an α chain) and an entire (full length) polypeptide of a second TCR chain (e.g., a β chain), or vice versa. In some embodiments, the polypeptides are directly linked to each other. In some embodiments, the scTCR may optionally comprise one or more linkers linking two or more polypeptides together. In some embodiments, the linker may be a synthetic compound linker, such as, for example, a chemical cross-linker. Non-limiting examples of suitable commercially available cross-linkers include N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0065] In some embodiments, the linker may be a peptide linker that joins two single chains together, as described herein. In some embodiments, the length and amino acid composition of the peptide linker sequence can be optimized to alter the orientation and / or proximity of the polypeptides relative to each other to achieve a desired activity of the constructs (e.g., TCR constructs) disclosed herein.

[0066] The constructs according to the present disclosure can also be provided in the form of a multimeric complex comprising at least two scTCR molecules, each of which is fused to at least one biotin moiety, or other interconnecting molecule / linker, and the scTCRs are interconnected by biotin-streptavidin interactions to allow the formation of said multimeric complex. Similar techniques known in the art for the generation of multimeric TCRs are also envisaged and included within the present disclosure. Thus, higher order multimeric complexes comprising three or more scTCRs of the present disclosure are also provided.

[0067] Suitable methods for generating fusion polypeptides are known in the art and include, for example, recombinant methods. In some embodiments, the constructs, TCRs (and functional fragments and derivatives thereof), and polypeptides of the present disclosure may be expressed as a single protein comprising a linker peptide linking the α and β chains and / or linking the γ and δ chains. In this regard, the constructs, TCRs (and functional fragments and derivatives thereof), and polypeptides of the present disclosure comprise the amino acid sequence of the variable region of the TCR of the present disclosure and may further comprise a linker peptide. In some embodiments, the linker peptide may effectively facilitate the expression of the construct or TCR (including its functional fragments and derivatives) in a host cell. In principle, the linker peptide may comprise any suitable amino acid sequence. Linker sequences for single chain TCR constructs are well known in the art. In some embodiments, such single chain constructs may further comprise one or two constant domain sequences. When a construct containing the linker peptide is expressed by a host cell, the linker peptide is also cleaved to produce separate α and β chains, and separate γ and δ chains.

[0068] In some embodiments, the TCR construct of the present disclosure comprises at least one TCR alpha or gamma and / or TCR beta or delta variable domain. Generally, the TCR construct comprises both TCR alpha and TCR beta variable domains, or both TCR gamma and TCR delta variable domains. In some embodiments, the TCR construct comprises an alpha beta / gamma delta heterodimer, or may be in a single chain format. In some embodiments, the fusion in adoptive therapy, i.e., an alpha beta or gamma delta heterodimeric TCR, may be transduced as a full-length chain, e.g., having both cytoplasmic and transmembrane domains. If necessary, there may be disulfide bonds introduced between the residues of the respective constant domains.

[0069] In some embodiments, the TCR constructs of the present disclosure are provided as single chain α or β, or γ and δ molecules, or as two-chain constructs composed of both α and β, or γ and δ chains. Thus, in some embodiments, the TCR construct is a single chain TCR construct comprising a CDR3β in its β chain having at least 70%, such as at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 1-106. In further or alternative embodiments, the TCR construct may further comprise a CDR1 and / or CDR2 domain sequence. In some embodiments, the TCR constructs of the present disclosure comprise at least one CDR sequence, preferably all three CDR sequences, i.e. CDR1, CDR2, and CDR3.

[0070] In some embodiments, the TCR constructs of the present disclosure are provided as two-chain constructs composed of both α and β chains, or both γ and δ chains. Thus, in some embodiments, the TCR constructs of the present disclosure are provided as two-chain constructs comprising both α and β chains, the β chain comprising a CDR3β having at least 70%, e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 1-106. In some embodiments, the TCR construct further comprises a CDR3α sequence in the α chain.

[0071] As outlined above, in some embodiments, the constructs of the present disclosure may be provided in the framework of an antibody construct or functional fragment thereof that specifically binds to an antigen as described herein. The antibody construct may be any type of immunoglobulin known in the art. For example, the antibody construct may be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody construct may be monoclonal or polyclonal. The antibody construct may be an antibody of natural origin, e.g., an antibody isolated and / or purified from a mammal, such as a human cell. Alternatively, the antibody construct may be a genetically engineered antibody, e.g., a humanized antibody or a chimeric antibody. The antibody construct may be in the form of a monomer or polymer. In some embodiments, the constructs disclosed herein may be in the form of an antigen-binding fragment (Fab), a single chain variable fragment (scFv), a single chain antibody, a single domain antibody (sdAb), a V-cell antibody, a fusion protein ... H Domain, V L Domain, V H The antibody construct is selected from the group consisting of an H domain, a bispecific antibody, or a functional fragment of either thereof.

[0072] B. nucleic acid In one aspect, provided herein are various nucleic acid molecules comprising nucleotide sequences encoding constructs of the present disclosure, such as expression cassettes, and expression vectors comprising these nucleic acid molecules operably linked to heterologous nucleic acid sequences, such as regulatory element sequences that enable in vivo expression of the constructs in a host cell or in vitro cell-free expression.

[0073] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to both RNA and DNA molecules, such as DNA molecules including cDNA, genomic DNA, synthetic DNA, and nucleic acid analogs, or nucleic acid molecules including RNA molecules. Nucleic acid molecules may be double-stranded or single-stranded (e.g., sense or antisense strands). Nucleic acid molecules may include unconventional or modified nucleotides. As used herein, the terms "polynucleotide sequence" and "nucleic acid sequence" interchangeably refer to the sequence of a polynucleotide molecule. The polynucleotide and polypeptide sequences disclosed herein are shown using standard letter abbreviations for nucleotide bases and amino acids as set forth in WIPO Standard ST.25 (1998), Appendix 2, Tables 1-6, 37 CFR §1.82, which is incorporated by reference.

[0074] The nucleic acid molecules of the present disclosure may be of any length, including nucleic acid molecules that are generally about 0.5 Kb to about 50 Kb, e.g., about 0.5 Kb to about 20 Kb, about 1 Kb to about 15 Kb, about 2 Kb to about 10 Kb, or about 5 Kb to about 25 Kb, e.g., about 10 Kb to 15 Kb, about 15 Kb to about 20 Kb, about 5 Kb to about 20 Kb, about 5 Kb to about 10 Kb, or about 10 Kb to about 25 Kb. In some embodiments, the nucleic acid molecule of the present disclosure is from about 1.5 Kb to about 50 Kb, from about 5 Kb to about 40 Kb, from about 5 Kb to about 30 Kb, from about 5 Kb to about 20 kb, or from about 10 Kb to about 50 Kb, e.g., from about 15 Kb to 30 Kb, from about 20 Kb to about 50 Kb, from about 20 Kb to about 40 Kb, from about 5 Kb to about 25 Kb, or from about 30 Kb to about 50 Kb.

[0075] In some embodiments disclosed herein, the nucleic acid molecule of the present disclosure comprises a nucleotide sequence encoding a construct comprising at least one complementarity determining region (CDR) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106. In some embodiments, the construct is a single-stranded construct or a double-stranded construct. In some embodiments, the construct is selected from the group consisting of: (a) a TCR; (b) an antibody; (c) a functional derivative or fragment of (a) or (b). In some embodiments, the construct is a TCR construct comprising a TCR alpha chain and a TCR beta chain operably linked to each other. In some embodiments, the construct is a TCR construct comprising a CDR3β in its β chain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1 to 106. In some embodiments, the construct further comprises a CDR3α sequence in its α chain.

[0076] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or expression vector. It will be appreciated that an expression cassette generally comprises a construct of genetic material that includes a coding sequence and sufficient regulatory information to direct proper transcription and / or translation of the coding sequence in a recipient cell in vivo and / or in vitro. In general, the expression cassette may be inserted into a vector and / or individual for targeting a desired host cell. Thus, in some embodiments, an expression cassette of the present disclosure comprises the coding sequence of the construct disclosed herein, which expression cassette is operably linked to any one or combination of expression control elements, such as a promoter, and optionally other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0077] In some embodiments, the nucleotide sequence is incorporated into an expression vector. As will be appreciated by those skilled in the art, the term "vector" generally refers to a recombinant polynucleotide construct designed for transmission between host cells, and vectors can be used for transformation purposes, such as introducing heterologous DNA into host cells. Thus, in some embodiments, a vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to cause replication of the inserted segment. In some embodiments, an expression vector can be an integrating vector.

[0078] In some embodiments, the expression vector may be a viral vector. As will be appreciated by those skilled in the art, the term "viral vector" is used broadly to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that typically contains a nucleic acid element derived from a virus that facilitates the transfer or integration of the nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid transfer. A viral particle typically contains various viral components in addition to the nucleic acid, and in some cases, host cell components. The term "viral vector" may refer to either a virus or a viral particle that can transfer a nucleic acid to a cell, or the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. In some embodiments, the viral vector is a baculovirus vector, a retrovirus vector, or a lentivirus vector. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof, including LTRs, that are primarily derived from the retrovirus genus, lentivirus.

[0079] Thus, further provided herein is a vector, plasmid, or virus that comprises one or more nucleic acid molecules that code for any of the constructs disclosed herein.The nucleic acid molecules may be included in a vector, and can induce their expression in, for example, the cell that is transformed / transduced by the vector.Vectors suitable for use in eukaryotic and prokaryotic cells are known in the art, commercially available, or easily prepared by those skilled in the art.

[0080] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic transfection, nucleoporation, hydrodynamic shock, and infection.

