WO2022066973A1 - Immunotherapy targeting pbk or oip5 antigens - Google Patents

Abstract

The present disclosure provides compositions and methods for targeting a PDZ- binding kinase (PBK) antigen and/or an Opa-interacting protein 5 (OIP5) antigen to, for example, treat or manage cancer. The compositions include, for example, binding proteins that are capable of binding to an antigemHLA complex. Also provided are polynucleotides and transgene constructs encoding binding proteins, such as a T cell receptor or a chimeric antigen receptor. Such transgene constructs can be transduced into an immune cell, such as a T cell, and used in immunotherapy in a subject having or at risk for a cancer associated with PBK and/or OIP5 expression. The present disclosure also provides immunogenic compositions comprising PBK and/or OIP5 peptide antigens, and related uses.

Description

IMMUNOTHERAPY TARGETING PBK OR OIP5 ANTIGENS STATEMENT REGARDING SEQUENCE LISTING The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 360056_47001WO_SEQUENCE_LISTING.txt. The text file is 56.6 KB, was created on September 23, 2021, and is being submitted electronically via EFS-Web. BACKGROUND Various malignancies including pediatric brain and spinal cancers have poor clinical outcomes, and recurrent disease following standard-of-care therapy is associated with extremely poor prognosis. Standard treatment regimens have involved surgery, radiation therapy, and/or chemotherapy; however, these approaches are not curative for all patients and can be accompanied by toxicities. Accordingly, new strategies are needed for targeting difficult-to-treat cancers. The present disclosure addresses such needs, and further provides other related advantages. BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1G show killing of target autologous lymphoblastoid cell line (LCL) by antigen-specific CD8⁺ T cells according to the present disclosure. (A-C) Percent lysis of peptide-pulsed target cells (autologous LCL with 1 μg/ml per peptide) in a 4-hour labeled chromium (⁵¹Cr) release assay ("CRA"; 20:1 donor effector cell:target cell ("E:T") ratio), minus background by CD8⁺ donor T cells from three different donors. † indicates signals that were later determined to be PBK/HLA-A*02 - specific lines. § indicates signals that were later determined to be PBK/HLA-B*07:02- specific lines. Wells with lysis >20% were expanded and re-tested. (D) CD14⁺ monocytes were isolated from donor PBMCs from Donor 1 and allowed to differentiate into monocyte-derived DCs. At Day -1/Day 0, CD8⁺ T cells were isolated and DCs were pulsed with peptide (control or experimental, as described herein). T cells and DCs were co-cultured at a 10:1 T cell:DC ratio. At Day 10, donor-derived CD8⁺ T cells were restimulated with peptide-pulsed autologous PBMCs. At Day 20 (shown), specific killing activity of T cells was measured using a split-well CRA. Ten (10) lines were specific for test peptides (PBK peptide = 6 lines; OIP5 peptide = 4 lines). From left to right in each triplet of bars: no peptide %, control peptides %, Experimental peptides %. (E) Killing activity of T cells following stimulation with peptide-pulsed autologous PBMCs. The eleven (11) PBK/HLA-A*02-specific cell lines shown responded to the entire PBK peptide subpool. The donor from which each line was derived is indicated. From left to right in each triplet of bars: po peptide, PBK peptide pool, irrelevant pool. (F) Killing activity of PBK/A*02:01-specific T cell lines against target cells pulsed with 9-mer peptides (peptides #208-#212). From left to right in each sextet of bars: no peptide, peptide #208, peptide #209, peptide #210, peptide #211, peptide #212. (G) Killing activity of T cells from Donor 1 following a second stimulation with peptide-pulsed matched PBMCs. Twenty (20) T cell lines retained function post Rapid Expansion Protocol (REP). Nine (9) lines were specific for control peptides and confirmed to be HA-1 specific. From left to right in each triplet of bars: no peptide %, control peptides %, CTA/H3.3%%.

Figure 2A shows: (top) specific lysis of target cells (control or pulsed with PBK peptides as indicated) by PBK-specific T cell lines using a CRA; (bottom) sequence information, HLA-type, and predicted HLA-binding of two (2) PBK peptide antigens. Peptide SLPLDENMTV (SEQ ID NO.:6) corresponds to #210 in the CRA graph, and peptide MMTLSIPHI (SEQ ID NO.:3) corresponds to #212 in the CRA graph. From left to right in each sextet of bars: PBK pool, no peptide, peptide #208, peptide #209, peptide#210, peptide #211, peptide #212.

Figure 2B shows specific lysis of target cells (control or pulsed with PBK peptides as indicated) by the eleven (11) PBK-specific T cell lines shown in Figure IE. Peptide SLPLDENMTV (SEQ ID NO.:6) corresponds to #210, and peptide MMTLSIPHI (SEQ ID NO:3) corresponds to #212; both nonamers are predicted to bind HLA. From left to right in each sextet of bars: no peptide, peptide #208, peptide #209, peptide#210, peptide #211, peptide #212.

Figures 3A-3B show lysis of target cells (pulsed with a PBK peptide having the amino acid sequence MMTLSIPHI (SEQ ID NO.:3)) by the indicated PBK-specific T cell lines in a CRA titration series. In Figures 3 A and 3B, the broken arrow indicates the line corresponding to Clone 9.3. Figure 3C shows killing activity of T cells following stimulation with peptide-pulsed autologous PBMCs. The seven (7) PBK/HLA- B*07:02-specific lines shown responded to the entire PBK peptide subpool. The donor from which the line was derived is indicated. From left to right in each triplet of bars: no peptide, PBK peptide pool, irrelevant pool. Figure 3D shows killing activity of T cells following stimulation with peptide-pulsed autologous PBMCs ("no peptide" control or pulsed with PBK peptides as indicated) by seven (7) PBK/HLA-B *07:02- specific lines. Peptide #362 has the sequence RPSAAHIVEA (SEQ ID NO.: 10); peptides #361 and #363-366 are other PBK peptides; all are predicted to bind HLA- B*07:02. From left to right in each group of seven bars: no peptide, peptide #361, peptide #362, peptide #363, peptide #364, peptide #365, peptide #366. Figure 3E shows lysis of target cells (pulsed with a PBK peptide having the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10)) by the indicated PBK-specific T cell lines in a CRA titration series. The broken arrow indicates the line corresponding to Clone 15.2. Figure 3F shows lysis of ATRT-311 (atypical teratoid rhabdoid tumor) pulsed with PBK peptide RPSAAHIVEA (SEQ ID NO.: 10) (control, with peptide, with IFN-y, or with peptide and IFN-y) by the indicated five (5) PBK-specific T cell lines. From left to right in each quartet of bars: no treament, + peptide, +IFNy, +IFNy + peptide.

Figure 4 shows (top) specific lysis of target cells (control or pulsed with PBK peptides as indicated) by donor OIP5-specific T cell lines using a CRA; (bottom) sequence information, HLA-type, and predicted HLA-binding of two (2) OIP5 peptide antigens. Peptide SLMKILSEV (SEQ ID NO.:4) corresponds to #216 in the CRA graph; peptide KSLMKILSEV (SEQ ID NO.: 5) corresponds to #223 in the CRA graph. From left to right within each group of bars (i.e., the group of bars above "6" on the x- axis, the group above " 15", the group above "17", the group above " 18"): OIP5 pool, no peptide, peptide #216, peptide #217, peptide #218, peptide #219, peptide #220, peptide #221, peptide #222, peptide #223, peptide #224, peptide #225.

Figures 5A and 5B show lysis of target cells pulsed with OIP5 peptide ((A) = SEQ ID NO:4); (B) = SEQ ID NO.:5) by the indicated OIP5-specific T cell lines in a titration series CRA. Figure 6 relates to an exemplary HLA-A*0201⁺ patient with high-grade glioma. Shown are clinical characteristics of patient. sET = supratentorial embryonal tumor. PNET = primary neuroectodermal tumor. EFS = event free survival. OS = overall survival. The overall survival probability of PNET⁺ patients is low (see e.g. Hwang el al., J. Clin. Oncol. 36(34):3388-3395 (2018)). The exemplary patient was misdiagnosed. HGGs within this cohort are resistant to most chemotherapies, drugs, and radiation. A tumor cell line ("PBT-05"; research.fhcrc.org/olson/en/btrl/org) was generated using tumor cells from this patient.

Figure 7 provides flow cytometry data showing that PBT-05 tumor cells have low expression of MHC Class I. Left-hand panels: MHC Class I-PE. Bottom panels: staining following 48h treatement with IFN-y.

Figures 8A and 8B show killing of (A) T cell donor-derived lymphoblastoid cells (LCL) with ("+#212") or without ("No pep") PBK peptide MMTLSIPHI (SEQ ID NO:3) and (B) PBT-05 tumor cells (control, with peptide, with IFN-y, or with peptide and IFN-y) by PBK-specific T cell clones of the present disclosure. In (B), from left to right within each quartet of bars: PBT-05, PBT-05 + peptide #212, PBT-05 + IFN-y, PBT-05 + IFN-y + peptide #212.

Figures 8C and 8D show killing of (C) T cell donor-derived lymphoblastoid cells (LCL) with ("+ peptide 212") or without ("No peptide") PBK peptide MMTLSIPHI (SEQ ID NO.:3) and (D) four (4) different HLA-A*02:01⁺ PBT cell lines that endogenously process and present peptide, by PBK-specific T cell by clone 9.3. Data are from 4-hour chromium release assays with a 20: 1 E:T ratio. In (D), PBT lines were untreated or were treated with IFN-y for 48 hours prior to T cell addition, as some lines expressed low levels of MHC Class I.

Figures 8E and 8F show killing, by PBK-specific T cell by clone 15.2, of (E) T cell donor-derived lymphoblastoid cells (LCL) with ("+ peptide 362") or without ("No peptide") PBK peptide RPSAAHIVEA (SEQ ID NO.: 10) and (F) two (2) different HLA-B*07:02+ PBT cell lines that endogenously process and present peptide. Data are from 4-hour chromium release assays with a 20:1 E:T ratio. In (F), PBT lines were untreated or were treated with IFN-y for 48 hours prior to T cell addition, as some lines expressed low levels of MHC Class I. Figure 8G shows PBK mRNA expression, as measured by qPCR, in several PBT (Primary Brain Tumor), T-ALL (T-cell acute lymphoblastic leukemia), T lymphocyte, ALCL (anaplastic large cell lymphoma), AML (acute myeloid leukemia), DLBCL (diffuse large B-cell lymphoma), B-ALL (B-cell acute lymphoblastic leukemia), B lymphocyte, and Burkitt’s Lymphoma lines. LCL cells were assayed as a negative control and used as a baseline to calculate fold change in expression.

Figure 8H shows PBK mRNA expression, as measured by RNASeq, in brain tumor tissues from atypical teratoid rhabdoid tumor (ATRT), medulloblastoma (Med), high-grade glioma (HGG), pineoblastoma (Pineo), and ependymoma (Epd).

Figure 81 shows exemplary PBK peptides and predicted HLA binding.

Figures 9A and 9B show killing of (A) T cell donor-derived lymphoblastoid cells (LCL) with or without OIP5 peptides (SEQ ID NOs.:4 and 5) and (B) PBT-05 tumor cells (control, with peptide, with IFN-y, or with peptide and IFN-y) by OIP5- specific T cell clones of the present disclosure. In (B), from left to right within each quartet of bars: PBT-05, PBT-05 + peptide #216/peptide #223, PBT-05 + IFN-y, PBT- 05 + IFN-y + peptide #216/peptide #223.

Figures 10A and 10B show killing of HLA-A*02:01⁺ PBT cell line GBM-511 treated with IFN-y and T cells in a killing assay using the Incucyte® bioimaging platform at the indicated effectortarget ratios.

Figures 11A-11H show killing of target tumor cells by PBK-specific T cell clone 9.3 and corresponding absolute target tumor cell counts. Percent lysis of peptide- pulsed target cells in a 48-hour killing assay (5: 1 donor effector celktarget cell ("E:T") ratio), minus background by CD8+ donor T cells, was determined using three biological replicates, each with four technical replicates. Assays were conducted with the experimental conditions "Clone 9.3" and "Clone 9.3 + IFN-y" ("IFNg treated" in 11B and 1 ID), and control conditions "No T cells" ("Untreated" in 1 IB and 1 ID) and "No T cells + IFN-y". Target cells tested include PBT-05 (11 A, 1 IB), ATRT 310 (11C, 1 ID), GBM (1 IE-1 IF), and as a control, HLA-A*02:01 -negative cell line GBM-110 (11G- 11H).

Figure 12A shows killing of lymphoblastoid cell lines (LCLs) and donor- derived cells pulsed with peptide MMTLSIPHI (SEQ ID NO.: 3) by PBK-specific T cell clone 9.3. Figure 12B shows killing of LCLs pulsed with peptide MMTLSIPHI (SEQ ID NO.: 3) or pulsed with a peptide variant containing distinct alanine substitutions, by PBK-specific T cell clone 9.3.

Figures 13A-13C show mRNA sequencing data analyzed using ANOVA (oneway analysis of variance). F values comparing normal brain tissue and primary brain tumor cohorts were calculated for PBK (13B, F = 1888.341), OIP5 (13C, F = 708.375), and, to demonstrate an exemplary low F value, SPAG9 (13A, F = 49.263).

Figure 14 shows killing, by PBK-specific T cell clone 9.3, of adult and pediatric lymphoma target cells pulsed with peptide MMTLSIPHI (SEQ ID NO. : 3). Adult lymphoma cell lines include JeKo-1 (Female, 78yo, Mantle cell lymphoma), NU-DUL- 1 (Male, 43yo, non-Burkitts type, early B-cell), and MA VER- 1 (Male, 77yo, Mantle cell lymphoma). Pediatric lymphoma cell lines include SU-DHL-1 (Male, lOyo, diffuse histiocytic lymphoma) and SR (Male, 1 lyo, undetermined origin). LCL cells were included as a control. From left to right, the pairs of bars ("No pep / With pep") correspond to: LCL, JeKo-1, NU-DUL-1, MAVER-1, SU-DHL-1, SR.

Figure 15 shows killing, by PBK-specific T cell clone 9.3, of adult and pediatric leukemia target cells pulsed with peptide MMTLSIPHI (SEQ ID NO.: 3). Adult lymphoma cell lines ML-2 (Male, 26yo, AML), OCI-AML3 (Male, 57yo, AML), TF-1 (Male, 35yo, erythroleukemia). Pediatric lymphoma cell lines include THP-1 (Male, lyo, AML), HSB-2 (Male, 11.5yo, ALL). LCL cells were included as a control. From left to right, the pairs of bars ("No pep / With pep") correspond to: LCL, M-2, OCI- AML3, TF-1, THP-1, HSB-2.

Figure 16A-16B shows killing activity against OIP5-specific T cell lines following stimulation with autologous PBMCs pulsed with (16A) OIP5 peptide #223 (KSLMKILSEV; SEQ ID NO.:5), irrelevant peptide, or no peptide; and (16B) OIP5 peptide #216 (SLMKILSEV; SEQ ID NO.:4), OIP5 peptide #223 (KSLMKILSEV; SEQ ID NO.:5), other OIP5 peptides, or no peptide. In Figure 16A, from left to right in each triplet of bars (i.e., at DI #10, D2 #4, etc.): no peptide, OIP5, irrelevant pool. In Figure 16B, from left to right in each quartet of bars (i.e., at DI #10, D2 #4, etc.): no peptide, peptide #216 (9mer), peptide #223, other OIP5 peptides. Figure 17 shows lysis of target cells pulsed with OIP5 peptide #216 (SLMKILSEV; SEQ ID NO.:4) by the indicated PBK-specific T cell lines in a CRA titration series.

Figures 18A-18B show killing of target cell lines (18A) PBT-05 and (18B) GBM-511 pulsed with OIP5 peptide #216 (SLMKILSEV; SEQ ID NO.:4) with or without IFN-y by the indicated OIP5-specific T cell lines. Killing of LCLs pulsed with OIP5 peptide #216 (SLMKILSEV; SEQ ID NO.:4) or no peptide is shown in parallel. In Figure 18A, from left to right in each group of seven bars (i.e., at LCL, LCL+pep, PBT-05, PBT-05+IFNy): D2 4.2, D2 6.1, D2 15.2, D2 17.5, D2 28.1, D3 FS27, D3 FS37. In Figure 18B, from left to right in each group of four bars (i.e., at LCL, LCL+pep, GBM-511, GBM-511+IFNy): D2 4.2, D2 28.1, D3 FS27, D3 FS37.

Figure 19 shows killing of LCLs pulsed with OIP5 peptide #216 (SLMKILSEV; SEQ ID NO.:4) or a peptide variant containing alanine substitutions as indicated, by PBK-specific T cell clone 9.3.

Figure 20 shows killing of target cells by the indicated OIP5-specific T cell lines. Target cells were pulsed with OIP5 peptide #216 (SLMKILSEV; SEQ ID NO.:4) and include LCLs and fibrolast cells from both male and female HLA-A2-matched donors. From left to right in each triplet of bars (i.e., at LCL, LCL + pep, Male A2+, etc.): D2 4.2, D2 28.1, D3 FS37.

Figure 21 shows OIP5 mRNA expression, as measured by qPCR, in several PBT (Primary Brain Tumor), T-ALL (T-cell acute lymphoblastic leukemia), T lymphocyte, ALCL (anaplastic large cell lymphoma), AML (acute myeloid leukemia), DLBCL (diffuse large B-cell lymphoma), B-ALL (B-cell acute lymphoblastic leukemia), B lymphocyte, and Burkitt’s Lymphoma lines. LCL cells were assayed as a negative control and used as a baseline to calculate fold change in expression.

Figures 22A and 22B show cytotoxicity of Clone 9.3 (with or without exogenous IFN-y) against GBM511 cells in a 24h killing assay (5 : 1 E:T). In Figure 22A, the higher curve is Clone 9.3 with IFN-y

Figure 23 provides additional cell lysis data from OIP5-specific T cell lines against target cells pulsed with no peptide, OIP5 peptide pool, or irrelevant peptide pool. From left to right in each triplet of bars (i.e., at 4, 6, 15, 17, 18, 28): no peptide, OIP5 peptide pool, irrelevant pool.

Figure 24 shows a scheme for an experiment investigating the ability of T cells expressing a T cell receptor comprising the variable domains of Clone 9.3 (or comprising the variable domains of a sequence-engineered variant Clone 9.3), as disclosed herein, to treat brain cancer in a mouse xenograft model.

DETAILED DESCRIPTION

The present disclosure generally relates to binding proteins specific for PBK or OIP5 antigens, and related compositions and uses. By way of background, immunotherapies such as T cell immunotherapies can be highly effective against hematologic malignancies, as exemplified by the impressive results of clinical trials evaluating therapeutic T cells genetically modified with chimeric antigen receptors (CARs) targeting CD 19 for acute lymphoblastic leukemia (ALL). However, CAR T cells can only target cell surface-expressed molecules, and identifying target cell surface antigens with disease-specific expression pattern can be a challenge. Alternative immunotherapy strategies include administration of T cells naturally expressing, or modified to express, T cell receptors (TCRs, including engineered TCRs) specific for peptide antigens derived from endogenous cell proteins and presented on the cell surface in association with HLA molecules.

Selecting an appropriate antigen target is important for effective T cell immunotherapy. For example, some antigens may have relatively low tumor-specific expression, and targeting such antigens may lead to undesirable killing of normal tissue and/or undesirably high levels of immune activity. Accordingly, ideal T cell antigen targets for immunotherapy are highly selectively expressed on tumors, and can be presented by widely expressed HLA alleles that are highly prevalent in the disease population.

The present disclosure provides antigens from PDZ-binding kinase (PBK) and Opa-interacting protein 5 (OIP5) that were surprisingly capable of eliciting an immune cell response and are expressed by certain cancers, including brain and spinal cancers. The present disclosure also provides immune cells that are capable of binding and promoting the killing of cells presenting such antigens. The present disclosure further provides binding proteins that are capable of binding to a peptide containing a PBK or OIP5 antigen (e.g., in complex with an HLA molecule, such as HLA-A*02:01 or HLA- B*07:01), and to isolated polynucleotides encoding the same. In certain embodiments, there are provided modified immune cells that comprise polynucleotides encoding binding proteins that are capable of binding to a peptide containing a PBK or OIP5 antigen of this disclosure, and methods of treating disease (e.g, cancer) using such modified immune cells as described herein.

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure.

In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g, "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include", "have", and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

In addition, it should be understood that the individual compounds, or groups of compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure. The term "consisting essentially of is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, or modules) "consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy -terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 1 0%, 8%, 6%, 5%, 4%, 3%, 2% or 1% ) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1% ) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid and non-naturally occurring amino acid polymers.

An "immunoglobulin superfamily binding protein" or "IgSF binding protein" refers to a cell surface or soluble protein that is involved in recognition of, binding to, and/or adhering to a target (e.g., cell, antigen, soluble factor) and comprises one or more immunologlobulin domain and/or immunoglobulin fold. IgSF binding proteins of the present disclosure comprise an antigen-recognition domain, such as is found in an antibody or antigen-binding fragment thereof (of any isotype), or in a T cell receptor or antigen-binding fragment thereof (e.g., an IgV region). IgSF proteins can possess a native binding specificity for a target, or can be engineered to have or enhance binding specificity and/or affinity for the target. Other IgSF proteins for use in the present disclosure include, for example, proteins comprising an IgCl domain, and IgC2 domain, an Igl domain; a killer-cell immunoglobulin-like receptor (KIR); a leukocyte immunoglobulin-like receptor (LILR); a cell adhesion molecule (CAM); and combinations of these. As used herein, an "immune system cell" means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, meagakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4⁺ T cell, a CD8⁺ T cell, a CD4- CD8 - double negative T cell, a

T cell, a regulatory T cell, a stem cell memory T cell, a natural killer cell, a natural killer T cell, and a dendritic cell. Macrophages and dendritic cells can be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

A "T cell" or "T lymphocyte" is an immune system cell that matures in the thymus and produces T cell receptors (TCRs). T cells can be naive ("TN"; not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased or no expression of CD45RO as compared to TCM (described herein)), memory T cells (TM) (antigen experienced and long-lived), including stem cell memory T cells, and effector cells (antigen-experienced, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, expresses CD62L, CCR7, CD28, CD95, CD45RO, and CD 127) and effector memory T cells (TEM, express CD45RO, decreased expression of CD62L, CCR7, CD28, and CD45RA). Effector T cells (TE) refers to antigen-experienced CD8⁺ cytotoxic T lymphocytes that express CD45RA, have decreased expression of CD62L, CCR7, and CD28 as compared to TCM, and are positive for granzyme and perforin. Helper T cells (TH) are CD4⁺ cells that influence the activity of other immune cells by releasing cytokines. CD4⁺ T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals. T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4⁺ CD25⁺ (Foxp3⁺) regulatory T cells and Tregl7 cells, as well as Tri, Th3, CD8⁺CD28‘, and Qa-1 restricted T cells.

"T cell receptor" (TCR) refers to an immunoglobulin superfamily member (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e. g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 433, 1997) capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having α and β chains (also known as TCR α and TCRβ, respectively), or y and 6 chains (also known as TCRy and TCRδ, respectively). In certain embodiments, polynucleotides encoding binding proteins of this disclosure, e.g., TCRs, can be codon optimized to enhance expression in a particular host cell, such as a cell of the immune system, a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten et al., Clin. Immunol. 119: 135, 2006). Exemplary T cells include CD4⁺ T cells, CD8⁺ T cells, and related subpopulations thereof (e.g., naive, central memory, effector memory).

Like certain other immunoglobulins (e.g., antibodies), the extracellular portion of TCR chains (e.g., a-chain, β-chain) contain two immunoglobulin domains, a variable domain (e.g., a-chain variable domain or V_α chain variable domain or Vp; typically amino acids 1 to 116 based on Kabat numbering (Kabat et al., " Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5^ ed.) at the N-terminus, and one constant domain (e.g., a-chain constant domain or C_α, typically 5 amino acids 117 to 259 based on Kabat, β-chain constant domain or Cp typically amino acids 117 to 295 based on Kabat) adjacent the cell membrane. Also, like antibodies, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al, EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). The source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit, or other mammal. The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR a-chain or β-chain (or y chain and 6 chain for y6 TCRs; an antibody heavy or light chain)) that is involved in binding of the immunoglobulin superfamily binding protein (e.g., TCR, antibody) to antigen. The variable domains of the a-chain and β-chain ( Vα and VP, respectively) of a native TCR generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. The Vα domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J); the Vβ domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (V-D-J). A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs that bind a particular antigen may be isolated using a Vα or Vβ domain from a TCR that binds the antigen to screen a library of complementa Vryα or Vβ domains, respectively.

The terms "complementarity determining region," and "CDR," are synonymous with "hypervariable region" or "HVR," and are known in the art to refer to sequences of amino acids within immunoglobulin (e.g., TCR) variable regions, which confer antigen specificity and/or binding affinity and are separated from one another by framework regions. In general, there are three CDRs in each TCR a-chain variable region (aCDRl, aCDR2, aCDR3) and three CDRs in each TCR β-chain variable region (PCDR1, PCDR2, PCDR3). In the case of antibodies (except for heavy-chain-only antibodies), both heavy chain and light chain variable regions are present, and each of these comprises three CDRs (CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, CDRL3).

In TCRs, CDR3 is thought to be the main CDR responsible for recognizing processed antigen. Typically, CDR1 and CDR2 mainly interact with the MHC. CDR1 and CDR2 are encoded within the variable gene segment of a TCR variable region-coding sequence, whereas CDR3 is encoded by the region spanning the variable and joining segments for Vα, or the region spanning variable, diversity, and joining segments for vp. Thus, if the identity of the variable gene segment of a Vα or Vβ is known, the sequences of their corresponding CDR1 and CDR2 can be deduced. Compared with CDR1 and CDR2, CDR3 is typically significantly more diverse in TCRs and antibodies because of the addition and loss of nucleotides during the recombination process. This is typically the case for CDR3P in TCRs and CDRH3 in antibodies.