[0081] Viral vectors that can be used in the present disclosure include, for example, baculovirus vectors, retrovirus vectors, adenovirus vectors and adeno-associated virus vectors, lentivirus vectors, herpes viruses, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY). For example, the chimeric receptors disclosed herein may be produced in eukaryotic cells, such as mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, such as the American Type Culture Collection; Manassas, VA. When selecting an expression system, care must be taken to ensure that the components are compatible with each other. Those of skill in the art are capable of making such judgments. Furthermore, those of skill in the art can refer to P. Jones, "Vectors: Cloning Applications", John Wiley and Sons, New York, NY, 2009, for guidance in selecting an expression system.

[0082] The nucleic acid molecules provided may contain sequences of natural origin or sequences that differ from sequences of natural origin, but due to the degeneracy of the genetic code, encode the same polypeptide, such as an antibody. These nucleic acid molecules may be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA, such as produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, the nucleic acid molecules may be double-stranded or single-stranded (e.g., either the sense strand or the antisense strand).

[0083] Nucleic acid molecules are not limited to sequences that code for a polypeptide (e.g., an antibody), but may also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence for a chimeric receptor). Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. Such nucleic acid molecules can be generated, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR). When the nucleic acid molecule is a ribonucleic acid (RNA), these molecules can be generated, for example, by in vitro transcription.

[0084] In another aspect, provided herein is a cell culture comprising at least one genetically engineered cell disclosed herein and a medium. In general, the medium may be any suitable medium for culturing the cells described herein. The techniques for transforming the various cells and species described above are known in the art and described in technical and scientific literature. Thus, a cell culture comprising at least one genetically engineered cell disclosed herein is also within the scope of this application. Suitable methods and systems for generating and maintaining cell cultures are known in the art.

[0085] C. Genetically engineered cells and cell cultures The recombinant nucleic acid of the present disclosure can be introduced into a cell, such as, for example, a human T-lymphocyte, to generate a genetically engineered cell comprising the nucleic acid molecule. Accordingly, some embodiments of the present disclosure relate to a method of generating a genetically engineered cell, the method comprising: (a) providing a host cell capable of protein expression; and (b) transducing the provided host cell with a recombinant nucleic acid of the present disclosure to generate a genetically engineered cell. Introduction of the nucleic acid molecule of the present disclosure into a cell can be accomplished by methods known to those skilled in the art, such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate deposition, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate deposition, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0086] Thus, in some embodiments, the nucleic acid molecule can be introduced into a host cell by viral or non-viral delivery vehicles known in the art to generate a genetically engineered cell. For example, the nucleic acid molecule can be stably integrated into the genome of the genetically engineered cell, replicated as an episome, or present in the genetically engineered cell as a minicircle expression vector for transient expression. Thus, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is present in the genetically engineered cell as a minicircle expression vector for transient expression. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the genetically engineered cell. Stable integration can be achieved using classical random genome recombination techniques, or by guide RNA-directed CRISPR / Cas9 genome editing, DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALEN ( t ranscription a ctivator- l ike e effector n This can be achieved using more precise techniques such as genome editing.

[0087] Nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acid into cells can be achieved by viral transduction. In a non-limiting example, baculovirus or adeno-associated virus (AAV) can be engineered to deliver nucleic acid to target cells via viral transduction. Several AAV serotypes have been reported, and all known serotypes can infect cells of multiple and diverse tissue types. AAV can transduce a wide range of species and tissues in vivo without toxicity, and generates relatively mild innate and adaptive immune responses.

[0088] Lentivirus-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentivirus vectors offer several attractive properties as gene delivery vehicles: (i) sustained gene delivery by stable vector integration into the host genome; (ii) ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important gene therapy and cell therapy target cell types; (iv) non-expression of viral proteins after vector transduction; (v) ability to deliver complex genetic elements such as polycistronic or intron-containing sequences; (vi) potentially safer integration site properties; and (vii) relatively easy system for vector manipulation and generation.

[0089] In some embodiments, a host cell may be genetically engineered (e.g., transduced, transformed, or transduced) with, for example, a vector construct of the present disclosure, which may be a viral vector or a vector for homologous recombination that contains a nucleic acid sequence homologous to a portion of the genome of the host cell, or an expression vector for expressing a polypeptide of interest. A host cell may be either an untransformed cell or a cell that is already transduced with at least one nucleic acid molecule.

[0090] In some embodiments, the genetically engineered cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in a test tube. In some embodiments, the genetically engineered cell is a eukaryotic cell. In some embodiments, the genetically engineered cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the genetically engineered cell is an immune system cell, e.g., a B cell, a monocyte, a NK cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (T H), cytotoxic T cells (T CTL ), a memory T cell, a γδ T cell, another T cell, a hematopoietic stem cell, or a hematopoietic stem cell precursor.

[0091] In some embodiments, the immune system cells are lymphocytes. In some embodiments, the lymphocytes are T lymphocytes. In some embodiments, the lymphocytes are T lymphocyte precursors. In some embodiments, the T lymphocytes are CD4+ T cells or CD8+ T cells. In some embodiments, the T lymphocytes are CD8+ T cytotoxic lymphocyte cells. Non-limiting examples of CD8+ T cytotoxic lymphocytes suitable for the compositions and methods disclosed herein include naive CD8+ cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, the T lymphocytes are CD4+ T helper lymphocyte cells. Suitable CD4+ T helper lymphocytes include, but are not limited to, naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

[0092] As outlined above, some embodiments of the present disclosure relate to various methods of producing the genetically engineered cells of the present disclosure, which include the steps of (a) providing a host cell capable of protein expression; and (b) transducing the provided host cell with a recombinant nucleic acid of the present disclosure to produce the genetically engineered cell. Non-limiting examples of embodiments of the methods of the present disclosure for producing the genetically engineered cell may further include one or more of the following features: In some embodiments, the cells are obtained by leukapheresis performed on a sample obtained from a subject, and the cells are transduced ex vivo. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or lipid nanoparticle. In some embodiments, these methods further include the steps of isolating and / or purifying the produced cells. Thus, the genetically engineered cells produced by the methods disclosed herein are also within the scope of the present disclosure.

[0093] In another aspect, provided herein is a cell culture comprising at least one genetically engineered cell disclosed herein and a medium. In general, the medium may be any suitable medium for culturing the cells described herein. The techniques for transforming the various cells and species described above are known in the art and described in technical and scientific literature. Thus, a cell culture comprising at least one genetically engineered cell disclosed herein is also within the scope of this application. Suitable methods and systems for generating and maintaining cell cultures are known in the art.

[0094] E. Pharmaceutical Compositions The constructs, nucleic acids, engineered cells, and / or cell cultures of the present disclosure may be incorporated into compositions, such as pharmaceutical compositions. Such compositions generally include one or more of the constructs, nucleic acids, engineered cells, and / or cell cultures provided and described herein, and a pharma- ceutically acceptable excipient, such as a carrier. In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to treat, prevent, ameliorate, or reduce or delay the onset of a disease, such as cancer.

[0095] Thus, one aspect of the present disclosure relates to a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and one or more of (a) the construct of the present disclosure; (b) the recombinant nucleic acid of the present disclosure; and (c) the genetically engineered cell of the present disclosure. In some embodiments, the pharmaceutical composition comprises (a) the construct of the present disclosure and (b) a pharma- ceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises (a) the recombinant nucleic acid of the present disclosure and (b) a pharma- ceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or lipid nanoparticle. In some embodiments, the pharmaceutical composition of the present disclosure comprises (a) the genetically engineered cell of the present disclosure and (b) a pharma- ceutically acceptable carrier.

[0096] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, such as sodium dodecyl sulfate. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is common to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0097] Sterile injectable solution can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or combination of the above listed components as required, followed by sterilization by filtration.Generally, dispersion is prepared by incorporating the active compound in a sterile medium that contains a basic dispersion medium and other components required from the above listed components.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which obtains the powder of active ingredient and any additional desired ingredient from the solution that has been previously sterile filtered.

[0098] Methods of the Disclosure Administration of any one of the therapeutic compositions described herein, such as constructs, nucleic acids, engineered cells, and pharmaceutical compositions, can be used to treat a subject in the treatment of such diseases, such as cancer, immune diseases, and chronic infections. In some embodiments, the constructs, nucleic acids, engineered cells, and pharmaceutical compositions described herein can be incorporated into therapeutic agents for use in methods of preventing and / or treating subjects suffering from, suspected of suffering from, or believed to be at risk of developing health disorders, such as proliferative diseases and bacterial infections. Examples of proliferative diseases include, but are not limited to, angiogenic diseases, metastatic diseases, tumorigenic diseases, neoplastic diseases, and cancer. In some embodiments, the proliferative disease is cancer.

[0099] Thus, in one aspect, some embodiments of the present disclosure relate to a method of preventing and / or treating a disease in a subject in need thereof, comprising administering to the subject a composition comprising one or more of: (a) a construct of the present disclosure; (b) a recombinant nucleic acid of the present disclosure; (c) a genetically engineered cell of the present disclosure; and (d) a pharmaceutical composition of the present disclosure. In some embodiments, the composition comprises a therapeutically effective amount or number of: (a) a construct of the present disclosure; (b) a recombinant nucleic acid of the present disclosure; (c) a genetically engineered cell of the present disclosure; and / or (d) a pharmaceutical composition of the present disclosure.

[0100] In some embodiments, pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended route of administration. Recombinant polypeptides of the present disclosure may be administered orally or by inhalation, but are more likely to be administered through parenteral routes. Examples of parenteral routes of administration include, for example, intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral administration may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate; and a tonicity adjuster such as sodium chloride or glucose. The pH may be adjusted (e.g., to a pH of about 7.2 to 7.8, such as 7.5) with an acid or base such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0101] Dosage, toxicity, and therapeutic efficacy of the recombinant polypeptides of interest of the present disclosure may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects is called the therapeutic index, and is defined as the ratio:LD 50 / ED 50 The therapeutic index can be expressed as: Generally, compounds that exhibit a high therapeutic index are suitable. Compounds that exhibit toxic side effects can be used, but care must be taken to design a delivery system that targets the compound to the affected tissue site in order to minimize potential damage to non-infected cells, thereby reducing side effects.