TCR and antibody variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, Chothia, Enhanced Chothia, EU, IMGT, and Aho), allowing equivalent residue positions to be annotated and for different molecules to be compared using, for example, ANARCI software tool (2016, Bioinformatics 15:298-300). A numbering scheme provides a standardized delineation of framework regions and CDRs in the variable domains. In certain embodiments, variable domains are numbered according to the IMGT numbering scheme (see, e.g., Lefranc et al., Dev. Comp. Immunol. 27:55, 2003, and imgt.org). In some embodiments, a CDR3 sequence is according to the IMGT junction definition.

In certain embodiments, a TCR is found on the surface of a T cell (or a T lymphocyte) and associates with a CD3 complex. "CD3"is a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., p. 172 and 178, 1999) that is associated with antigen signaling in T cells. In mammals, the complex comprises a CD3y chain, a CD3δ chain, two CD3ε chains, and a homodimer of CD3ζ chains. The CD3y, CD3β, and CD3ε chains are related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD3P, and CD3ε chains are negatively charged, which is believed to allow these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD3P, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine based activation motif or IT AM, whereas each chain has three IT AMs. Without wishing to be bound by theory, it is believed that the ITAMs are important for the signaling capacity of a TCR complex. CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.

As used herein, "TCR complex" refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3y chain, a CD3β chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD3P chain, two CD3ε chains, a homodimer of CD3ζ chains, a TCRy chain, and a TCRP chain.

A "component of a TCR complex", as used herein, refers to a TCR chain (i.e., TCRa, TCRp, TCRy or TCRδ), a CD3 chain (i.e., CD3y, CD3δ, CD3ε or CD3ζ) , or a complex formed by two or more TCR chains or CD3 chains (e.g, a complex of TCRa and TCRP, a complex of TCRy and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3y and CD3ε, or a sub-TCR complex of TCRa, TCRP, CD3y, CD3δ, and two CD3ε chains).

"Chimeric antigen receptor" (CAR) refers to a fusion protein that is engineered to contain two or more naturally occurring amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell. CARs can include an extracellular portion comprising an antigen-binding domain (e.g, obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived or obtained from a TCR specific for a cancer antigen, a scFv derived or obtained from an antibody, or an antigen-binding domain derived or obtained from a killer immunoreceptor from an NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing co-stimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3).220 (2016), Stone et al., Cancer Immunol. Immunother., 63(11): 1163 (2014), and Walseng et al., Scientific Reports 7: 10713 (2017), which CAR constructs and methods of making the same are incorporated by reference herein). In some embodiments, CARs of the present disclosure that specifically bind to an antigen (e.g., in the context of a peptide:HLA complex) comprise a TCR Vα domain and a Vβ domain.

As used herein, "fusion protein" or "fusion polypeptide" refers to a protein that, in a single chain, has at least two distinct domains, sequences, motifs, wherein the domains, sequences, or motifs are not naturally found together (e.g., in the specified arrangement, order, or number, or at all) in a protein. In certain embodiments, a fusion protein comprises at least two distinct domains or motifs that are not naturally found together in a single peptide or polypeptide. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, or the like, or such fusion proteins can be synthesized.

"Antigen" or "Ag" as used herein refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells (e.g., T cells), or both. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen, or that endogenously (e.g., without modification or genetic engineering by human intervention) express a mutation or polymorphism that is immunogenic.

Certain antigens associated with various cancers are known as cancer/testis antigens (CTAs). These antigens have normal expression restricted to male germ cells in the testis (and, in some cases, ovary and trophoblast), but not in adult somatic tissues), but have high expression in cancers. Exemplary CTAs are annotated in the CTdatabase (cta.lncc/br). As disclosed herein, PBK (PDZ-binding kinase; see, e.g., UniProtKB Q96KB5) and OIP5 (OPA-interacting protein 5; see, e.g., UniProtKB 043482) are CTAs.

The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three dimensional structural characteristics, as well as specific charge characteristics. As used herein, the terms "PBK antigen" or "PDZ-binding kinase antigen" refer to a naturally or synthetically produced peptide portion of a PBK protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, up to about 20 to 25 amino acids. The amino acid sequence of human PBK is provided in SEQ ID NO: 1. In some embodiments, a PBK antigen comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, or a variant thereof having one, two, or three amino acid substitutions. In some embodiments, a PBK antigen comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10, or a variant thereof having one, two, or three amino acid substitutions. In some embodiments, "PBK antigen" may be used interchangeably with "PBK peptide" or "PBK peptide antigen" or "PBK antigen peptide".

As used herein, the terms "OIP5 antigen" or "Opa-interacting protein 5" refer to a naturally or synthetically produced peptide portion of a OIP5 protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, up to about 20 to 25 amino acids. The amino acid sequence of human OIP5 is provided in SEQ ID NO:2. In some embodiments, a OIP5 antigen comprises or consists of the amino acid sequence set forth in SEQ ID NO:4 or 5, or a variant thereof having one, two, or three amino acid substitutions. In some embodiments, "OIP5 antigen" may be used interchangeably with " OIP5 peptide" or " OIP5 peptide antigen" or " OIP5 antigen peptide".

In some embodiments, a PBK or OIP5 antigen comprises a length of about 9 amino acids, about 10 amino acids, about 11 amino acids, about 12 amino acids, about 13 amino acids, about 14 amino acids, about 15 amino acids, about 16 amino acids, about 17 amino acids, about 18 amino acids, about 19 amino acids, about 20 amino acids, about 21 amino acids, about 22 amino acids, about 23 amino acids, about 24 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, or more. In some embodiments, a PBK or OIP5 antigen comprises a length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

Principles of antigen processing by antigen presenting cells (APC) (such as dendritic cells, macrophages, lymphocytes or other cell types), and of antigen presentation by APC to T cells, including major histocompatibility complex (MHC)- restricted presentation between immunocompatible (e.g., sharing at least one allelic form of an MHC gene that is relevant for antigen presentation) APC and T cells, are well established (see, e.g., Murphy, Janeway’s Immunobiology (8^th Ed.) 2011 Garland Science, NY; chapters 6, 9 and 16). For example, processed antigen peptides originating in the cytosol (e.g., tumor antigen, intracellular pathogen) are generally from about 7 amino acids to about 11 amino acids in length and will associate with class I MHC (HLA) molecules, whereas peptides processed in the vesicular system (e.g., bacterial, viral) will vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC (HLA) molecules.

"Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to a cell surface of all nucleated cells. MHC class I molecules are heterodimers having a membrane spanning α chain (with three a domains) and a non- covalently associated β2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8⁺ T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4⁺ T cells. Human MHC is referred to as human leukocyte antigen (HLA). HLAs corresponding to "class I" MHC present peptides from inside the cell and include, for example, HLA-A, HLA-B, and HLA-C. Alleles include, for example, HLA A*02:01; HLA-A*03:01; HLA-A*l l:01; HLA- B*07:02; HLA-B*40:01; HLA-B*44:02; or HLA-B *44: 03. HLAs corresponding to "class II" MHC present peptides from outside the cell and include, for example, HLA- DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

As used herein, the term "CD8 co-receptor" or "CD8" means the cell surface glycoprotein CD8, either as an alpha-alpha homodimer or an alpha-beta heterodimer. The CD8 co-receptor assists in the function of cytotoxic T cells (CD8⁺) and functions through signaling via its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21 :630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1 :81- 88, 2004). There are five (5) known CD8 betα chains (see UniProtKB identifier Pl 0966) and a single known CD8 alphα chain (see UniProtKB identifier P01732). "CD4" is an immunoglobulin co-receptor glycoprotein that assists the TCR in communicating with antigen-presenting cells (see, Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002)). CD4 is found on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and includes four immunoglobulin domains (DI to D4) that are expressed at the cell surface. During antigen presentation, CD4 is recruited, along with the TCR complex, to bind to different regions of the MHCII molecule (CD4 binds MHCII P2, while the TCR complex binds MHCII α1/β1). Without wishing to be bound by theory, it is believed that close proximity to the TCR complex allows CD4-associated kinase molecules to phosphorylate the immunoreceptor tyrosine activation motifs (IT AMs) present on the cytoplasmic domains of CD3. This activity is thought to amplify the signal generated by the activated TCR in order to produce various types of T helper cells.

Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. For example, the term "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab'2 fragment. Thus, the term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multi specific, e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof (IgGl, IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

The terms "VL" or "VL" and " VH" or "VH" refer to the variable binding region or domain from an antibody light chain and an antibody heavy chain, respectively. In certain embodiments, a VL is a kappa (K) class (also "VK" herein). In certain embodiments, a VL is a lambda (λ) class. Like TCR variable domains, the variable domains of antibodies comprise CDRs and framework regions (FRs). There are three CDRs in each antibody variable domain (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRHs and CDRLs, respectively). In certain embodiments, an antibody VH comprises four FRs and three CDRs as follows: FR1- HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen-binding site through their respective CDRs.

TCR-mimic antibodies are antibodies (of any isotype e.g., IgG (1, 2, 3, 4), IgE, IgD, IgA, IgM) that are capable of binding to a peptide :MHC complex (/.<?., recognizing the peptide:MHC complex and binding thereto). In some embodiments, TCR-mimic antibodies possess antigen-specific, major histocompatibility complex-compatibility or -restriction similar to that of T-cell receptors. TCR-mimic antibodies may be prepared by the hybridoma methodology described by Kohler et al., Nature 256 :495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). TCR-mimic antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), and Noy et al. Expert Rev. Anticancer Ther. 5(3 ): 523-536 (2005); these techniques are incorporated herein in their entireties), for example. TCR-mimic antibodies may also be obtained using methods disclosed in PCT Publication No. WO 2004/076677A2. Antigenbinding fragments of TCR-mimic antibodies (e.g., a CDR, a VH, a VL, a Fab, a Fd, or the like) are also contemplated.

An" altered domain" or "altered protein" refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild type TCRα chain, TCRβ chain, TCRa constant domain, TCRP constant domain) of at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%).

In certain embodiments, binding proteins according to the present disclosure comprise variant sequences as compared to a reference or wild-type sequence (e.g., a variant TCR CDR3P as compared to the parent or wild-type TCR CDR3P of a T cell clone as described herein). As used herein, a "variant" amino acid sequence, peptide, or polypeptide, refers to a an amino acid sequence (or peptide or polypeptide) having one or two amino acid substitutions, deletions, or insertions as compared to a reference or wild-type amino acid sequence. In certain embodiments, a variant amino acid sequence, peptide, or polypeptide, retains substantially the same functionality (e.g., binding specificity and affinity for a peptide:HLA complex) as the reference or wildtype molecule; for example, a variant TCR CDR3P as disclosed herein retains about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or 100% of the antigen-binding specificity or affinity as compared to the parent or wild-type TCR CDR3p.

Altered domains or altered proteins or derivatives can include those based on all possible codon choices for the same amino acid and codon choices based on conservative amino acid substitutions. For example, the following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (ala; A), serine (ser; S), threonine (thr; T); 2) aspartic acid (asp; D), glutamic acid (glu; E); 3) asparagine (asn; N), glutamine (gin; Q); 4) arginine (arg; R), lysine (lys; K); 5) Isoleucine (ile; I), leucine (L), methionine (met; M), valine (val; V); and 6) phenylalanine (phe; F), tyrosine (tyr; Y), tryptophan (trp; W). (See also WO97/09433 at page 10, Lehninger, Biochemistry, 2^nd Edition, Worth Publishers, Inc., NY, NY, pp. 71-77, 1975; Lewin Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p.8, 1990; Creighton, Proteins, W.H. Freeman and Company 1984). In addition, individual substitutions, deletions or additions that alter, add or delete, a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservative substitutions." As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, polynucleotides, and fragments thereof generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and also to fragments generated by any of ligation, scission, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. Nucleic acid molecules can be either single stranded or double stranded.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such a nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).

As used herein, the terms "recombinant", "engineered", and "modified" refer to a cell, microorganism, nucleic acid molecule, polypeptide, protein, plasmid, or vector that has been modified by introduction of an exogenous nucleic acid molecule, or refers to a cell or microorganism that has been genetically engineered by human intervention — that is, modified by introduction of of a heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive, where such alterations or modifications can be introduced by genetic engineering. Human-generated genetic alterations can include, for example, modifications introducing nucleic acid molecules (which may include an expression control element, such as a promoter) encoding one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material. Exemplary modifications include those in coding regions or functional fragments thereof of heterologous or homologous polypeptides from a reference or parent molecule.

As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s). In certain embodiments, a mutation is a substitution of one or three codons or amino acids, a deletion of one to about 5 codons or amino acids, or a combination thereof.

A "conservative substitution" is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97/09433 at page 10; Lehninger, Biochemistry, 2^nd Edition; Worth Publishers, Inc. NY, NY, pp.71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

The term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule. A "transgene" or "transgene construct" refers to a construct that contains two or more genes operably linked in an arrangement that is not found in nature. The term "operably-linked" (or "operably linked" herein) refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably- linked with a coding sequence when it can affect the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other. In some embodiments, the genes present in a transgene are operably linked to an expression control sequence (e.g., a promoter).

A construct (e.g., a transgene) can be present in a vector (e.g., a bacterial vector, a viral vector) or can be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors can be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that can include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acid molecules to which they are linked (expression vectors). Vectors useful in the compostions and methods of this disclosure are described further herein.

The term "expression", as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process can include transcription, post-transcriptional control, post- transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof.