[0102] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use. The compounds can be administered at doses up to ED50 with little or no toxicity. 50 Preferably, the dose is within a circulating concentration range that includes the IC50 / IC10 determined in cell culture. This dose may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the disclosure, the therapeutically effective amount can be estimated initially from cell culture assays. The dose can be determined based on the IC50 / IC10 determined in cell culture. 50 The compound may be formulated in animal models to achieve a circulating plasma concentration range that includes (e.g., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Plasma concentrations may be measured, for example, by high performance liquid chromatography.

[0103] A therapeutically effective amount (e.g., an effective dose) of a recombinant polypeptide of the present disclosure will depend on the polypeptide selected. For example, a single dose ranging from about 0.001-0.1 mg / kg (per kg patient body weight) may be administered, and in some embodiments, about 0.005, 0.01, or 0.05 mg / kg may be administered. In some embodiments, 600,000 IU / kg is administered (IU may be determined by lymphocyte proliferation bioassay and expressed in international units (i.e., IU)). The composition may be administered from one or more times per day to one or more times per week. One of skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease, previous treatment history, general health, and / or age of the subject, may affect the dosage and timing required to effectively treat the subject. Additionally, treatment of a subject with a therapeutically effective amount of a recombinant polypeptide of the present disclosure may include a single treatment or may include a series of treatments. In some embodiments, the composition is administered every 8 hours for 5 days, followed by a rest period of 2 to 14 days, eg, 9 days, followed by an additional 5 days of administration every 8 hours.

[0104] Administration of genetically engineered cells to a subject In some embodiments, the methods of treatment disclosed herein include administering an effective amount or number of genetically engineered cells to a subject in need of such treatment. This administration can be accomplished using any method of injection delivery known in the art. For example, the genetically engineered cells can be injected directly into the individual's bloodstream or otherwise administered to the individual.

[0105] In some embodiments, the methods disclosed herein include administering the genetically engineered cells to a subject by a method or route that results in partial localization of the introduced cells at a desired site to produce a desired effect, where the term "administering" is used interchangeably with the terms "introducing," "injecting," and "implanting." The genetically engineered cells or their differentiated progeny may be administered by any suitable route that results in delivery to a desired location within an individual where at least a portion of the administered cells or cellular components remain viable. The survival period of the cells after administration to a subject may be as short as a few hours, e.g., 24 hours, a few days, or as long as several years or even the lifespan of the individual, i.e., long-term engraftment.

[0106] When provided prophylactically, the genetically engineered cells described herein may be administered to a subject prior to any symptoms of the disease or condition being treated. Thus, in some embodiments, prophylactic administration of the genetically engineered cell population prevents the onset of symptoms of the disease or condition.

[0107] When provided therapeutically in some embodiments, the genetically engineered cells are provided at the onset of a symptom or sign of a disease or condition, for example at (or after) the onset of a disease or condition.

[0108] For use in the various embodiments described herein, an effective amount or number of the genetically engineered cells disclosed herein is at least 10 2 cells, at least 5 x 10 2 cells, at least 10 3 cells, at least 5 x 10 3 cells, at least 10 4cells, at least 5 x 10 4 cells, at least 10 5 cells, at least 2 x 10 5 cells, at least 3 x 10 5 cells, at least 4 x 10 5 cells, at least 5 x 10 5 cells, at least 6 x 10 5 cells, at least 7 x 10 5 cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 1 x 10 6 cells, at least 2 x 10 6 cells, at least 3 x 10 6 cells, at least 4 x 10 6 cells, at least 5 x 10 6 cells, at least 6 x 10 6 cells, at least 7 x 10 6 cells, at least 8 x 10 6 cells, at least 9 x 10 6 The genetically engineered cells may be derived from one or more donors or from an autologous source. In some embodiments, the genetically engineered cells are expanded in culture prior to administration to a subject in need of treatment.

[0109] In some embodiments, delivery of a genetically engineered cell composition (e.g., a composition comprising a plurality of genetically engineered cells according to any of the cells described herein) to an individual by a given method or route results in at least partial localization of the cell composition at a desired site. The composition comprising genetically engineered cells can be administered by any suitable route that results in effective treatment in the individual, for example, administration of at least a portion of the composition, e.g., at least 1×10 3When the cells are delivered to the desired site over a period of time, delivery to the desired location within the individual is achieved. Modes of administration include injection, infusion, and infusion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. Delivery by injection or infusion is the standard mode of administration for cell delivery.

[0110] In some embodiments, the genetically engineered cells are administered systemically, e.g., by infusion or injection, e.g., a population of genetically engineered cells is not administered directly to a target site, tissue, or organ, but rather is administered so that it enters the circulatory system of an individual and thereby undergoes metabolic and other similar biological processes.

[0111] The efficacy of a treatment comprising any of the compositions provided herein for the treatment of a disease or condition can be determined by a medical professional. However, those skilled in the art will appreciate that a treatment is deemed effective if any one or all of the signs, symptoms, or markers of the disease are improved or alleviated. Efficacy can also be measured by inhibiting the deterioration of a subject (e.g., the progression of the disease is stopped or at least delayed) as assessed by reducing the need for hospitalization or medical intervention. Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of a disease in a subject or animal (including, in some non-limiting examples, a human or mammal) and includes (1) preventing the disease, e.g., stopping or slowing the progression of a condition; or (2) relieving the disease, e.g., causing regression of a condition; and (3) preventing or reducing the likelihood of the onset of a condition.

[0112] As discussed above, a therapeutically effective number of genetically engineered cells refers to a number of genetically engineered cells sufficient to promote or provide a therapeutic benefit in the treatment or management of a disease, such as cancer, or to delay or reduce one or more symptoms associated with the disease when administered to a subject suffering from, suspected of suffering from, or at risk for the disease. In some embodiments, an effective number includes a number sufficient to prevent or delay the onset of a disease symptom, alter the course of a disease symptom (e.g., without limitation, delay the progression of a disease symptom), or reverse a disease symptom. In some embodiments, an effective number includes a number sufficient to inhibit tumor growth or metastasis of cancer in an individual. In some embodiments, an effective number includes a number sufficient to increase cytokine production and inhibit (e.g., kill) cancer cells or infected cells.

[0113] In some embodiments of the disclosed methods, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual suffers from or is suspected of suffering from a condition associated with a proliferative disorder or disease, such as cancer. The term "cancer" generally refers to the presence of cells that possess characteristics typical of cells that cause cancer, such as unregulated proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological properties. Although cancer cells are often observed to aggregate into tumors, such cells may be present alone in an animal subject or may be non-tumorigenic cancer cells, such as leukemia cells. Thus, the term "cancer" may encompass associations with solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "cancer" includes pre-malignant cancers as well as malignant cancers. In some embodiments, the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.

[0114] Examples of diseases suitable for treatment with the compositions and methods of the present disclosure include diseases related to cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. In some embodiments, the proliferative disorder is cancer. Examples of cancers that can be suitably diagnosed, prevented, and / or treated with the compositions and methods of the present disclosure include lung cancer. In principle, there is no particular limitation on the lung cancers that can be suitably diagnosed, prevented, and / or treated with the compositions and methods disclosed herein. Examples of suitable lung cancers include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, non-small cell carcinoma, adenosquamous carcinoma, small cell lung carcinoma, large cell carcinoma, neuroendocrine carcinoma of the lung, and non-small cell lung cancer (NSCLC). Further lung cancers that may be suitably diagnosed, prevented, and / or treated by the compositions and methods disclosed herein include, but are not limited to, undifferentiated non-small cell carcinoma, non-small cell carcinoma not otherwise specified, lung squamous cell carcinoma, bronchioloalveolar carcinoma, sarcomatoid carcinoma, pleomorphic carcinoma, carcinosarcoma, pulmonary blastoma, metastatic carcinoma of unknown primary, primary lung lymphoepithelioma-like carcinoma, and benign neoplasms of the lung. In some embodiments, the cancer is NSCLC. In some embodiments, the lung cancer is NSCLC selected from the group consisting of squamous cell carcinoma, adenocarcinoma, large cell carcinoma, carcinoid tumor, pleomorphic carcinoma, salivary gland carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, and unclassified carcinoma. In some embodiments, the NSCLC is squamous cell carcinoma. In some embodiments, the NSCLC is adenocarcinoma. In some embodiments, the NSCLC is large cell carcinoma. In some embodiments, the NSCLC comprises stage I NSCLC. In some embodiments, the NSCLC comprises stage II NSCLC.

[0115] In some embodiments, the cancer is a multi-drug resistant cancer or a recurrent cancer. The compositions and methods disclosed herein are believed to be suitable for both non-metastatic and metastatic cancers. Thus, in some embodiments, the cancer is a non-metastatic cancer. In other embodiments, the cancer is a metastatic cancer. In some embodiments, the composition administered to the subject inhibits metastasis of the cancer in the subject. In some embodiments, the composition administered inhibits tumor growth in the subject.

[0116] In another aspect, provided herein is a method of aiding in the prevention and / or treatment of a disease in a subject in need thereof, comprising administering to the subject a first therapy comprising one or more constructs, recombinant nucleic acids, engineered cells, or pharmaceutical compositions disclosed herein, and administering to the subject at least one additional therapy, wherein the first therapy and the at least one additional therapy together prevent and / or treat the disease in the subject. In some embodiments, the method comprises administering to the subject a first therapy comprising an effective number of engineered cells disclosed herein, wherein the engineered cells treat the disease.