The term "introduced" in the context of inserting a nucleic acid molecule into a cell, means "transfection", or "transformation", or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule can be incorporated into the genome of a cell (e.g., a chromosome, a plasmid, a plastid, or a mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g, transfected mRNA).

As used herein, "heterologous" or "exogenous" nucleic acid molecule, construct or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but can be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of the heterologous or exogenous nucleic acid molecule, construct or sequence can be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule is added (i.e., not endogenous or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, transduction, electroporation, or the like, wherein the added molecule can integrate into the host genome or exist as extra- chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can be present in multiple copies. In addition, "heterologous" refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity. It will be appreciated that in the case of a host cell that comprises a heterologous polynucleotide, the polynucleotide is "heterologous" to progeny of the host cell, whether or not the progeny were themselves manipulated (e.g., transduced) to contain the polynucleotide. Such a host cell may be referred-to as a "modified" host cell, whether the subject host cell was itself modified to comprise the polynucleotide, or whether an ancestor cell of the subject host cell was modified to comprise the polynucleotide.

As described herein, more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cel 1.

As used herein, the term "endogenous" or "native" refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell. For example, an endogenous control sequence from a first gene (e.g., a promoter, translational attenuation sequences) can be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.

The term "homologous" or "homolog" refers to a molecule or activity found in or derived from a host cell, species or strain. For example, a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene, and can optionally have an altered expression level, a different sequence, an altered activity, or any combination thereof.

"Sequence identity," as used herein, refers to the percentage of amino acid residues in one sequence that are identical with the amino acid residues in another reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The percentage sequence identity values can be generated using the NCBI BLAST 2.0 software as defined by Altschul et al. (1997), Nucl. Acids Res. 25:3389-3402, with the parameters set to default values.

PBK- and OIP5-Binding Proteins and Modified Host Cells

In certain aspects, the present disclosure provides binding proteins that are capable of binding (e.g., specifically binding) to a PBK antigen or an OIP5 antigen, such as in the context of a peptide:HLA complex. In some embodiments, the HLA comprises HLA-A*02:01 and/or the PBK antigen or OIP5 antigen comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs:3-6 and 10.

The terms "PBK-specific binding protein" and "OIP5-specific binding protein," as used herein, respectively refer to a protein or polypeptide (such as, for example, a TCR, scTCR, scTv, CAR, or TCR-mimic antibody or antigen-binding fragmen thereof) that is capable of binding to (e.g., specific binding) to a PBK peptide antigen:HLA complex (including, in some embodiments, wherein an alanine-substituted variant PBK peptide antigen is present in the peptdide antigen:HLA complex) or a OIP5 peptide antigen:HLA complex. In some embodiments, the PBK-specific or OIP5-specific binding protein does not bind a peptide that does not contain the PBK or OIP5 peptide and does not bind to an HLA complex containing such a peptide. A host cell (such as, for example, an immune cell) that encodes and/or expresses a PBK- or OIP5-specific binding protein of this disclosure (i.e., heterologously or otherwise) is, in some contexts, referred to as a "PBK-specific" or "OIP5-specific" cell.

Binding proteins of this disclosure, such as TCRs, scTCRs, CARs, scTvs, and TCR-mimic antibodies, will contain a binding domain that is capable of binding to a target (e.g., peptide:HLA complex). A "binding domain" (also referred to as a "binding region" or "binding moiety"), as used herein, refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein) that possesses the ability to specifically and non-covalently associate, unite, or combine with a target (e.g., an antigenic peptide or peptide:MHC complex). A binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e. complex comprising two or more biological molecules), or other target of interest.

In certain embodiments, a binding protein comprises one or more variable domain from an immungolublin superfamily binding protein. In some embodiments, a binding proteim comprises a T cell receptor (TCR) a-chain variable domain (Vα) and/or a TCR β-chain variable domain (Vβ). In some embodiments, a binding proteim comprises one or more variable domain from a TCR-mimic antibody (see e.g. Kurosawa et al., Sci Reports 9:9827 (2019); Trenevska et al. Front. Immunol. (2017) doi.org/10.3389/fimmu.2017.01001; Dahan & Reiter, Expert Rec. Mol. Med. 14 e6 (2012) doi.org/10.1017/erm.2012.2; Chang et al. Exper Opin Biol Ther 16:979-987 (2016) doi.org/10.1080/14712598.2016.1176138); Noy et al., Expert Rev. Anticancer Ther. 5(3):523-236 (2005)).

Certain binding domains include immunoglobulin variable regions or single chain constructs comprising the same (e.g., single chain TCR (scTCR), scTv, CAR).

In some embodiments, binding comprises specific binding. As used herein in the context of a binding interaction, "specifically binds" or "specific for" refers to an association or union of a binding protein (e.g, TCR receptor) or a binding domain (or fusion protein thereof) to a target molecule with an affinity or K_a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10⁵ M'¹ (which equals the ratio of the on-rate [k_0n]to the off-rate [k_Off] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding proteins or binding domains (or fusion proteins thereof) may be classified as "high affinity" binding proteins or binding domains (or fusion proteins thereof) or as "low affinity" binding proteins or binding domains (or fusion proteins thereof). "High affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a K_a of at least 10⁷ M^-1, at least 10⁸ M^-1, at least 10⁹ M^-1, at least IO¹⁰ M^-1, at least 10¹¹ M^-1, at least 10¹² M^-1, or at least 10¹³ M'¹. "Low affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a K_a of up to 10⁷ M^-1, up to 10⁶ M^-1, up to 10⁵ M'¹. Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10'⁵ M to 10'¹³ M).

In certain embodiments, a binding protein of the present disclosure binds to a PBK- or OIP5 antigen peptide (or a peptide:HLA complex that comprises such a peptide) with a Kd of less than about 10^-8 M, less than about 10'⁹ M, less than about 10" ¹⁰ M, less than about 10'¹¹ M, less than about 10'¹² M, or less than about 10'¹³ M, or with an affinity that is about the same as, at least about the same as, or is greater than at or about the affinity exhibited by an exemplary binding protein provided herein, such as any of the PBK-specific or OlP-specific TCRs provided herein, for example, as measured by the same assay. In certain embodiments, a PBK-specific or OlP-specific binding protein comprises a PBK-specific or OlP-specific immunoglobulin superfamily binding protein or binding portion thereof.

In certain embodiments, a receptor or binding domain may have "enhanced affinity," which refers to selected or engineered receptors or binding domains with stronger binding to a target antigen than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a K_a (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a Kd (dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (koff) for the target antigen that is less than that of the wild type binding domain, or a combination thereof.

A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or binding protein affinities, such as peptide:MHC multimer/tetramer staining, Western blot, ELISA, analytical ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Dolton et al. , Immunology 146: 11-22, 2015, Scatchard et al., Ann. NY Acad. Sci. 51 :660, 1949; Wilson, Science 20295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent; all incorporated herein by reference). Binding domains can also be identified by screening e.g. T cells, B cells, plasma cells, PBMCs, or hybridomas for reactivity against/binding to a PBK or OIP5 antigen as provided herein. For example, these cells or their supernatants may be exposed to antigen presenting cells that express or have been pulsed with an antigen of interest. Binding proteins can also be raised by introducing an antigen of interest into a suitable host, such as a mouse, rabbit, camel, non-human primate, or shark to which the antigen is foreign, then isolating T cells, NK- T cells, NK cells, B cells, splenocytes, plasma cells, or the like, from the host and determining whether the isolated cells express a binding protein specific for the antigen.

In certain embodiments, a binding protein or fusion protein (e.g., TCR, scTCR,

CAR, scTv, TCR-mimic antibody or antigen-binding fragment) of the present disclosure is expressed by a host cell (e.g., by a T cell, NK cell, or NK-T cell heterologously expressing the binding protein or fusion protein), preferably at the cell surface when the binding protein is capable of functioning as a receptor at the cell surface. Avidity of such a host cell for a PBK or OIP5 peptide antigen or PBK or OIP5 peptide antigen:HLA complex can be determined by, for example, exposing the host cell to the peptide, or to a peptide:HLA complex (e.g., organized as a tetramer), or to an antigen-presenting cell (APC) that presents the peptide to the host cell, optionally in a peptide:HLA complex, and then measuring an activity of the host cell, such as, for example, production or secretion of cytokines (e.g., IFN-y; TNFα); intracellular mobilization of calcium in the host cell; increased phosphorylation of T cell signaling proteins; increased expression of host cell signaling or activation components (e.g., CD137 (4-1BB)); proliferation of the host cell; or killing of the APC (e.g., using a labeled-chromium release assay or an IncuCyte® assay). Activity of TCR-mimic antibodies can be assessed using standard antibody assays, such as for example ELISA,

BLI, SPR, an effector function assay such as using a target cell and an immune effector cell (e.g. expressing FcyRs driving expression of a cell activation reporter element), or the like.

The term "functional avidity" refers to a biological measure or activation threshold of an in vitro immune cell (e.g., T cell, NK cell, NK-T cell) response to a given concentration of a ligand (e.g. antigen), wherein the biological measures can include cytokine production (e.g., IFNy production, IL-2 production, etc.), cytotoxic activity, activation, and proliferation. For example, T cells that biologically (immunologically) respond in vitro to a low antigen dose by producing cytokines, being cytotoxic, expressing an activation marker, or proliferating are considered to have high functional avidity, while T cells having lower functional avidity require higher amounts of antigen before an immune response, similar to that obtained by the higher-avidity T cells, is elicited. It will be understood that functional avidity is different from affinity and avidity. Affinity refers to the strength of any given bond between a binding protein and its antigen/ligand. Some binding proteins are multivalent and bind to multiple antigens - in this case, the strength of the overall connection is the avidity.

Numerous correlations exist between the functional avidity and the effectiveness of an immune response. Some ex vivo studies have shown that distinct T cell functions (e.g., proliferation, cytokine production (e.g. as measured or detected using e.g. ELISA, Luminex (e.g. Luminex xMAP®), etc.) can be triggered at different thresholds (see, e.g., Betts et al., J. Immunol. 172:6401, 2004; Langenkamp et al., Eur. J. Immunol. 32:2046, 2002). Factors that can affect functional avidity include (a) the affinity of a TCR for the pMHC-complex, that is, the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 1672511, 2001), (b) expression levels of the TCR and the CD4 or CD8 co-receptors, and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273: 104, 1996), as well as expression levels of molecules that attenuate T cell function and TCR signaling.

The concentration of antigen needed to induce a half-maximum response between the baseline and maximum response after a specified exposure time is referred to as the "half maximal effective concentration" or "EC₅₀". The EC₅₀ value is generally presented as a molar (moles/liter) amount, but it is often converted into a logarithmic value as follows - logio(EC₅₀). For example, if the EC₅₀ equals 1 pM (10‘⁶ M), the logio(EC₅₀) value is -6. Another value used is pEC₅₀, which is defined as the negative logarithm of the EC₅₀ (-logio(EC₅₀)). In the above example, the EC₅₀ equaling 1 pM has a pEC50 value of 6. In certain embodiments, the functional avidity of a binding protein of this disclosure will be a measure of its ability to promote IFNy production by immune cells (e.g., T cells, NK-T cells, NK cells), which can be measured using assays known in the art and/or described herein. "High functional avidity" TCRs or binding domains thereof refer to those TCRs or binding domains thereof having a EC₅₀ of at least 10'⁴ M, at least about 10'⁵ M, or at least about 10'⁶ M.

Also contemplated are fusion proteins comprising a scTCR or scTv of the present disclosure linked to the constant domain of an antibody (e.g., IgG (1, 2, 3, 4), IgE, IgD, IgA, IgM, and variants thereof) or a fragment thereof (e.g., a fragment that, in some embodiments, retains binding to one or more Fc receptors, to Clq, to Protein A, to Protein G, or any combination thereof), and including immunoglobulin heavy chain monomers and multimers, such as Fc dimers; see, e.g., Wong et al., J. Immunol. 198: 1 Supp. (2017). Variant Fc polypeptides comprising mutations that enhance, reduce, or abrogate binding to or by, e.g., FcRn or other Fc receptors, are known and are contemplated within this disclosure.

In some embodiments, a binding protein is provided that is capable of binding to a peptide containing a PDZ-binding kinase (PBK) antigen:HLA complex, wherein the PBK antigen comprises or consists of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3) or RPSAAHIVEA (SEQ ID NO : 10).

In some embodiments, a binding protein is provided that is capable of binding to a MMTLSIPHI (SEQ ID NO:3):HLA-A*02:01 complex. In some embodiments, a binding protein is provided that is capable of binding to a RPSAAHIVEA (SEQ ID NO.: 10):HLA-B*07:02 complex. In other embodiments, a binding protein is provided that is capable of binding to a OPA-interacting protein 5 (OIP5) antigen:HLA complex, wherein the OIP5 antigen comprises or consists of the amino acid sequence SLMKILSEV (SEQ ID NO. :4) or KSLMKILSEV (SEQ ID NO. :5). In some embodiments, a binding protein is provided that is capable of binding to a SLMKILSEV (SEQ ID NO:4):HLA-A*02:01 complex. Exemplary PBK-specific binding proteins include TCR 9.3 and TCR 15.2. These TCRs are also referred-to herein by reference to the T cell clones from which they were obtained, i.e. Clone 9.3 and Clone 15.2, respectively. In some embodiments, a binding protein comprises one or more CDR and/or one or more variable domain, or a functional variant thereof, from TCR 9.3 (specific for MMTLSIPHI (SEQ ID NO.:3)). In some embodiments, a binding protein comprises one or more CDR and/or one or more variable domain, or a functional variant thereof, from TCR 15.2 (specific for RPSAAHIVEA (SEQ ID NO : 10)).