[0117] Further examples of diseases suitable for treatment with the compositions and methods of the present disclosure include diseases associated with immune checkpoint inhibition. In these examples, the methods of the present disclosure may be checkpoint inhibition immunotherapy. In some embodiments, the checkpoint inhibition immunotherapy includes the use of one or more inhibitors of checkpoint receptors, such as, for example, PD-1 / PD-L1, CTLA-4, IDO, TIM3, LAG3, TIGIT, BTLA, VISTA, ICOS, KIR, and CD39. In some embodiments, the checkpoint receptor is an inhibitory checkpoint receptor selected from the group consisting of PD-1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, CD5, CD132, IDO, KIR, LAG3, TIM-3, TIGIT, and VISTA. In some embodiments, the checkpoint receptor is a stimulatory checkpoint receptor selected from the group consisting of CD27, CD28, CD40, OX40, GITR, ICOS, and CD137. In some embodiments, the checkpoint blockade immunotherapy includes anti-PD1 checkpoint therapy. In some embodiments, the checkpoint blockade immunotherapy includes anti-PD1-L1 checkpoint therapy.

[0118] As described in more detail below, various constructs of the present disclosure can bind to antigens derived from viral pathogens. In some embodiments of the present disclosure, provided herein are methods for diagnosing, preventing, and / or treating malignancies associated with microbial infection. In some embodiments, the malignancies are associated with bacterial infection.

[0119] In some embodiments, provided herein are methods for diagnosing, preventing, and / or treating malignancies associated with viral infection. In some embodiments, this malignancy is associated with infection by Epstein-Barr Virus (EBV), originally recognized through its association with Burkitt's lymphoma, but has since been associated with a significant range of lymphoproliferative lesions, and malignant lymphomas of B-cell, T-cell, and NK-cell origin. Examples of EBV-associated malignancies that may be suitably diagnosed, prevented, and / or treated using the compositions and methods disclosed herein include Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphoma, nasopharyngeal carcinoma, gastric carcinoma, post-transplant lymphoproliferative disorder, and B-lymphoproliferative disorder. Additional EBV-associated malignancies that may be suitably diagnosed, prevented, and / or treated using the compositions and methods disclosed herein include, but are not limited to, T-cell lymphoproliferative disorders, NK-cell lymphoproliferative disorders, NK-cell lymphomas, T-cell lymphomas, NK-cell lymphomas, T-cell leukemias, leiomyosarcoma, and lymphoepithelioma-like carcinomas.

[0120] Additional Treatments As discussed above, some embodiments of the present disclosure provide a method for preventing or treating a disease in a subject, which comprises administering to the subject a composition disclosed herein as a single therapy (e.g., monotherapy).In some embodiments of the present disclosure, the composition is administered to the subject individually as a first therapy, or in combination with at least one additional therapy, for example, at least 1, 2, 3, 4, or 5 additional therapies.Suitable therapies that can be administered in combination with the composition of the present disclosure include, but are not limited to, chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, and surgery.

[0121] Non-limiting examples of therapeutics suitable for combination with the methods disclosed herein include anti-CTLA4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-CD20 antibodies, anti-CD40 antibodies, anti-DR5 antibodies, anti-CD1d antibodies, anti-TIM3 antibodies, anti-SLAMF7 antibodies, anti-KIR receptor antibodies, anti-OX40 antibodies, anti-HER2 antibodies, anti-ErbB-2 antibodies, anti-EGFR antibodies, cetuximab, rituximab, trastuzumab, and pembrolizumab. Additional therapeutics suitable for combination with the methods disclosed herein include, but are not limited to, radiation therapy, such as single-dose radiation, fractionated radiation, focal radiation, and whole organ irradiation. Also suitable for combination with the methods disclosed herein include IL-12, IFNα, GM-CSF, chimeric antigen receptors, adoptively transferred T cells, anti-cancer vaccines, and oncolytic viruses.

[0122] In some embodiments, the first therapy and the at least one additional therapy are applied concomitantly. In some embodiments, the first therapy and the at least one additional therapy are applied simultaneously. In some embodiments, the first therapy and the at least one additional therapy are applied sequentially. In some embodiments, the first therapy is applied before the at least one additional therapy. In some embodiments, the first therapy is applied after the at least one additional therapy. In some embodiments, the first therapy is applied before and / or after the at least one additional therapy. In some embodiments, the first therapy and the at least one additional therapy are applied in alternation. In some embodiments, the first therapy and the at least one additional therapy are applied concomitantly within a single formulation.

[0123] In yet another aspect, various methods of obtaining the constructs disclosed herein are provided herein, comprising: (a) identifying a plurality of TCRs associated with a health disorder; (b) determining the sequence of CDR3β present in each of the identified TCRs; and (c) generating a construct comprising the CDR3β sequence determined in (b). In some embodiments, the method further comprises identifying one or more cognate antigens commonly recognized by the CDR3β sequences. In some embodiments, the disease is associated with a proliferative disease. In some embodiments, the proliferative disease is cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the disease is a malignancy associated with a viral infection. In some embodiments, the disease is a malignancy associated with infection by Epstein-Barr Virus (EBV). In some embodiments, the disease is associated with immune checkpoint inhibition.

[0124] kit Also provided herein are various kits for carrying out the methods described herein. In particular, some embodiments of the present disclosure provide kits for diagnosing a disease in a subject. Some other embodiments relate to kits for preventing a disease in a subject in need thereof. Some other embodiments relate to kits for methods of treating a disease in a subject in need thereof. For example, some embodiments provide kits that include one or more of the constructs, recombinant nucleic acids, genetically engineered cells, or pharmaceutical compositions provided and described herein, as well as written instructions for making and using them.

[0125] In some embodiments, the kits of the present disclosure further comprise one or more means useful for administering any one of the provided constructs, recombinant nucleic acids, genetically engineered cells, or pharmaceutical compositions to an individual. For example, in some embodiments, the kits of the present disclosure further comprise one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled catheters) that are used to administer any one of the provided constructs, recombinant nucleic acids, genetically engineered cells, or pharmaceutical compositions to an individual. In some embodiments, the kits may have one or more additional therapeutic agents that can be administered simultaneously or sequentially with other kit components for a desired purpose, such as diagnosing, preventing, or treating a disease in a subject in need thereof.

[0126] Any of the above kits may further comprise one or more additional reagents, which may be selected from the following: dilution buffers, reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control constructs, positive control constructs, and reagents suitable for in vitro generation of the constructs.

[0127] In some embodiments, the components of the kit may be in separate containers. In other embodiments, the components of the kit may be combined in a single container.

[0128] In some embodiments, the kit may further include instructions for use of the components of the kit to carry out the methods disclosed herein. For example, the kit may include a package insert containing information regarding the pharmaceutical compositions and dosage forms in the kit. Generally, the information will assist patients and physicians in effectively and safely using the enclosed pharmaceutical compositions and dosage forms. For example, the following information regarding the combinations disclosed herein may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, side effects, overdose, appropriate dosages and administration, method of delivery, appropriate storage conditions, reference materials, manufacturer / distributor information, and intellectual property information.

[0129] The instructions for carrying out the method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. The instructions may be present in the kit as a package insert on the label of the kit's container or a component thereof (e.g., associated with a package or subpackage), etc. The instructions may be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, floppy disk, flash drive, etc. In some cases, the actual instructions may not be present in the kit, but a means for obtaining the instructions from a remote source (such as the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or downloaded. As with the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

[0130] All publications and patent applications mentioned in this disclosure are herein 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.

[0131] Any reference cited herein is not admitted to constitute prior art. The discussion of the references states what their authors assert, but applicants do not exercise their right to challenge the accuracy and pertinence of the cited documents. Although many sources of information, including scientific journal articles, patent documents, and textbooks, are referenced herein, it is understood that this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0132] The general method descriptions provided herein are for illustrative purposes only: other alternatives and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are intended to be within the spirit and scope of this application.

[0133] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not intended to limit the scope of the disclosure or claims in any way. EXAMPLES

[0134] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are described, for example, in Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (with supplements since 2014); Bollag, DM et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. Nonviral Vectors for Gene Therapy. San Diego: Academic Press;Kaplitt, MG et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press;Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques."Manual of Immunology: A Comprehensive Textbook of Techniques." San Diego, CA: Academic Press;Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley;Mullis, KB, Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher;Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press;Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (with supplements since 2014);and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. "Gene Transfer and Expression in Mammalian Cells." Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference.

[0135] Example 1

[0136] Clinical samples Procedures for human tissue and blood collection were approved by the Stanford University Institutional Review Board (IRB 15166 and IRB 21319). Inclusion criteria included: adult patients (age: 18 years or older); confirmed or suspected NSCLC diagnosis; primary tumor: >2 cm; and consented to the study. Patients receiving neoadjuvant therapy or with underlying pulmonary infection, inflammatory, or fibrotic disease were excluded. A total of 21 patients with surgically resectable NSCLC treated at Stanford were included in the study. DNA was extracted from peripheral blood PBMCs (Qiagen) for HLA binding.

[0137] Example 2

[0138] Tissue processing Tissues were processed within 2 hours after surgery. Tissues were divided, one section for cell suspension and the other for histology. Tissues were minced and subsequently digested with collagenase III (200 IU / mL) and DNase (100 U / mL) (Worthington Biochemical) in RPMI for 40 minutes and passed through a 70 μm filter to generate a cell suspension. Sections for histology were fixed in 4% paraformaldehyde and transferred to 70% ethanol solution the following day.

[0139] Example 3

[0140] FACS analysis T cells were isolated from tumor single cell suspensions by antibody staining followed by cell sorting on a five-laser FACSAria Fusion (Stanford University FACS facility) purchased with funding from the Parker Institute for Cancer Immunotherapy. To assess viability, tumor cell suspensions were stained in PBS with Zombie Aqua dye (Biolegend). This was followed by staining in PBS with 2% FBS containing Fc blocking solution (Biolegend) and the following antibodies: anti-CD4 (OKT4, Biolegend), anti-CD8 (SK1, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD45 (H130, Biolegend), anti-CD25 (BC96, Biolegend), anti-PD-1 (EH12.2H7, Biolegend), anti-CD137 (4B4-1, BD Biosciences), and anti-HLA-DR (L243, Biolegend). CD3+CD45+ AquaZombie cells were index-sorted directly into 96-well plates preloaded with 4 μL of capture buffer, flash frozen on dry ice and stored at -80°C.

[0141] Example 4

[0142] GLIPH2 analysis and construction of T cell specificity groups The GLIPH2 algorithm was implemented to construct T cell specificity clusters using 778,938 distinct CDR3β sequences from the MD Anderson Cancer Center NSCLC data set

[25] . Briefly, by comparing with a reference data set of 273,920 distinct CDR3β sequences (both CD4 and CD8) from 12 healthy individuals, GLIPH2 first found clusters of CDR3β sequences that share either global or local motifs as previously described

[24] . The results of CDR3β clusters with shared sequence motifs were accompanied by multiple statistics to facilitate the extraction of reliable specificity clusters, including bias in Vβ gene usage, CDR3β length distribution (related to local motifs only), cluster size, HLA allele usage, and clonal amplification. To construct reliable specificity groups with the NSCLC data set, we prioritized TCR specificity groups with at least three distinct CDR3β members from at least three different patients with significant bias in Vβ gene usage and CDR3β clonal amplification compared to the reference data set, leading to the detection of 4,226 specificity groups that formed the basis for further analysis across studies.

[0143] Example 5

[0144] Annotation of specificity groups by tetramer-derived CDR3β sequences To annotate putative specificity groups from lung cancer patients, we performed a combined GLIPH analysis using both MD Anderson lung cancer patient CDR3β sequences and widely available tetramer-derived CDR3β sequences. To do so, we first performed an independent GLIPH analysis with a total of 10,051 CDR3β sequences from the tetramer data set to identify tetramer-derived CDR3β sequences that could potentially form TCR specificity groups. This resulted in 395 specificity groups containing 1,561 CDR3β sequences. These 1,561 CDR3β sequences were then combined with 778,938 CDR3β sequences from the MD Anderson lung cancer data set for the GLIPH2 analysis described above. Any specificity group that contained at least one CDR3β sequence from the tetramer data was considered "annotated" and was assigned specificity and HLA restriction according to the associated tetramer sequence. Notably, in all cases where CDR3β sequences from multiple tetramers were found in a particular specificity group, there was only one major tetramer-defined specificity / HLA involved.

[0145] Example 6

[0146] Preparation of single-cell RNA-seq (scRNA-Seq) samples using the Smart-seq2 method Complete transcriptomes from FACS-sorted T cells at the single-cell level were generated following the procedure previously reported

[54] with some modifications. First-strand cDNA was then generated using Takara's SMARTScribe Reverse Transcriptase kit according to the manufacturer's instructions (Takara Bio). Notable modifications from the Smart-Seq2 RT step of the previous report include: 2 mM dNTPs and 2 μM oligo-dT were included in the capture buffer; 1 M betaine and an additional 6 mM MgCl2 were included in the RT reaction buffer. cDNA samples were then amplified for 22–25 cycles using the KAPA Library Quantification kit (Roche). 1 μL of amplified cDNA (total of 25 / well) was used for single-cell TCR sequencing, thereby avoiding the RT step as previously reported

[35] . To proceed with scRNA-Seq, full-length cDNA samples were first washed with 0.6–0.8 volumes of pre-calibrated AMPure XP beads (Beckman Coulter) to eliminate DNA fragments smaller than 500 base pairs. An automated liquid handling device, Biomek FXP Automated Workstation (Beckman Coulter), was used to remove cell-to-cell variability. The quality of the purified full-length cDNA was verified with an AATI Fragment Analyzer (Agilent). The measurements from the Fragment Analyzer were subsequently used to normalize the cDNA input values ​​by a Mantis liquid handling device (Formulatrix). The cDNA samples were then integrated into 384-well plates (LVSD) by a Mosquito X1 liquid handling device (TTP labtech). After transfer, Illumina sequencing libraries were prepared using a Mosquito HTS liquid handling device (TTP labtech). Complete transcriptome libraries were generated using the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096) using only 0.4 μL of cDNA per well (23 μL in total).Custom i5 and i7 unique 8 bp index primers (IDT) were used to multiplex 384 wells in one sequencing run. Libraries were amplified on a C1000 Touch® thermocycler with a 384-well reaction module (Bio-rad). Pooled libraries were examined on an Agilent 2100 Bioanalyzer (Stanford PAN Facility) and double-ended read sequences (150 bp x 2) were obtained on a Hiseq 4000 Sequencing System purchased with funding from the National Institutes of Health (NIH; S10OD018220) for the Stanford Functional Genomics Facility (SFGF).

[0147] Example 7

[0148] Single-cell sequencing of TCR α / β chains For scRNA-Seq sample preparation, T single cells were sorted and captured as described above. Following first strand cDNA synthesis (Takara) and amplification (Roche), 1 μL of amplified cDNA (total of 25 μL / well) was used for single cell TCR sequencing, thus avoiding the RT step as previously reported

[35] . Nested PCR was performed using TCRα / β primers carrying multiple barcodes that allow sequencing of accumulated CDR3α / β in a single Miseq run. Both end sequence reads were combined, demultiplexed, and mapped to human TCR controls from the international ImMunoGeneTics information system® (IMGT) using a dedicated script as previously reported.

[0149] Example 8

[0150] Data analysis of scRNA-Seq results Sequencing reads were first demultiplexed and sorted into individual fastq files corresponding to the full-length transcriptome of each individual T cell. Reads were mapped using STAR aligner (2.7.1a) with default parameters to the human genome control GRCh38 (v21) from the UCSC genome browser. Mapped reads were sorted and indexed using SAMtools software (1.4). Gene expression was first quantified by counting reads mapped to genes with htseq-count (HTSeq 0.9.1) using the following settings: --stranded=no --type=exon --idattr=gene_name --mode=intersection-nonempty. All single-cell T cell states were analyzed with the R package Seurat (3.1.4) using raw read counts unless otherwise stated. To derive the TCR repertoire from the scRNA-Seq results, reads mapping to both the TCRα and TCRβ genes were first reconstructed using the TraCeR algorithm as previously described

[36] . The reconstructed DNA sequences were then submitted to IMGT to call gene compartment utilization and CDR3 amino acid sequences through HighV-QUEST.

[0151] Example 9

[0152] GLIPH2 analysis on CDR3β sequences from the TRACERx NSCLC group Raw fastq files (n=202) of bulk CDR3β nucleotide sequences from the NSCLC TRACERx (TRAcking Cancer Evolution through therapy (Rx)) cohort were downloaded from the Short Read Archive as reported [ref. 26]. CDR3β amino acid sequences, V gene usage, and clonal numbers were then derived using a dedicated pathway constructed in a previous report [ref. 35]. To quantify the percentage of tumor-enriched specificity groups shown in Figure 1B, we first performed a combined GLIPH2 analysis combining CDR3β sequences from the MD Anderson cohort (n=778,938) with bulk CDR3β sequences from each tumor sample in the TRACERx cohort. The top 20 clonal amplifications belonging to the 449 tumor-enriched specificity groups as well as the total percentage of the remaining CDR3β clonotypes were then derived for each tumor (n=202).

[0153] Example 10

[0154] Soluble biotinylated TCR α / β chains for yeast selection Soluble TCR α / β chains used for yeast selection were generated as previously reported [Reference 33]. Briefly, synthetic gene blocks (gBlocks®) of N-terminally truncated TCR α chain V or TCR β chain V and modified C gene fragments were assembled into baculovirus pAcGP67a constructs (BD Biosciences) using Gibson assembly (New England BioLabs). Finally, baculovirus plasmids were co-transduced into SF9 cells (ATCC) using Bestbac 2.0 (Expression systems) with FuGENE® 6 (Promega) to generate crude viral supernatant (P0). Virus was then added at a dilution of 1:500 at 1 × 10 6Higher titer virus (P1) was generated by passage in 30–50 mL cultures at a density of 1 × 10 cells / mL. To generate soluble TCR α / β chains, up to 4 L of High Five cells (Hi5, Thermo Fisher Scientific) were cultured at 2 × 10 cells / mL at a dilution of 1:500–1:1000. 6 Cells were incubated at a density of 1000 ng / mL and infected with P1 baculovirus for 1 week prior to protein purification. Recombinant TCRα / β chains were bound to Ni-NTA resin (QIAGEN) in Hi5 cell medium for 3 h at room temperature, washed with 20 mM imidazole in 1×HBS pH 7.2, and eluted in 200 mM imidazole in 1×HBS pH 7.2. After buffer exchange into 1×HBS pH 7.2 using a 30 kDa filter (Millipore), purified proteins were biotinylated overnight at 4° C. with birA ligase in the presence of 100 μM biotin, 40 mM bicine (pH 8.3), 10 mM ATP, and 10 mM magnesium acetate. Biotinylated proteins were purified by size-exclusion chromatography using an AKTA Purifier Superdex 200 column (GE Healthcare) and examined on SDS-PAGE gels to confirm stoichiometry and biotinylation with excess streptavidin.

[0155] Example 11

[0156] Lentiviral TCR transduction Fusion gene fragments of the TCR α chain, P2A linker, and TCR β chain were purchased from IDT and cloned into the MCS of the EF1a-MCS-GFP-PGK-Pro lentiviral vector

[23] . The day before transfection, HEK-293T cells were cultured at 7.5 × 10 6Cells were seeded at a density of 10 cells per well into 10 cm dishes containing 10 mL of DMEM. 293T cells were co-transfected with 3.3 μg of lentiviral plasmid, 2.5 μg of gag-pol plasmid, and 0.83 μg of VSV-G envelope plasmid premixed with 33 μL of PEI in 120 μL of Opti-MEM (ThermoFisher Scientific). Media was replenished after 24 hours, and viral supernatants were harvested after 24 and 48 hours. TCR-deficient Jurkat cells (as shown below) were transduced with viral supernatants, TCR expression was assessed by flow cytometry, and TCR-expressing cells were sorted based on expression of GFP, CD3, and transduced TCR α / β chains.