Exemplary OIP5-specific binding proteins include TCR 4.2 and TCR 28.1. These TCRs are also referred-to herein by reference to the T cell clones from which they were obtained, i.e. Clone 4.2 and Clone 28.1, respectively. In some embodiments, a binding protein comprises one or more CDR and/or one or more variable domain, or a functional variant thereof, from TCR 4.2 (specific for SLMKILSEV (SEQ ID NO.:4)). In some embodiments, a binding protein comprises one or more CDR and/or one or more variable domain, or a functional variant thereof, from from TCR 28.1 (specific for SLMKILSEV (SEQ ID NO.:4)).

Tables 1-4 below provide certain features of PBK-specific TCRs 9.3 (and variants) and 15.2, and of OIP5-specific TCRs 4.2 and 28.1. Table 1 provides antigenspecificity, an HLA with which the antigen is compatible, and gene usage of the TCRs. Table 2 provides CDR amino acid sequences (IMGT junction definition). Table 3 provides variable domain amino acid sequences. Table 4 provides variable domain amino acid sequences of certain engineered variants of TCR 9.3.

Table 1.

*non-limiting, means simply that the indicated peptide is capable of complexing with the indicated HLA.

Table 2.

Table 3.

Table 4. Vα

In some embodiments, an isolated binding protein is provided that is capable of binding to a PDZ-binding kinase (PBK) peptide antigen:HLA complex, wherein the PBK peptide antigen comprises or consists of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3), and wherein, optionally, the binding comprises specific binding. In some embodiments, the HLA comprises HLA-A*02:01.

In other embodiments, an isolated binding protein is provided that is capable of binding to a PDZ-binding kinase (PBK) peptide antigen:HLA complex, wherein the PBK peptide antigen comprises or consists of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10), and wherein, optionally, the binding comprises specific binding. In some embodiments, the HLA comprises HLA-B*07:02.

In certain embodiments, a binding protein comprises an immunoglobulin superfamily variable domain. In some embodiments, a binding protein comprises a TCR a-chain variable domain ( Vα) and/or a TCR β-chain variable domain (VP). In some embodiments, a binding protein comprises a heavy chain variable domain (VH) and/or a light chain variable domain (VL) of a TCR-mimic antibody.

In certain embodiments, a binding protein comprises: (i) the amino acid sequence of SEQ ID NO.:34, wherein SEQ ID NO.:34 is optionally a complementarity determining region (CDR3)a; and/or (ii) the amino acid sequence of SEQ ID NO.:37, wherein SEQ ID NO.:37 is optionally a CDR3p. In certain further embodiments, the binding protein comprises: (iii) the amino acid sequence of SEQ ID NO.: 32, wherein SEQ ID NO.:32 is optionally a CDRla; (iv) the amino acid sequence of SEQ ID NO.:33, wherein SEQ ID NO.:33 is optionally a CDR2a; (v) the amino acid sequence of SEQ ID NO.:35, wherein SEQ ID NO.:35 is optionally a CDR1P; and/or (vi) the amino acid sequence of SEQ ID NO.:36, wherein SEQ ID NO.:36 is optionally a CDR2p.

In certain embodiments, a binding protein comprises: (1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:32- 34, respectively; and (2) a TCR Vβ comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:35-37, respectively.

In certain embodiments, a binding protein comprises: (a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs.:56, 64, 66, and 67; and/or (b) a TCR VP amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs.:57 and 65.

In certain embodiments, a binding protein comprises the framework amino acid sequence set forth in any one of SEQ ID NOs. : 19-31, or any combination of these.

In certain embodiments, a binding protein comprises the TCR Vα and TCR VP amino acid sequences set forth in SEQ ID NOs.: (i) 56 and 57, respectively; (ii) 56 and 65, respectively; (iii) 64 and 57, respectively; (iv) 64 and 65, respectively; (v) 66 and 57, respectively; (vi) 66 and 65, respectively; (vii) 67 and 57, respectively; or (viii) 67 and 65, respectively.

In certain embodiments, a binding protein comprises the amino acid sequences set forth in SEQ ID NOs.: (i) 11 and 12, respectively; (ii) 13 and 12, respectively; (iii) 15 and 12, respectively; (iv) 16 and 12, respectively; (v) 11 and 14, respectively; (vi) 13 and 14, respectively; (vii) 15 and 14, respectively; or (viii) 16 and 14, respectively.

In certain embodiments, a binding protein comprises: (i) the amino acid sequence of SEQ ID NO.:40, wherein SEQ ID NO.:40 is optionally a complementarity determining region (CDR3)a; and/or (ii) the amino acid sequence of SEQ ID NO.:43, wherein SEQ ID NO.:43 is optionally a CDR3p. In certain further embodiments, the binding protein comprises: (iii) the amino acid sequence of SEQ ID NO.: 38, wherein SEQ ID NO.:38 is optionally a CDRla; (iv) the amino acid sequence of SEQ ID NO.:39, wherein SEQ ID NO.:39 is optionally a CDR2a; (v) the amino acid sequence of SEQ ID NO.:41, wherein SEQ ID NO.:41 is optionally a CDR1P; and/or (vi) the amino acid sequence of SEQ ID NO.:42, wherein, optionally, SEQ ID NO.:42 is optionally a CDR2p.

In certain embodiments, a binding protein comprises: (1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:38- 40, respectively; and (2) a TCR Vβ comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:41-43, respectively.

In certain embodiments, a binding protein comprises: (a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:58; and/or (b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO : 59.

In certain embodiments, a binding protein comprises the TCR Vα and TCR VP amino acid sequences set forth in SEQ ID NOs.:58 and 59, respectively.

Engineering in TCR variable domain framework regions has been shown to improve protein expression, without reducing or materially reducing binding function, in some instances (Thomas et al., Nature Communications 10:4451 (2019); doi.org/10.1038/s41467-019-12441-w; which framework mutations are incorporated herein by reference). Such mutations can be employed, optionally in combination with cysteine mutations (e.g. T48C (Ca) and S79C or S57C (CP)) in the TCR constant regions, to potentially improve expression and function.

Amino acids associated with "dominant" (vs. endogenous TCR) expression of engineered TCRs include:

• Vα: o In framework region 1, a T at IMGT position 5, a Q at IMGT position 8, a V at IMGT position 19, a T at IMGT position 20, and/or a T at IMGT position 24; o In framework region 2, a L at IMGT position 39, a M at IMGT position 50, and/or a R at IMGT position 55; and o In framework region 3, a A at IMGT position 66, a S at IMGT position 86, and a L at IMGT position 96.

. VP: o In framework region 1, a R at IMGT position 9 and/or a Y at IMGT position 10; and o In framework region 2, a Q at IMGT position 43. Thus, introducing one or more of the above amino acids into a TCR V-region at the indicated position(s) (if not already present in the native amino acid sequence) can generate a variant with potentially improved expression and optionally function.

In certain embodiments, a binding protein comprises: (i) a CDR3P according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; (ii) a CDR3α according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; (iii) a Vα domain having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) amino acid identity to the Vα domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; or (iv) a Vβ domain having at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) amino acid identity to the VP domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3, provided that (a) at least three or four of the CDRs have no mutations; (b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and (c) the encoded binding protein retains its ability to bind to a peptide comprising a MMTLSIPHLHLA complex.

In further embodiments, a binding protein further comprises a CDRip, a CDR2P, a CDRla, or a CDR2a according to a TCR of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a TCR of a T cell from Line 34 or 40 from Donor 2, or according to a TCR of a T cell from line 4, 11, or 16 from Donor 3.

In some embodiments, a binding protein comprises: (i) a CDR3P according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; (ii) a CDR3 α aacording to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; (iii) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; or (iv) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5, provided that (a) at least three or four of the CDRs have no mutations; (b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and (c) the binding protein retains its ability to bind to a peptide comprising a SEQ ID NO.: 10:HLA complex. In further embodiments, the encoded binding protein further comprises a CDRip, a CDR2P, a CDRla, or a CDR2a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5.

TCRs and T cell clones of presently disclosed PBT- or OIP5-specific T cell lines are identifiable, and have sequences which are determinable, using known methods. See, e.g., Bleakley et al., Blood 115:4923-4933, 2010; Warren et al., Blood 91(6):2197-2207 (1998); Walter et al., N. Engl. J. Med. 333(16): 1038-1044; PCT Publication No. WO 2018/058002 (Example 1), which methods and related reagents are incorporated herein by reference. For example, full TCR regions can be identified using 5' first-strand complementary DNA (cDNA) amplification and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using a SMARTer RACE cDNA Amplification Kit (Clontech Laboratories). Briefly, cDNA is synthesized from RNA using 5' CDS Primer A, SMARTer IIA oligo, and SMARTScribe Reverse Transcriptase. The cDNA is then used to perform a RACE- PCR reaction using Phusion High-Fidelity DNA Polymerase and gene-specific primers for the TCR a (hTCR_Calpha-R 5'- CAGCCGCAGCGTCATGAGCAGATTA-3'(SEQ ID NO: 7)) or TCR p chain (hTCR Cbl-R 5'- CCACTTCCAGGGCTGCCTTCAGAAATC-3' (SEQ ID NO:8) and hTCR_Cb2-R 5'- TGGGATGGTTTTGGAGCTAGCCTCTGG-3' (SEQ ID NO:9)). RACE-PCR products are purified and sequenced to identify TCR a and β chains. TCR variable, diversity, and joining regions can be defined using IMGT/V-QUEST software.

TCRs can be constructed by pairing the TRA and TRB sequences encoding the dominant chains in each PBT-specific or OIP5-specific T cell clone. TRA and TRB sequences are confirmed by PCR using a forward primer from the 5’ end of the appropriate V region and reverse primers from the TRA or TRB constant region(s), followed by Sanger sequencing. In any of the herein disclosed embodiments, the encoded Va domain comprises no change in amino acid sequence of CDR1 (as compared to a reference Va domain of an exemplary T cell clone provided herein), the encoded Vp domain comprises no change in amino acid sequence of CDR1, or the CDR1 of the encoded Va domain and the CDR1 of the encoded Vp domain comprise no change in amino acid sequence. In further embodiments, the encoded Va domain comprises no change in amino acid sequence of CDR2, the encoded Vp domain comprises no change in amino acid sequence of CDR2, or the CDR2 of the encoded Va domain and the CDR2 of the encoded Vp domain comprise no change in amino acid sequence.

In certain embodiments, the HLA comprises HLA-A*02:01. In other embodiments, the HLA comprises HLA-B *07:02. In any of the herein disclosed embodiments, the binding protein comprises a TCR, a single-chain TCR (scTCR), a scTv, a chimeric antigen receptor (CAR), a TCR-mimic antibody, or any combination thereof. Methods for producing engineered TCRs are described in, for example, Bowerman et al., Mol. Immunol., 46(15):3000 (2009), the techniques of which are herein incorporated by reference. Methods for making CARs are well known in the art and are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Publication No. 2010/065818; U.S. Patent No. 8,822,647; PCT Publication No. WO 2014/031687; U.S. Patent No. 7,514,537; and Brentjens et al., 2007, Clin. Cancer Res. 13:5426, the techniques of which are herein incorporated by reference.

In certain embodiments, a PBK-specific or OIP5-specific binding domain alone (i.e., without any other portion of the binding protein) can be soluble and can bind to the antigen or antigen:HLA complex with a Kd of less than about 10_-8 M, less than about 10'⁹ M, less than about 10'¹⁰ M, less than about 10'¹¹ M, less than about 10'¹² M, or less than about 10'¹³ M. In particular embodiments, a PBK-specific or OIP5-specific binding domain includes an antigen-specific scTCR (e.g., single chain αβTCR proteins such as Vα-L-Vβ, VP-L-Vα, Vα-Cα-L-Vα, or Vα-L-VP-CP, wherein Vα and Vβ are TCRa and P variable domains respectively, Ca and Cβ are TCRa and P constant domains, respectively, and L is a linker). In certain embodiments, the binding protein further comprises a TCR P polypeptide constant domain (CP), a TCR a polypeptide constant domain (Ca), or both. A Vβ and a Cβ together comprise TCR P polypeptide or chain. A Vα and a Ca together comprise TCR a polypeptide or chain. In some embodiments, the Cβ comprises or consists of an amino acid sequence having at least at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:69 or 70, and/or the Ca comprises or consists of an amino acid sequence having at least at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:68.

In certain embodiments, the Cβ and/or the Ca comprises one or more non-native amino acid at a position such that when the the Cβ and the Ca associate to form a dimer, a non-native disulfide bond is formed between the Cβ and the Ca, wherein, optionally, the non-native amino acid comprises a cysteine in the Cβ and/or a cysteine in the Ca. In some embodiments, the binding protein comprises a TCR Cβ and a TCR Ca, wherein the TCR Cβ comprises a cysteine amino acid in place of a native serine at amino acid position 57 (e.g., GV(S→ C)TD) and the TCR Ca comprises a cysteine amino acid in place of a native threonine at amino acid position 48 (e.g., DK(T→ C)VL; see. e.g., Cohen et al., Cancer Res. 67(8):3898-3903 (2007)).