[0157] Example 12

[0158] Retroviral TCR transduction For retroviral-mediated expression of TCR27 and TCR28 in primary T cells, the TCRα chain, P2A linker, and TCRβ chain were PCR amplified from lentiviral vectors (as shown above) and cloned into the MCS of MSGV1-based retroviral vectors (obtained from Steve Rosenberg laboratory) using In-Fusion Cloning (Takara). For retroviral-mediated expression of TCR27 and TCR28 in primary T cells, fusion gene fragments of the TCRα chain, P2A linker, and TCRβ chain were purchased from IDT and cloned into the MCS of MSGV1-based retroviral vectors.

[0159] Example 13

[0160] cell culture The Jurkat 76 T cell line, deficient in both TCRα and TCRβ, was obtained from Dr. Shao-An Xue (Department of Immunology, University College London). Jurkat and primary T cells were grown in complete RPMI (ThermoFisher) containing 10% FBS, 25 mM HEPES, 290 μg / mL L-glutamine, 100 U / mL penicillin, 100 U / mL streptomycin, 1 mM sodium pyruvate, and 1× non-essential amino acids. T2 cells were grown in IMDM (Fisher Scientific) containing 20% ​​FBS, 290 μg / mL L-glutamine, 100 U / mL penicillin, and 100 U / mL streptomycin.

[0161] Example 14

[0162] Ex vivo stimulation of Jurkat T cell clones Jurkat 76 cells expressing an exogenous TCR of interest were sorted and co-cultured with T2 cells in complete RPMI as detailed above. Peptides were dissolved in DMSO at a stock concentration of 20 mM and diluted to a final concentration of 2 mM. After 18 hours of stimulation, cells were washed and stained with anti-CD3 (OKT3, Biolegend), anti-CD69 (FN50, Biolegend), and anti-TCRα / β (IP26, Biolegend) antibodies. Cells were acquired using a FACS Fortessa (BD Biosciences), an automated high-throughput sampler, and data were analyzed using FlowJo software (Treestar).

[0163] Example 15

[0164] Whole-exome sequencing Whole-exome sequencing of tumor DNA and corresponding germline leukocyte DNA was performed as previously described

[57] with the addition of 75 ng of sheared genomic DNA for library preparation with the KAPA HyperPrep kit (Roche) with modifications to the manufacturer's instructions. Library prepared samples were captured with the SeqCap EZ MedExome kit (NimbleGen) according to the manufacturer's instructions. Sequence data were demultiplexed and mapped to hg19 using a dedicated bioinformatics pathway as previously described

[58] . Variants were called using default parameters using VarScan 2

[59] , Mutect

[60] , and Strelka

[61] . Variants called by at least two methods were then filtered for 1) a mutant allele frequency of at least 2.5%, 2) a depth of at least 30x in both tumor and germline samples, 3) germline reads equal to zero, and 4) a population allele frequency of less than 0.1% in the genome assembly database

[62] .

[0165] Example 16

[0166] statistical analysis Unless otherwise stated, all statistical analyses performed in finding reliable specificity groups using GLIPH2 were performed using Fisher's exact test using a correlation table with the CDR3β query population (specificity group) and the reference population

[24] . The Poisson test was used to determine representativeness bias in the comparison of different CDR3β sequences or specificity groups between tumor and non-diseased lungs. The Student's t-test was used to evaluate the results from all in vitro measurements. Statistical significance was defined as a p-value < 0.05.

[0167] Example 17

[0168] Establishment of specific populations of tumor-infiltrating T cells in human lung cancer This example describes the results of experiments performed to identify T cells that recognize shared tumor antigens in lung cancer.

[0169] To identify T cells that recognize shared tumor antigens in lung cancer, we used GLIPH2 to establish specificity groups. As previously described

[23] , TCR clonotypes that are likely to share specificity are divided into groups based on short amino acid sequence motifs embedded within the variable CDR3β region of the TCR. The improved GLIPH2 offers the advantage of analyzing large T cell repertoire data sets and identifying specificity groups carrying local or global sequence motifs with much higher power

[24] . We adapted the GLIPH2 algorithm to a recently published T cell repertoire data set

[25] consisting of 778,938 distinct CDR3β sequences from 178 HLA-typed non-small cell lung cancer (NSCLC) patients with surgically resectable disease (see e.g. Table 1 below). Notably, T cell clonotypes from bulk sequencing were derived from both surgically resected tumors as well as non-diseased lung (see e.g. Table 1). [Table 1]

[0170] Using this data set, 435 tumor-enriched specificity groups from NSCLC patients were established after adapting a series of criteria including enrichment of Vβ genes as well as clonal amplification indicative of T cell antigen recognition (Figures 1A and 3A). To identify specificity groups associated with antigens shared across these patients, the specificity groups were further constructed to include at least three different CDR3β sequences from at least three patients. The portion of clonotypes that are members of the previously defined specificity groups as described above was established. A significantly higher percentage of the most amplified TCR clonotypes in tumors were found to belong to the tumor-enriched specificity groups (Figure 1B). In contrast, a much lower proportion of TCR clonotypes from the patients' non-diseased lungs belonged to the tumor-enriched specificity groups (see, for example, Figure 3B). The 435 tumor-enriched specificity groups were then determined to be associated with lung cancer and not simply normal lung tissue or other types of lung disease. In a validation panel of 7,363,492 clonotypes from 202 tumor samples representing 68 NSCLC patients

[26] , a significantly higher proportion of the top amplified TCR clonotypes in tumors compared to their non-amplified counterparts belonged to 435 tumor-enriched specificity groups (Figure 1B). In contrast, a lower proportion of TCR clonotypes belonging to tumor-enriched specificity groups was observed in lung tissue from healthy donors and COPD patients (without diagnosed cancer), regardless of clonal amplification (Figure 1B). In summary, the experimental data described herein identify a set of specificity groups predicted to recognize shared tumor antigens across NSCLC patients.

[0171] Example 18

[0172] Computational validation of TCR specificity groups using HLA tetramer sequences To validate the specificity groups established by GLIPH2, publicly available CDR3β sequences from various HLA tetramer databases were included in combination with CDR3β sequences from MD Anderson Cancer Center for a collaborative GLIPH2 analysis [References 23, 27, 28]. The CDR3β sequences available from the tetramer data set encompass primarily viral specificities and have been experimentally shown to bind epitopes in the context of their respective HLA. This allowed us to annotate some specificity groups with sequences from the tetramer database associated with experimentally established antigen specificities and HLA restrictions. This collaborative analysis allowed us to annotate 396 specificity groups (Figure 1C). Of these specificity groups, 71 were clonally expanded and annotated as 10 different tetramers (Figure 3C). As expected, we found that clonotypes with predicted specificities for influenza (Flu), Epstein-Barr virus (EBV), or cytomegalovirus (CMV) antigens were not preferentially localized in tumors compared to non-diseased lung (Figures 4A-4B). Further network analysis organized these tetramer-annotated specificities sharing some identical CDR3β sequence members into communities (Figures 1C and 3D-3G). Specificities belonging to a given community were consistently annotated as identical HLA tetramers (Figures 1C, 3D, and 3E), indicating that some antigen specificities are likely associated with the same specificity and HLA restriction, despite sharing different sequence motifs.

[0173] Example 19

[0174] Experimental validation of GLIPH2 predicted specificity To experimentally validate peptide-MHC specificity, sequences of TCRα / β pairs from single T cells are required. Therefore, we performed TCR sequencing (TCR-seq) of single cells from 15 early-stage NSCLC patients treated at Stanford University. Tumor-infiltrating T cells were prepared from surgically resected specimens and sorted by FACS prior to sequencing (see, for example, Figure 5). In total, 4,704 CDR3α and CDR3β sequence pairs were sequenced and combined with CDR3β sequences from MD Anderson Cancer Center for further analysis.

[0175] Example 20

[0176] Expansion of EBV-specific T cell clones in patients responding to immune checkpoint blockade To determine whether pathogen-specific T cells can affect clinical responses to anti-PD1 checkpoint immunotherapy, we analyzed the TCR repertoires of two NSCLC patients who experienced a clinical response to treatment (see, e.g., Figure 6A). CDR3α / β repertoire pairs from pre- and post-treatment blood samples were sequenced and used to identify 102 CDR3β clonotypes amplified in post-treatment samples (see, e.g., Figure 2A). Of these amplified clones, 41 belonged to 99 specificity groups identified in the CDR3β repertoire of tumor-infiltrating T cells (total n=66,094, see, e.g., Figure 3A). These specificity groups were then annotated using tetramer CDR3β sequences, and 11 (total n=99) were found, including three amplified CDR3β clones predicted to recognize EBV and Flu antigens (Figure 2B). To verify the speculation of specificity, two Jurkat cell clones expressing TCRα / β chains predicted to recognize EBV antigens and a T2 cell line expressing wild-type B*35 (Figures 2B and 6B) were formed. Indeed, by co-culture with T2-B*35 cells, both Jurkat-TCR27 and Jurkat-TCR28 cells were observed to respond to the predicted EBV peptides (see, e.g., Figure 2C). Notably, these EBV-specific specificity groups were not only amplified after treatment, but also showed a maldistribution in the tumor compared to the adjacent lung, thus suggesting potential cross-reactivity against unknown TAAs (see, e.g., Figure 6C). In the past, it was believed that the common pathogen-specific T cells found in tumors were "bystander cells" and not specific for TAAs. The experimental data described herein demonstrated that T cell specificity for TAAs and pathogen-derived antigens is not mutually exclusive. Furthermore, these pathogen-specific T cells within tumors displayed an effector phenotype that was not exhausted or stressed and lacked CD39 expression.These data suggested that cross-reactive T cells may play a role in suppressing cancer progression by instituting anti-PD1 checkpoint blockade.

[0177] While certain alternative embodiments of the present disclosure have been disclosed, it should be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. Accordingly, there is no intention to be strictly limited to the abstract and disclosure presented herein.

[0178] [Table 2-1] [Table 2-2] [Table 2-3]

[0179] References 1.Kawakami, Y., et al., Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci USA, 1994. 91(9): p. 3515-9. 2.Coulie, PG, et al., A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med, 1994. 180(1): p. 35-42. 3.van der Bruggen, P., et al., A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science, 1991. 254(5038): p. 1643-7. 4.Coulie, PG, et al., A mutated intron sequence codes for an antigenic peptide recognized by cytolytic T lymphocytes on a human melanoma. Proc Natl Acad Sci USA, 1995. 92(17): p. 7976-80. 5.Wolfel, T., et al., A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science, 1995. 269(5228): p. 1281-4. 6.Murray, RJ, et al., Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J Exp Med, 1992. 176(1): p. 157-68. 7.Koziel, MJ, et al., HLA class I-restricted cytotoxic T lymphocytes specific for hepatitis C virus. Identification of multiple epitopes and characterization of patterns of cytokine release. J Clin Invest, 1995. 96(5): p. 2311-21. 8.Rehermann, B., et al., The cytotoxic T lymphocyte response to multiple hepatitis B virus polymerase epitopes during and after acute viral hepatitis. J Exp Med, 1995. 181(3): p. 1047-58. 9.Tran, E., et al., Immunogenicity of somatic mutations in human gastrointestinal cancers. Science, 2015. 350(6266): p. 1387-90. 10.Gros, A., et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients. Nat Med, 2016. 22(4): p. 433-8. 11.Zacharakis, N., et al., Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med, 2018. 24(6): p. 724-730. 12.Schumacher, T. N., W. Scheper, and P. Kvistborg, Cancer Neoantigens. Annu Rev Immunol, 2019. 37: p. 173-200. 13.Rosenberg, SA and ME Dudley, Adoptive cell therapy for the treatment of patients with metastatic melanoma. Curr Opin Immunol, 2009. 21(2): p. 233-40. 14.Hinrichs, CS and SA Rosenberg, Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev, 2014. 257(1): p. 56-71. 15.de Vos van Steenwijk, PJ, et al., An unexpectedly large polyclonal repertoire of HPV-specific T cells is poised for action in patients with cervical cancer. Cancer Res, 2010. 70(7): p. 2707-17. 16.Piersma, SJ, et al., Human papilloma virus specific T cells infiltrating cervical cancer and draining lymph nodes show remarkably frequent use of HLA-DQ and -DP as a restriction element. Int J Cancer, 2008. 122(3): p. 486-94. 17.Evans, EM, et al., Infiltration of cervical cancer tissue with human papillomavirus-specific cytotoxic T-lymphocytes. Cancer Res, 1997. 57(14): p. 2943-50. 18.Triozzi, PL and AP Fernandez, The role of the immune response in merkel cell carcinoma. Cancers (Basel), 2013. 5(1): p. 234-54. 19.Simoni, Y., et al., Bystander CD8(+) T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature, 2018. 557(7706): p. 575-579. 20.Rosato, PC, et al., Virus-specific memory T cells populate tumors and can be repurposed for tumor immunotherapy. Nat Commun, 2019. 10(1): p. 567. 21.Scheper, W., et al., Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat Med, 2019. 25(1): p. 89-94. 22.Yu, W., et al., Clonal Deletion Prunes but Does Not Eliminate Self-Specific alphabeta CD8(+) T Lymphocytes. Immunity, 2015. 42(5): p. 929-41. 23.Glanville, J., et al., Identifying specificity groups in the T cell receptor repertoire. Nature, 2017. 547(7661): p. 94-98. 24.Huang, H., et al., Analyzing the CD4+ T cell response repertoire to M. tuberculosis using GLIPH2 and whole-genome antigen screening. Nat Biotechnol, 2020. 25.Reuben, A., et al., Comprehensive T cell repertoire characterization of non-small cell lung cancer. Nat Commun, 2020. 11(1): p. 603. 26.Joshi, K., et al., Spatial heterogeneity of the T cell receptor repertoire reflects the mutational landscape in lung cancer. Nat Med, 2019. 25(10): p. 1549-1559. 27.Shugay, M., et al., VDJdb: a curated database of T-cell receptor sequences with known antigen specificity. Nucleic Acids Res, 2018. 46(D1): p. D419-D427. 28.Song, I., et al., Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8(+) T cell epitope. Nat Struct Mol Biol, 2017. 24(4): p. 395-406. 29. Sidney, J., et al., HLA class I supertypes: a revised and updated classification. BMC Immunol, 2008. 9: p. 1. 30.Harjanto, S., LF Ng, and JC Tong, Clustering HLA class I superfamilies using structural interaction patterns. PLoS One, 2014. 9(1): p. e86655. 31.Robins, HS, et al., Overlap and effective size of the human CD8+ T cell receptor repertoire. Sci Transl Med, 2010. 2(47): p. 47ra64. 32.Arstila, TP, et al., A direct estimate of the human alphabeta T cell receptor diversity. Science, 1999. 286(5441): p. 958-61. 33.Gee, MH, et al., Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes. Cell, 2018. 172(3): p. 549-563 e16. 34.Kheir, F., et al., Detection of Epstein-Barr Virus Infection in Non-Small Cell Lung Cancer. Cancers (Basel), 2019. 11(6). 35.Han, A., et al., Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat Biotechnol, 2014. 32(7): p. 684-92. 36.Stubbington, MJT, et al., T cell fate and clonality inference from single-cell transcriptomes. Nat Methods, 2016. 13(4): p. 329-332. 37.Guo, X., et al., Global characterization of T cells in non-small-cell lung cancer by single-cell sequencing. Nat Med, 2018. 24(7): p. 978-985. 38.Joglekar, AV, et al., T cell antigen discovery via signaling and antigen-presenting bifunctional receptors. Nat Methods, 2019. 16(2): p. 191-198. 39.Li, G., et al., T cell antigen discovery via trogocytosis. Nat Methods, 2019. 16(2): p. 183-190. 40.Kula, T., et al., T-Scan: A Genome-wide Method for the Systematic Discovery of T Cell Epitopes. Cell, 2019. 178(4): p. 1016-1028 e13. 41.Sewell, AK, Why must T cells be cross-reactive? Nat Rev Immunol, 2012. 12(9): p. 669-77. 42.McCarthy, EF, The toxins of William B. Coley and the treatment of bone and soft-tissue sarcomas. Iowa Orthop J, 2006. 26: p. 154-8. 43.Morales, A., D. Eidinger, and A. W. Bruce, Intracavitary Bacillus Calmette-Guerin in the treatment of superficial bladder tumors. J Urol, 1976. 116(2): p. 180-3. 44.Gopalakrishnan, V., et al., Gut microbiome modulates response to anti-PD-1 immunotherapy in melanoma patients. Science, 2018. 359(6371): p. 97-103. 45.Routy, B., et al., Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science, 2018. 359(6371): p. 91-97. 46.Vetizou, M., et al., Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota. Science, 2015. 350(6264): p. 1079-84. 47.Sivan, A., et al., Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy. Science, 2015. 350(6264): p. 1084-9. 48.Matson, V., et al., The commensal microbiome is associated with anti-PD-1 efficacy in metastatic melanoma patients. Science, 2018. 359(6371): p. 104-108. 49.Riquelme, E., et al., Tumor Microbiome Diversity and Composition Influence Pancreatic Cancer Outcomes. Cell, 2019. 178(4): p. 795-806 e12. 50.Emerson, RO, et al., Immunosequencing identifies signatures of cytomegalovirus exposure history and HLA-mediated effects on the T cell repertoire. Nat Genet, 2017. 49(5): p. 659-665. 51.Abazeed, ME, et al., Integrative radiogenomic profiling of squamous cell lung cancer. Cancer Res, 2013. 73(20): p. 6289-98. 52.Barbie, DA, et al., Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature, 2009. 462(7269): p. 108-12. 53.Altman, JD and MM Davis, “MHC-peptide tetramers to visualize antigen-specific T cells.” Curr Protoc Immunol, 2003. Chapter 17: p. Unit 17 3. 54.Picelli, S., et al., Full-length RNA-seq from single cells using Smart-seq2. Nat Protoc, 2014. 9(1): p. 171-81. 55. Rueden, CT, et al., ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics, 2017. 18(1): p. 529. 56.Schindelin, J., et al., Fiji: an open-source platform for biological-image analysis. Nat Methods, 2012. 9(7): p. 676-82. 57.Hellmann, MD, et al., Circulating tumor DNA analysis to assess risk of progression after long-term response to PD-(L)1 blockade in NSCLC. Clin Cancer Res, 2020. 58.Newman, AM, et al., An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med, 2014. 20(5): p. 548-54. 59. Koboldt, DC, et al., VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res, 2012. 22(3): p. 568-76. 60.Cibulskis, K., et al., Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat Biotechnol, 2013. 31(3): p. 213-9. 61. Saunders, CT, et al., Strelka: accurate somatic small-variant calling from sequenced tumor-normal sample pairs. Bioinformatics, 2012. 28(14): p. 1811-7. 62.Lek, M., et al., Analysis of protein-coding genetic variation in 60,706 humans. Nature, 2016. 536(7616): p. 285-91.

Claims

1. A construct comprising at least one complementarity determining region (CDR) having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-106.

2. 2. The construct of claim 1, wherein at least one CDR has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-56.

3. 2. The construct of claim 1, wherein the at least one CDR has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 57-106.

4. The construct according to any one of claims 1 to 3, wherein the construct is a single-stranded construct or a double-stranded construct.

5. The construct of any one of claims 1 to 4, wherein the construct is selected from the group consisting of: (a) a T cell receptor (TCR); (b) an antibody; and (c) a functional derivative or fragment of (a) or (b).