In certain embodiments, a binding protein comprises two components, such as a chain and a β chain, which can associate on the cell surface to form a functional binding protein. The two associated components may comprise mature proteins. In certain embodiments, an antigen-binding fragment of a TCR comprises a single chain TCR (scTCR), which comprises both the TCR Va and TCR Vp domains, but only a single TCR constant domain (C_aor Cp).

In further embodiments, an antigen-binding fragment of a TCR or a chimeric antigen receptor or TCR-mimic antibody is chimeric (e.g., comprises amino acid residues or motifs from more than one donor or species), humanized (e.g, comprises residues from a non-human organism that are altered or substituted so as to reduce the risk of immunogenicity in a human), or human. Binding proteins of the present disclosure can be expressed by a host cell, e.g. whether as a cell surface molecule (e.g. TCR, scTCR, CAR) or a soluble molecule (e.g. TCR-mimic antibody, scTv (see e.g. Novoty et al. PNAS SS(19):8646-8650 (1991)).

In certain embodiments, a binding protein of the present disclosure comprises one or more junction amino acids. "Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., 2 to about 10) amino acid residues between two adjacent motifs, regions or domains of a polypeptide, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can result from the design of a construct that encodes a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein), or from cleavage of, for example, a self-cleaving peptide adjacent one or more domains of an encoded binding protein of this disclosure (e.g., a P2A peptide disposed between a TCR a-chain and a TCR β-chain, the self-cleavage of which can leave one or more junction amino acids in the a-chain, the TCR β-chain, or both).

In any of the embodiments described herein, a polypeptide of this disclosure can comprise a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside the cell. A signal peptide may be removed from the polypeptide during or once localization or secretion is completed. Polypeptides that have a signal peptide are referred to herein as a "pre-protein" and polypeptides having their signal peptide removed are referred to herein as "mature" proteins or polypeptides. Non-limiting examples of leader peptides the amino acid sequences shown in SEQ ID NOs.:71-78.

In any of the herein disclosed embodiments, a polynucleotide encoding the binding protein (and, optionally, accessory protein or proteins) is codon optimized. Codon optimization can be performed using, e.g., the GenScript® OptimumGene™ tool. Codon-optimized sequences include sequences that are partially codon-optimized (i.e., one or more of the codons is optimized for expression in the host cell) and those that are fully codon-optimized. Any suitable host cell cell may encode a binding protein of this disclosure, or be engineered to include a heterologous polynucleotide encoding a binding protein of this disclosure. In some embodiments, an immune cell is preferred (e.g., a T cell, a NK cell, a NK-T cell, a B cell, or a plasma cell). In some embodiments, an immune cell comprises a CD4⁺T cell, a CD8⁺ T cell, or both. Methods for transfecting/transducing T cells with desired nucleic acids have been described (e.g., U.S. Patent Application Pub. No. US 2004/0087025) as have adoptive transfer procedures using T cells of desired target-specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 772:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011/0243972; US 2011/0189141; Leen et al, Ann. Rev. Immunol. 25:243, 2007), such that adaptation of these methodologies to the presently disclosed embodiments is contemplated, based on the teachings herein.

In certain embodiments, polynucleotides encoding binding proteins of this disclosure, e.g., TCRs, can be codon optimized to enhance expression in a particular host cell, such as a cell of the immune system, a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell. Exemplary T cells include CD4⁺ T cells, CD8⁺ T cells, and related subpopulations thereof (e.g., naive, central memory, effector memory, stem cell memory).

Any appropriate method can be used to transfect or transduce the cells, for example, the T cells, or to administer the polynucleotides or compositions of the present methods. Known methods for delivering polynucleotides to host cells include, for example, use of cationic polymers, lipid-like molecules, and certain commercial products such as, for example, IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome- mediated transfection, receptor-mediated transduction, microprojectile bombardment, transposon-mediated transfer, and the like. Still further methods of transfecting or transducing host cells employ vectors, described in further detail herein. Other methods of introducing a binding protein-encoding polynucleotide into a host cell or host cell genome include gene engineering strategies such as e.g., CRISPR/Cas systems. A host cell of the present disclosure may comprise a single polynucleotide that encodes a binding protein as described herein, or the binding protein may be encoded by more than one polynucleotide. In other words, components or portions of a binding protein may be encoded by two or more polynucleotides, which may be contained on a single nucleic acid molecule or may be contained on two or more nucleic acid molecules.

In certain embodiments, a host cell (e.g., an immune cell such as a T cell, optionally a CD8+ T cell, a CD4+ T cell, or both) is capable of killing at least about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or more, of antigen- presenting cells (e.g., dendritic cells or PBMCs) when the immune cells and the antigen-presenting cells are present at at least about a 20: 1 (including 20: 1) ratio and wherein the peptide antigen according to SEQ ID NO: 3 or 10 was added to a culture comprising the antigen-presenting cells at about 1,000 ng/mL, about 100 ng/mL, or about 10 ng/mL, as measured in a 4-hour labeled chromium release assay. In further embodiments, a modified immune cell is capable of killing at least about 10%, about 20%, about 30%, or more of the antigen-presenting cells wherein the peptide antigen is added to the antigen-presenting cells at about 1 pg/mL.

In certain embodiments, a host cell (e.g., an immune cell such as a T cell, optionally a CD8+ T cell, a CD4+ T cell, or both) is capable of killing at least about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or more, of antigen- presenting cells (e.g., dendritic cells or PBMCs) when the host cells and the antigen- presenting cells are present at least about a 20: 1 (including 20: 1) ratio and wherein a peptide antigen according to SEQ ID NO:4 or 5 was added to a culture comprising the antigen-presenting cells at about 1,000 ng/mL, about 100 ng/mL, or about 10 ng/mL, as measured in a 4-hour labeled chromium release assay. In further embodiments, a host cell is capable of killing at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or more of the antigen-presenting cells wherein the peptide antigen was added to the antigen-presenting cells at about 10 pg/mL.

In certain embodiments, a host cell (e.g., an immune cell such as a T cell, optionally a CD8+ T cell, a CD4+ T cell, or both) of the present disclosure specifically kills at least about 60%, about 70%, about 80%, about 90%, or more of lymphoblastoid cells (e.g., autologous LCLs) in a presence of peptide antigen (SEQ ID NO:3, 4, 5, or 10) in a 4-hour labeled chromium (⁵¹Cr) release assay, wherein the modified immune cell and the LCLs are present at at least about a 20: 1 ratio (including 20: 1) and wherein the peptide antigen is added to the LCLs at about Ipg/mL.

In certain embodiments, a host cell (e.g., an immune cell such as a T cell, optionally a CD8+ T cell, a CD4+ T cell, or both) is capable of killing at least about 40%, about 50%, about 60%, or more, of glioma cells (e.g., high grade glioma (Grade 3 or Grade 4 tumor according to World Health Organization (WHO) scale)) when the host cells and the glioma cells are present at at least about a 20: 1 (including 20: 1) ratio and wherein a peptide antigen according to SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 was added to a culture comprising the antigen-presenting cells at about Ipg/mL, as measured in a 4-hour labeled chromium release assay. In further embodiments, the host cell (e.g., an immune cell such as a T cell, optionally a CD8+ T cell, a CD4+ T cell, or both) is capable of killing at least about 60%, about 70%, about 80%, about 90%, about 99%, or more or more of the glioma cells when exogenous interferongamma is added to the glioma cells and the host cells (e.g., an immune cell such as a T cell, optionally a CD8+ T cell, a CD4+ T cell, or both)

In certain embodiments, a modified immune cell of the present disclosure can kill a target glioma cell (e.g., a PBT-05 cell, wherein killing can be determined by, for example, a chromium release assay) in the absence of an exogenous peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3 or 10, and optionally in the presence of an exogenous cytokine, such as, for example, IFN-y. In certain embodiments, a modified immune cell of the present disclosure can kill a target glioma cell in the presence of an exogenous peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3, and optionally in the presence of an exogenous cytokine, such as, for example, IFN-y. In certain embodiments, a modified immune cell of the present disclosure can specifically kill a target glioma cell derived from an ATRT (e.g., ATRT-310; ATRT-311) or glioblastoma multiforme (GBM; e.g., GMB-511) cell line, in the absence of an exogenous peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3, and optionally in the presence of an exogenously added cytokine, such as, for example, IFN-y. In certain embodiments, a host cell of the present disclosure is capable of specifically killing at least about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of target glioma cells present in a sample in the absence of an exogenous peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3, and optionally in the presence of an exogenously added cytokine, such as, for example, IFN-y, as measured in a labeled chromium (⁵¹Cr) release assay of about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20, about 22, or about 24 hours, wherein the modified immune cell and the target glioma cells are present at at an effector :target ratio of about 1 : 1, about 2: 1, or about 4: 1.

In any of the foregoing embodiments, a host cell (e.g., immune cell such as a T cell, NK-T cell, or NK cell) may modified to reduce or eliminate expression of one or more endogenous genes that encode a polypeptide involved in immune signaling or other related activities. Examples of gene knockouts include those that encode PD-1, LAG-3, CTLA4, TIM3, an HL A molecule, a TCR molecule, or the like. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may be recognized as foreign by an allogeneic recipient receiving the host (e.g. modified immune) cells, which may result in elimination of the host (e.g. modified immune) cells (e.g., an HLA allele), or may downregulate the immune activity of the host (e.g. modified immune) cells (e.g., PD-1, LAG-3, CTLA4), or may interfere with the binding activity of a heterologously expressed binding protein of the present disclosure (e.g., an endogenous TCR of a modified T cell that binds a non-PBK or OIP5 antigen and thereby interferes with the modified immune cell binding a cell that expresses a PBK or OIP5 antigen).

Accordingly, decreasing or eliminating expression or activity of such endogenous genes or proteins can improve the activity, tolerance, or persistence of the modified immune cells in an autologous or allogeneic host setting, and may allow for universal administration of the cells (e.g., to any recipient regardless of HLA type). In certain embodiments, a modified immune cell is a donor cell (e.g., allogeneic) or an autologous cell. In certain embodiments, a modified immune cell of this disclosure comprises a chromosomal gene knockout of one or more of a gene that encodes PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g., a gene that encodes an al macroglobulin, an α2 macroglobulin, an α3 macroglobulin, a β1 microglobulin, or a β2 microglobulin), or a TCR component (e.g., a gene that encodes a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119(24):5697 (2012); and Torikai et al., Blood 722(8):1341 (2013), the gene-editing techniques, compositions, and adoptive cell therapies of which are herein incorporated by reference in their entirety).

As used herein, the term "chromosomal gene knockout" refers to a genetic alteration or introduced inhibitory agent in a host cell that prevents (e.g., reduces, delays, suppresses, or abrogates) production, by the host cell, of a functionally active endogenous polypeptide product. Alterations resulting in a chromosomal gene knockout can include, for example, introduced nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletion, and strand breaks, as well as the heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

In certain embodiments, a chromosomal gene knock-out or gene knock-in is made by chromosomal editing of a host cell. Chromosomal editing can be performed using, for example, endonucleases. As used herein "endonuclease" refers to an enzyme capable of catalyzing cleavage of a phosphodiester bond within a polynucleotide chain. In certain embodiments, an endonuclease is capable of cleaving a targeted gene thereby inactivating or "knocking out" the targeted gene. An endonuclease may be a naturally occurring, recombinant, genetically modified, or fusion endonuclease. The nucleic acid strand breaks caused by the endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, a donor nucleic acid molecule may be used for a donor gene "knock-in", for target gene "knock-out", and optionally to inactivate a target gene through a donor gene knock in or target gene knock out event. NHEJ is an error- prone repair process that often results in changes to the DNA sequence at the site of the cleavage, e.g., a substitution, deletion, or addition of at least one nucleotide. NHEJ may be used to "knock-out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs. As used herein, a "zinc finger nuclease" (ZFN) refers to a fusion protein comprising a zinc finger DNA-binding domain fused to a non-specific DNA cleavage domain, such as a Fokl endonuclease. Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and amino acids at certain residues can be changed to alter triplet sequence specificity (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 255: 1917-1934, 1999). Multiple zinc finger motifs can be linked in tandem to create binding specificity to desired DNA sequences, such as regions having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of a site-specific DNA double strand break (DSB) in the genome, and targeted integration of a transgene comprising flanking sequences homologous to the genome at the site of DSB is facilitated by homology directed repair. Alternatively, a DSB generated by a ZFN can result in knock out of target gene via repair by non-homologous end joining (NHEJ), which is an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, made using a ZFN molecule.