6. The construct according to any one of claims 1 to 5, wherein the construct is a TCR construct comprising a TCR α chain and a TCR β chain operably linked to each other.

7. The construct of any one of claims 1 to 6, wherein the construct is a TCR construct comprising a CDR3β within its β chain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1 to 106.

8. The construct of claim 7 , further comprising a CDR3α sequence within its α chain.

9. The constructs include antigen-binding fragments (Fab), single chain variable fragments (scFv), single chain antibodies, single domain antibodies (sdAbs), V H Domain, V L Domain, V H The construct according to any one of claims 1 to 8, which is an antibody construct selected from the group consisting of an H domain, a bispecific antibody, or a functional fragment of either thereof.

10. A recombinant nucleic acid comprising a nucleic acid sequence encoding the construct of any one of claims 1 to 9.

11. The nucleic acid of claim 10 , wherein the nucleic acid sequence is operably linked to a heterologous nucleic acid sequence.

12. The nucleic acid molecule according to any one of claims 10 to 11, further configured as an expression cassette or vector.

13. The nucleic acid of claim 12 , wherein the vector is a plasmid vector or a viral vector.

14. The nucleic acid of claim 13 , wherein the viral vector is derived from a lentivirus, adenovirus, adeno-associated virus, baculovirus, or retrovirus.

15. a) a construct according to any one of claims 1 to 9; and / or b) A genetically engineered cell comprising a recombinant nucleic acid according to any one of claims 10 to 14.

16. The genetically engineered cell of claim 15 , wherein the genetically engineered cell is a eukaryotic cell.

17. 17. The genetically engineered cell of claim 16, wherein the eukaryotic cell is a mammalian cell.

18. 20. The genetically engineered cell of claim 17, wherein the mammalian cell is a human cell.

19. The genetically engineered cell of any one of claims 15 to 18, wherein the cell is an immune cell.

20. The immune cells include B cells, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, basophils, eosinophils, neutrophils, dendritic cells, macrophages, regulatory T cells, helper T cells (T H ), cytotoxic T cells (T CTL 20. The genetically engineered cell of claim 19, which is a T cell, a memory T cell, a γδ T cell, other T cell, a hematopoietic stem cell, or a hematopoietic stem cell precursor.

21. 21. The genetically engineered cell of claim 20, wherein the immune cell is a lymphocyte.

22. 22. The genetically engineered cell of claim 21, wherein the lymphocyte is a T lymphocyte or a T lymphocyte precursor.

23. 23. The genetically engineered cell of claim 22, wherein the T lymphocyte is a CD4+ T cell or a CD8+ T cell.

24. 24. The genetically engineered cell of any one of claims 22 to 23, wherein the T lymphocytes are CD8+ T cytotoxic lymphocyte cells selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells.

25. 24. The genetically engineered cell of any one of claims 22-23, wherein the T lymphocytes are CD4+ T helper lymphocyte cells selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

26. 1. A method for producing a genetically engineered cell, comprising: a) providing a host cell capable of protein expression; and b) transducing the provided host cell with a recombinant nucleic acid according to any one of claims 10 to 14 to produce a genetically engineered cell.

27. 27. A genetically engineered cell produced by the method of claim 26.

28. A cell culture comprising at least one genetically engineered cell according to any one of claims 15 to 25 and 27 and a culture medium.

29. a pharma- ceutically acceptable carrier, and a) a construct according to any one of claims 1 to 9; b) a recombinant nucleic acid according to any one of claims 10 to 14; and / or c) A pharmaceutical composition comprising a genetically engineered cell according to any one of claims 15 to 25 and 27.

30. 30. The pharmaceutical composition of claim 29, comprising the recombinant nucleic acid of any one of claims 10 to 14 and a pharma- ceutically acceptable carrier.

31. 31. The pharmaceutical composition of claim 30, wherein the recombinant nucleic acid is encapsulated in a viral capsid or lipid nanoparticle.

32. The pharmaceutical composition of claim 29, wherein the composition comprises a genetically engineered cell according to any one of claims 15 to 25 and 27.

33. A pharmaceutical composition according to any one of claims 29 to 32 for preventing and / or treating a disease in a subject in need thereof.

34. The pharmaceutical composition of claim 33, wherein the disease is associated with immune checkpoint inhibition.

35. The pharmaceutical composition according to any one of claims 33 to 34, wherein the prevention and / or treatment is aimed at checkpoint inhibition immunotherapy.

36. The pharmaceutical composition of claim 35, wherein the checkpoint inhibition immunotherapy is anti-PD1 checkpoint therapy or anti-PD1-L1 checkpoint therapy.

37. The pharmaceutical composition according to any one of claims 33 to 36, wherein the disease is associated with lung cancer selected from the group consisting of adenocarcinoma, squamous cell carcinoma, small cell carcinoma, non-small cell carcinoma, adenosquamous carcinoma, small cell lung carcinoma, large cell carcinoma, neuroendocrine carcinoma of the lung, non-small cell lung carcinoma (NSCLC), undifferentiated non-small cell carcinoma, other non-small cell carcinoma not otherwise specified, lung squamous cell carcinoma, bronchioloalveolar carcinoma, sarcomatoid carcinoma, pleomorphic carcinoma, carcinosarcoma, pulmonary blastoma, metastatic carcinoma of unknown primary, primary pulmonary lymphoepithelioma-like carcinoma, and benign neoplasms of the lung.

38. 38. The pharmaceutical composition of claim 37, wherein the lung cancer is an NSCLC selected from the group consisting of squamous cell carcinoma, adenocarcinoma, large cell carcinoma, carcinoid tumor, pleomorphic carcinoma, salivary gland carcinoma, adenosquamous carcinoma, sarcomatoid carcinoma, and unclassified carcinoma.

39. The pharmaceutical composition of claim 38, wherein the NSCLC comprises stage I NSCLC or stage II NSCLC.

40. The pharmaceutical composition according to any one of claims 37 to 39, wherein the cancer is a non-metastatic cancer, a metastatic cancer, a multidrug resistant cancer, or a recurrent cancer.

41. 41. The pharmaceutical composition of claim 40, which inhibits tumor growth or cancer metastasis in the subject.

42. 42. The pharmaceutical composition of claim 41, wherein the disease is a malignant tumor associated with a viral infection.

43. 43. The pharmaceutical composition of claim 42, wherein the disease is a malignant tumor associated with infection by Epstein-Barr virus (EBV).

44. 44. The pharmaceutical composition of claim 43, wherein the malignant tumor is associated with EBV infection and is selected from the group consisting of Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphoma, nasopharyngeal carcinoma, gastric cancer, post-transplant lymphoproliferative disorders, B-lymphoproliferative disorders, T / NK lymphoproliferative disorders, T / NK lymphoma / leukemia, leiomyosarcoma, and lymphoepithelioma-like carcinoma.

45. The pharmaceutical composition according to any one of claims 33 to 44, wherein the subject is a mammal.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the mammal is a human.

47. The pharmaceutical composition according to any one of claims 33 to 46, administered to a subject individually as a first therapy or in combination with at least one additional therapy.

48. 48. The pharmaceutical composition of claim 47, wherein the at least one additional therapy is selected from the group consisting of chemotherapy, radiation therapy, immunotherapy, hormone therapy, toxin therapy, targeted therapy, or surgery.

49. The pharmaceutical composition of any one of claims 47 to 48, wherein the at least one additional therapy is selected from the group consisting of anti-CTLA4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-CD20 antibody, anti-CD40 antibody, anti-DR5 antibody, anti-CD1d antibody, anti-TIM3 antibody, anti-SLAMF7 antibody, anti-KIR receptor antibody, anti-OX40 antibody, anti-HER2 antibody, anti-ErbB-2 antibody, anti-EGFR antibody, cetuximab, rituximab, trastuzumab, pembrolizumab, radiation therapy, single dose irradiation, fractionated irradiation, focal irradiation, whole organ irradiation, IL-12, IFNα, GM-CSF, chimeric antigen receptor, adoptive transfer T cell, anti-cancer vaccine, and oncolytic virus.

50. The pharmaceutical composition of any one of claims 47 to 49, wherein the first therapy and the at least one additional therapy are administered concomitantly.

51. The pharmaceutical composition of any one of claims 47 to 50, wherein the first therapy is administered simultaneously with the at least one additional therapy.

52. 51. The pharmaceutical composition of any one of claims 47 to 50, wherein the first therapy and the at least one additional therapy are administered sequentially.

53. 53. The pharmaceutical composition of claim 52, wherein the first therapeutic approach is administered prior to the at least one additional therapeutic approach.

54. 53. The pharmaceutical composition of claim 52, wherein the first therapeutic regimen is administered after the at least one additional therapeutic regimen.

55. 53. The pharmaceutical composition of claim 52, wherein the first therapeutic approach is administered before and / or after the at least one additional therapeutic approach.

56. 56. The pharmaceutical composition of any one of claims 47 to 55, wherein the first therapy and the at least one additional therapy are administered in alternation.

57. 49. The pharmaceutical composition of any one of claims 47 to 48, wherein the first therapy and the at least one additional therapy are administered together in a single formulation.

58. A kit for diagnosing, preventing, and / or treating a disease in a subject in need thereof, the system comprising: a) a construct according to any one of claims 1 to 9; b) a recombinant nucleic acid according to any one of claims 10 to 14; c) a genetically engineered cell according to any one of claims 15 to 25 and 27; and / or d) A kit comprising a pharmaceutical composition according to any one of claims 29 to 32.

59. A method for obtaining the construct of claim 1, comprising the steps of: a) identifying multiple T cell receptors (TCRs) associated with a health disorder; b) determining the sequence of the CDR3β present in each of the identified TCRs; c) generating a construct comprising the CDR3β sequence determined in b).

60. 60. The method of claim 59, further comprising identifying one or more cognate antigens that are commonly recognized by the CDR3β sequences.

Images