As used herein, a "transcription activator-like effector nuclease" (TALEN) refers to a fusion protein comprising a TALE DNA-binding domain and a DNA cleavage domain, such as a Fokl endonuclease. A "TALE DNA binding domain" or "TALE" is composed of one or more TALE repeat domains/units, each generally having a highly conserved 33-35 amino acid sequence with divergent 12th and 13th amino acids. The TALE repeat domains are involved in binding of the TALE to a target DNA sequence. The divergent amino acid residues, referred to as the Repeat Vαriable Diresidue (RVD), correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined such that an HD (histine-aspartic acid) sequence at positions 12 and 13 of the TALE leads to the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) to A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide. Non-canonical (atypical) RVDs are also known (see, e.g., U.S. Patent Publication No. US 2011/0301073, which atypical RVDs are incorporated by reference herein in their entirety). TALENs can be used to direct site-specific double-strand breaks (DSB) in the genome of T cells. Nonhom ologous end joining (NHEJ) ligates DNA from both sides of a double-strand break in which there is little or no sequence overlap for annealing, thereby introducing errors that knock out gene expression. Alternatively, homology directed repair can introduce a transgene at the site of DSB providing homologous flanking sequences are present in the transgene. In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a TALEN molecule.

As used herein, a "clustered regularly interspaced short palindromic repeats/Cas" (CRISPR/Cas) nuclease system refers to a system that employs a CRISPR RNA (crRNA)-guided Cas nuclease to recognize target sites within a genome (known as protospacers) via base-pairing complementarity and then to cleave the DNA if a short, conserved protospacer associated motif (PAM) immediately follows 3’ of the complementary target sequence. CRISPR/Cas systems are classified into three types (i.e., type I, type II, and type III) based on the sequence and structure of the Cas nucleases. The crRNA-guided surveillance complexes in types I and III need multiple Cas subunits. Type II system, the most studied, comprises at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA comprises a duplex forming region. A crRNA and a tracrRNA form a duplex that is capable of interacting with a Cas9 nuclease and guiding the Cas9/crRNA:tracrRNA complex to a specific site on the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from a PAM. Cas9 nuclease cleaves a double-stranded break within a region defined by the crRNA spacer. Repair by NHEJ results in insertions and/or deletions which disrupt expression of the targeted locus. Alternatively, a transgene with homologous flanking sequences can be introduced at the site of DSB via homology directed repair. The crRNA and tracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, e.g., Jinek et al., Science 337:816-21, 2012). Further, the region of the guide RNA complementary to the target site can be altered or programed to target a desired sequence (Xie et al., PLOS One 9:el00448, 2014; U.S. Pat. Appl. Pub. No. US 2014/0068797, U.S. Pat. Appl. Pub. No. US 2014/0186843; U.S. Pat. No. 8,697,359, and PCT Publication No. WO 2015/071474; each of which is incorporated by reference). In certain embodiments, a gene knockout comprises an insertion, a deletion, a mutation or a combination thereof, and made using a CRISPR/Cas nuclease system.

Exemplary gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, CAS9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.

Exemplary gRNA sequences and methods of using the same to knock out endogenous genes that encode immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), the gRNAs, Cas9 DNAs, vectors, and gene knockout techniques of which are hereby incorporated by reference in their entirety.

Alternative Cas nucleases may be used, including but not limited to, Cas 12, Cas 13, and Cas 14 nucleases, and variants thereof. For example, Cas nucleases disclosed in WO 2019/178427, which is hereby incorporated by reference in its entirety (including the Cas nucleases, CRISPR-Cas systems, and related methods disclosed therein), may be utilized.

As used herein, a "meganuclease," also referred to as a "homing endonuclease," refers to an endodeoxyribonuclease characterized by a large recognition site (double stranded DNA sequences of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structure motifs: LAGLID ADG, GIY- YIG, HNH, His-Cys box and PD-(DZE)XK. Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, Pl-Sce, 1-SceIV, I-Csml, I-PanI, LScell, LPpoI, 1-SceIII, LCrel, I- TevI, I-TevII and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Patent Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res . 25:3379-3388, 1997; Dujon et al., Gene 82: 115-118, 1989; Perler et al., Nucleic Acids Res. 22:1125- 1127, 1994; Jasin, Trends Genet. 72:224-228, 1996; Gimble et al., J. Mol. Biol. 263: 163-180, 1996; Argast et al., J. Mol. Biol. 280:345-353, 1998). In certain embodiments, naturally occurring meganucleases may be used to promote site-specific genome modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, an HLA-encoding gene, or a TCR component-encoding gene. In other embodiments, an engineered meganuclease having a novel binding specificity for a target gene is used for site-specific genome modification (see, e.g., Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952-62, 2003; Chevalier et al., Molec. Cell 70:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49- 66, 2007; U.S. Patent Publication Nos. US 2007/0117128; US 2006/0206949; US 2006/0153826; US 2006/0078552; and US 2004/0002092). In further embodiments, a chromosomal gene knockout is generated using a homing endonuclease that has been modified with modular DNA binding domains of TALENs to make a fusion protein known as a megaTAL. MegaTALs can be utilized to not only knock-out one or more target genes, but to also introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.

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

A chromosomal gene knockout can be confirmed directly by DNA sequencing of the host immune cell following use of the knockout procedure or agent. Chromosomal gene knockouts can also be inferred from the absence of gene expression (e.g., the absence of an mRNA or polypeptide product encoded by the gene) following the knockout. In some embodiments, a polynucleotide encoding a binding protein is heterologous to the host cell and is comprised in an endogenous TCR gene locus of the host cell.

In another aspect, compositions are provided that comprise (e.g. an effective amount of) a binding protein, polynucleotide, vector, or host cell of the present disclosure, and a pharmaceutically acceptable carrier, diluent, or excipient.

Also provided herein are unit doses that comprise an effective amount of a modified immune cell or of a composition comprising the modified immune cell. In certain embodiments, a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4⁺ T cells, combined with (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8⁺ T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells (i.e., has less than about 50%, less than about 40%, less than about 30%, less then about 20%, less than about 10%, less than about 5%, or less then about 1% the population of naive T cells present in a unit dose as compared to a patient sample having a comparable number of PBMCs).

In some embodiments, a unit dose comprises (i) a composition comprising at least about 50% modified CD4⁺ T cells, combined with (ii) a composition comprising at least about 50% modified CD8⁺ T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In further embodiments, a unit dose comprises (i) a composition comprising at least about 60% modified CD4⁺ T cells, combined with (ii) a composition comprising at least about 60% modified CD8⁺ T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In still further embodiments, a unit dose comprises (i) a composition comprising at least about 70% engineered CD4⁺ T cells, combined with (ii) a composition comprising at least about 70% engineered CD8⁺ T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 80% modified CD4⁺ T cells, combined with (ii) a composition comprising at least about 80% modified CD8⁺ T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 85% modified CD4⁺ T cells, combined with (ii) a composition comprising at least about 85% modified CD8⁺ T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 90% modified CD4⁺ T cells, combined with (ii) a composition comprising at least about 90% modified CD8⁺ T cells, in about a 1 : 1 ratio, wherein the unit dose contains a reduced amount or substantially no naive T cells.

It will be appreciated that a unit dose of the present disclosure may comprise a modified immune cell as described herein (i.e.., expressing a binding protein specific for a PBK or OIP5 antigen) and a modified immune cell expressing a binding protein specific for a different antigen (e.g., a different PBK or OIP5 antigen, or an antigen from a different protein or target, such as, for example, BCMA, CD3, CEACAM6, c- Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O- acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gpl30, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., including MAGE-A1, MAGE- A3, and MAGE-A4), mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD 137, TWKAK -R, HLA, tumor- or pathogen- associated peptide bound to HL A, hTERT peptide bound to HLA, tyrosinase peptide bound to HLA, WT-1 peptide bound to HLA, LTpR, LIFRp, LRP5, MUC1, OSMRp, TCRa, TCRp, CD 19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCHI, WT-1, HA’-H, Robol, a-fetoprotein (AFP), Frizzled, 0X40, PRAME, and SSX-2. or the like). For example, a unit dose can comprise modified CD8⁺ T cells expressing a binding protein that specifically binds to a PBK-HLA complex and modified CD4⁺ T cells (and/or modified CD8⁺ T cells) expressing a binding protein (e.g., a CAR) that specifically binds to a PSMA antigen. It will also be appreciated that any of the immune cells disclosed herein (e.g., cells specific for a PBK antigen and cells specific for a OIP5 antigen) may be administered in a combination therapy.

In any of the embodiments described herein, a unit dose comprises equal, or approximately equal numbers of engineered CD45RA" CD3⁺ CD8⁺ and modified CD45RA' CD3⁺ CD4⁺ TM cells.

Also provided are host cells for expressing a TCR-mimic antibody or antigenbinding fragment according to the present disclosure, as well as host cells that comprise or containing a vector or polynucleotide encoding a TCR-mimic an antibody or antigenbinding fragment.

Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In certain embodiments, the cells are B cells (e.g., immortalized and/or engineered to encode the antibody or antigenbinding fragment), plasma cells, or hematopoietic progenitor cells. In certain embodiments, the cells are a mammalian cell line such as CHO cells (e.g., DHFR- CHO cells (Urlaub et al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells. NSO cells, human liver cells, e.g. Hepa RG cells, myeloma cells or hybridoma cells. Other examples of mammalian host cell lines include mouse sertoli cells (e.g., TM4 cells); monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Mammalian host cell lines suitable for antibody production also include those described in, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. . K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

In certain embodiments, a host cell is a prokaryotic cell, such as an E. coli. The expression of peptides in prokaryotic cells such as E. coli is well established (see, e.g., Pluckthun, A. Bio/Technology 9:545-551 (1991). For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523.

In particular embodiments, a cell may be transfected with a vector according to the present description. Transfection can be accomplished using methods such as, for example, electroporation, lipofection, e.g., based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine, etc. In certain embodiments, the introduction is non-viral.

Moreover, host cells of the present disclosure may be transfected stably or transiently with a vector according to the present disclosure, e.g. for expressing an antibody, or an antigen-binding fragment thereof, according to the present disclosure. In such embodiments, the cells may be stably transfected width the vector as described herein. Alternatively, cells may be transiently transfected with a vector according to the present disclosure encoding an antibody or antigen-binding fragment as disclosed herein. In any of the presently disclosed embodiments, a polynucleotide may be heterologous to the host cell.

Accordingly, the present disclosure also provides recombinant host cells that express an antibody or antigen-binding fragment of the present disclosure. For example, the cell may be of a species that is different to the species from which the antibody was fully or partially obtained (e.g., CHO cells expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not express the antibody or antigen-binding fragment in nature. Moreover, the host cell may impart a post-translational modification (PTM; e.g., glysocylation or fucosylation) on the antibody or antigen-binding fragment that is not present in a native state of the antibody or antigen-binding fragment (or in a native state of a parent antibody from which the antibody or antigen binding fragment was engineered or derived). Such a PTM may result in a functional difference (e.g., reduced immunogenicity). Accordingly, an antibody or antigen-binding fragment of the present disclosure that is produced by a host cell as disclosed herein may include one or more post-translational modification that is distinct from the antibody (or parent antibody) in its native state (e.g., a human antibody produced by a CHO cell can comprise a more post-translational modification that is distinct from the antibody when isolated from the human and/or produced by the native human B cell or plasma cell).

Insect cells useful expressing an antibody or antigen-binding fragment include, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWTOl “Mimic™” cells. See, e.g., Palmberger et al., J. Biotechnol. 753(3-4): 160-166 (2011). Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expressing protein-encoding vectors, and include fungi and yeast strains with "humanized" glycosylation pathways, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat.

Biotech. 22: 1409-1414 (2004); Li et a!., Nat. Biotech. 24:210-215 (2006).

Plant cells can also be utilized as hosts for expressing an antibody or antigenbinding fragment of the present disclosure. For example, PLANTIBODIES™ technology (described in, for example, U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429) employs transgenic plants to produce antibodies.

In certain embodiments, the host cell comprises a mammalian cell. In particular embodiments, the host cell is a CHO cell, a HEK293 cell, a PER.C6 cell, a Y0 cell, a Sp2/0 cell, a NS0 cell, a human liver cell, a myeloma cell, or a hybridoma cell.

Polynucleotides, Transgenes and Vectors

In further aspects, the present disclosure provides an isolated polynucleotide that encodes a binding protein as described herein (e.g., a PBK-specific or OIP5-specific TCR, scTCR, scTv, CAR, TCR-mimic antibody (and optionally further comprises constant domains or other components as described herein)), and may additionally encoded a safety switch protein, a selection marker, a CD8 co-receptor β-chain, or a CD8 co-receptor a-chain, or any combination thereof, provided that at least a portion of the isolated polynucleotide is codon-optimized for expression in a host cell (e.g., an engineered immune cell as disclosed herein).

In particular, any of the aforementioned heterologous polynucleotides comprised in the modified immune cells (e.g., encoding any of the binding proteins of the present disclosure) may also or alternatively be provided in an isolated form. In some embodiments, the polynucleotide is codon-optimized for expression in a host cell.

In certain embodiments, a heterologous polynucleotide encoding a TCR Vα or a-chain and a heterologous polynucleotide encoding a TCR Vβ or β-chain are contained in a single open reading frame comprised in host cell, wherein the single open reading frame further comprises a polynucleotide encoding a self-cleaving peptide disposed between the Vα (or α chain)-encoding polynucleotide and the Vβ (or β-chain)-encoding polynucleotide.

An isolated polynucleotide of this disclosure may further comprise a polynucleotide encoding a safety switch protein, a selection marker, a CD8 co-receptor betα chain, or a CD8 co-receptor alphα chain as disclosed herein, or may comprise a polynucleotide encoding any combination thereof.

In certain embodiments, a polynucleotide encoding two or more components or portions of a binding protein of the present disclosure comprises the two or more coding sequences operatively associated in a single open reading frame. Such an arrangement can advantageously allow coordinated expression of desired gene products, such as, for example, contemporaneous expression of alpha- and beta-chains of a TCR, such that they are produced in about a 1 :1 ratio. In certain embodiments, two or more substituent gene products of a binding protein of this disclosure, such as a TCR (e.g., alpha- and beta-chains), are expressed as separate molecules and associate post-translationally. In further embodiments, two or more substituent gene products of a binding protein of this disclosure are expressed as a single peptide with the parts separated by a cleavable or removable segment. For instance, self-cleaving peptides useful for expression of separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, a Porcine teschovirus-1 2 A (P2A) peptide, a Thoseaasigna virus 2A (T2A) peptide, an Equine rhinitis A virus (ERAV) 2A (E2A) peptide, and a Foot-and-Mouth disease virus 2A (F2A) peptide.

In some embodiments, a polynucleotide comprises DNA, RNA (optionally mRNA), or both. In certain embodiments, a polynucleotide comprises DNA.

A host cell of the present disclosure may comprise a single polynucleotide that encodes a binding protein as described herein, or the binding protein may be encoded by more than one polynucleotide. In other words, components or portions of a binding protein may be encoded by two or more polynucleotides, which may be contained on a single nucleic acid molecule or may be contained on two or more nucleic acid molecules.

In certain embodiments, a polynucleotide encoding two or more components or portions of a binding protein of the present disclosure comprises the two or more coding sequences operatively associated in a single open reading frame.

In further embodiments, a binding protein is expressed as part of a transgene construct that encodes one or more additional accessory protein, such as a safety switch protein, a tag, a selection marker, a CD8 co-receptor β-chain, a-chain or both, or any combination thereof. Polynucleotides and transgene constructs useful for encoding and expressing binding proteins and accessory components (e.g., one or more of a safety switch protein, a selection marker, CD8 co-receptor β-chain, or a CD8 co-receptor a- chain).

In certain embodiments, a safety switch protein can be targeted using a cognate drug or other compound to selectively modulate the activity (e.g., lessen or ablate) of such cells when desirable. Safety switch proteins used in this regard include, for example, a truncated EGF receptor polypeptide (huEGFRt) that is devoid of extracellular N terminal ligand binding domains and intracellular receptor tyrosine kinase activity but retains the native amino acid sequence, type I transmembrane cell surface localization, and a conformationally intact binding epitope for pharmaceuticalgrade anti-EGFR monoclonal antibody, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 775: 1255-1263, 2011), a caspase polypeptide (e.g., iCasp9; Straathof et al., Blood 705:4247-4254, 2005; Di Stasi et al, N. Engl. J. Med. 365:1673- 1683, 2011; Zhou and Brenner, Exp. Hematol. pii:S0301-472X(16)30513-6. doi: 10.1016/j. exphem.2016.07. Oi l), RQR8 (Philip et al., Blood 124: 1277-1287 , 2014), a 10 amino acid tag of the human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Set. USA 705:623-628, 2008), as discussed herein, and a marker/safety switch polypeptide, such as RQR (CD20 + CD34; Philip et al., 2014).

Other accessory components useful for therapeutic cells comprise a tag or selection marker that allows the cells to be identified, sorted, isolated, enriched, or tracked. For example, marked immune cells having desired characteristics (e.g., an antigen-specific TCR and a safety switch protein) can be sorted away from unmarked cells in a sample and more efficiently activated and expanded for inclusion in a therapeutic product of desired purity.

As used herein, the term "selection marker" comprises a nucleic acid construct that confers an identifiable change to a cell permitting detection and positive selection of immune cells transduced with a polynucleotide comprising a selection marker. RQR is a selection marker that comprises a major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, an RQR-encoding polynucleotide comprises a polynucleotide that encodes the 16 amino acid CD34 minimal epitope. In some embodiments, such as certain embodiments provided in the examples herein, the CD34 minimal epitope is incorporated at the amino terminal position of the CD8 stalk domain (Q8). In further embodiments, the CD34 minimal binding site sequence can be combined with a target epitope for CD20 to form a compact marker/ suicide gene for T cells (RQR8) (Philip et al., 2014, incorporated by reference herein). This construct allows for the selection of immune cells expressing the construct, with for example, CD34 specific antibody bound to magnetic beads (Miltenyi) and that utilizes clinically accepted pharmaceutical antibody, rituximab, that allows for the selective deletion of a transgene expressing engineered T cell (Philip et al., 2014).

Further exemplary selection markers also include several truncated type I transmembrane proteins normally not expressed on T cells: the truncated low-affinity nerve growth factor, truncated CD 19, and truncated CD34 (see for example, Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011; Mavilio et al., Blood 83:1988-1997, 1994; Fehse et al., Mol. Ther. 1:448-456, 2000; each incorporated herein in their entirety). One feature of CD 19 and CD34 is the availability of the off-the-shelf Miltenyi CliniMACs™ selection system that can target these markers for clinical-grade sorting. However, CD 19 and CD34 are relatively large surface proteins that may tax the vector packaging capacity and transcriptional efficiency of an integrating vector. Surface markers containing the extracellular, non signaling domains or various proteins (e.g., CD 19, CD34, LNGFR) also can be employed. Any selection marker may be employed and should be acceptable for Good Manufacturing Practices. In certain embodiments, selection markers are expressed with a polynucleotide that encodes a gene product of interest (e.g., a binding protein of the present disclosure, such as a TCR or CAR). Further examples of selection markers include, for example, reporters such as GFP, EGFP, β-gal or chloramphenicol acetyltransferase (CAT). In certain embodiments, a selection marker, such as, for example, CD34 is expressed by a cell and the CD34 can be used to select enrich for, or isolate (e.g, by immunomagnetic selection) the transduced cells of interest for use in the methods described herein. As used herein, a CD34 marker is distinguished from an anti-CD34 antibody, or, for example, a scFv, TCR, or other antigen recognition moiety that binds to CD34.

In certain embodiments, a selection marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD 19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.

Host cells comprising a heterologous polynucleotide encoding a binding protein of the present disclosure may, in certain embodiments, further comprise a heterologous polynucleotide encoding a CD8 co-receptor protein, or a beta-chain or alpha-chain component thereof. By way of background, inclusion of CD4⁺ T cells in an immunotherapy cell product can provide antigen-induced IL-2 secretion and augment persistence and function of transferred cytotoxic CD8⁺ T cells (see, e.g., Kennedy et al., Immunol. Rev. 222: 129 (2008); Nakanishi et al., Nature 462(7272):510 (2009)). In certain circumstances, a class I-restricted TCR in a CD4⁺ T cells may require the transfer of a CD8 co-receptor to enhance sensitivity of the TCR to class I HLA peptide complexes. CD4 co-receptors differ in structure to CD8 and cannot effectively substitute for CD8 co-receptors (see, e.g., Stone & Kranz, Front. Immunol. 4'244 (2013); see also Cole et al., Immunology 737(2): 139 (2012). Thus, another accessory protein for use in the compositions and methods of this disclosure comprises a CD8 co- receptor or component thereof.

An encoded CD8 co-receptor includes, in some embodiments, a β-chain (see, e.g., UniProtKB identifiers Pl 0966-1, Pl 0966-2, Pl 0966-3, Pl 0966-4, Pl 0966-6, P10966-7, P10966-8, and P10966-9). In further embodiments, the encoded CD8 co- receptor is a recombinant CD8 co-receptor further comprising a RQR polypeptide. Without wishing to be bound by theory, it is believed that distance from the host cell surface is important for RQR polypeptides to function as selection markers/safety switches (Philip et al., 2010 (supra)). In some embodiments, the encoded RQR polypeptide is contained in a β-chain, an a-chain, or both, of the encoded CD8 coreceptor. In specific embodiments, a host cell or a polynucleotide comprises a polynucleotide encoding iCasp9 and a polynucleotide encoding a recombinant CD8 coreceptor protein that comprises a β chain containing a RQR polypeptide and further comprises a CD8 a-chain.

In further embodiments, a polynucleotide or a host cell comprises a polynucleotide encoding iCasp9 and a polynucleotide encoding a recombinant CD8 coreceptor protein that comprises an a-chain containing a RQR polypeptide and further comprises a CD8 β-chain. In some embodiments, both of the encoded CD8 a-chain and the encoded CD8 β-chain contain a RQR polypeptide.

A host cell may be efficiently transduced to contain, and may efficiently express, a single polynucleotide that encodes the binding protein, safety switch protein, selection marker, and CD8 co-receptor protein. For example, in some embodiments, a host cell of the present disclosure comprises a heterologous polynucleotide that encodes, from 5' to 3', ([an iCasp9 polypeptide]-[a porcine teschovirus 2A (P2A) peptide]-[a TCR β chain]-[a P2A peptide]-[a TCR α chain]-[a P2A peptide]-[a CD8 P- chain comprising an RQR polypeptide]-[a P2A peptide]-[a CD8 a-chain]).

In some embodiments, a polynucleotide or host cell comprises polynucleotide encoding a binding protein can further comprise: (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor a chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii). Without being bound by theory, in certain embodiments, coexpression or concurrent expression of a binding protein and a CD8 co-receptor protein or portion thereof functional to bind to an HLA molecule may improve one or more desired activity of a host cell (e.g., immune cell, such as a T cell, optionally a CD4⁺ T cell) as compared to expression of the binding protein alone. It will be understood that the binding protein-encoding polynucleotide and the CD8 co-receptor polypeptide- encoding polynucleotide may be present on a single nucleic acid molecule (e.g., in a same expression vector), or may be present on separate nucleic acid molecules in a host cell.

In certain further embodiments, a polynucleotide or host cell comprises a polynucleotide comprising: (a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b). In further embodiments, a polynucleotide comprises a polynucleotide that encodes a self-cleaving peptide and is disposed between: (1) the polynucleotide encoding a binding protein (e.g., TCR of the present disclosure) and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co- receptor α chain; and/or (2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co- receptor β chain.

In still further embodiments, a polynucleotide can comprise, operably linked inframe: (i) (pnCD8a)-(pnSCPl)-(pnCD8P)-(pnSCP2)-(pnTCR); (ii) (pnCD8P)- (pnSCPl)-(pnCD8a)-(pnSCP2)-(pnTCR); (iii) (pnTCR)-(pnSCPl)-(pnCD8a)- (pnSCP2)-(pnCD8p); (iv) (pnTCR)-(pnSCPl)-(pnCD8p)-(pnSCP2)-(pnCD8a); (v) (pnCD8a)-(pnSCPl)-(pnTCR)-(pnSCP2)-(pnCD8P); or (vi) (pnCD8P)-(pnSCPl)- (pnTCR)-(pnSCP2)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnCD8β is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnTCR is the polynucleotide encoding a TCR, and wherein pnSCPl and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptides are optionally the same or different (e.g, P2A, T2A, F2A, E2A).

In some embodiments, the encoded TCR comprises a TCRα chain and a TCRβ chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRα chain and the polynucleotide encoding a TCRβ chain. In some embodiments, the polynucleotide comprises, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)- (pnTCRP)-(pnSCP3)-(pnTCRa); (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)- (pnTCRP)-(pnSCP3)-(pnTCRa); (iii) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)- (pnTCRa)-(pnSCP3)-(pnTCRP); (iv) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)- (pnTCRa)-(pnSCP3)-(pnTCRP); (v) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)- (pnCD8a)-(pnSCP3)-(pnCD8P); (vi) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)- (pnCD8P)-(pnSCP3)-(pnCD8a); (vii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)- (pnCD8a)-(pnSCP3)-(pnCD8P); or (viii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)- (pnCD8P)-(pnSCP3)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnTCRa is the polynucleotide encoding a TCR α chain, wherein pnTCRP is the polynucleotide encoding a TCR β chain, and wherein pnSCPi, pnSCP2, and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptides are optionally the same or different.

In certain embodiments, a heterologous polynucleotide encoding a TCR Vα or a-polypeptide and a heterologous polynucleotide encoding a TCR Vβ or P-polypeptide are contained in a single open reading frame comprised in the host cell, wherein the single open reading frame further comprises a polynucleotide encoding a self-cleaving peptide disposed between the Vα (or a polypeptide)-encoding polynucleotide and the VP (or P- polypeptide)-encoding polynucleotide.

In any of the embodiments described herein, an isolated polynucleotide is codon-optimized for expression in an immune cell, such as a T cell.

Also provided herein are vectors that comprise a polynucleotide or transgene construct of the instant disclosure. Some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome (e.g., lentiviral vector, retroviral vector). Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors"). According to related embodiments, it is further understood that, if one or more agents (e.g., polynucleotides encoding binding proteins as described herein) are co administered to a subject, that each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent or the same agent) may be introduced to a cell or cell population or administered to a subject.

In certain embodiments, polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from lentiviral vector or a y-retroviral vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, and spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

"Retroviruses" are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses. "Lentiviral vector," as used herein, means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

In certain embodiments, the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors. In other embodiments, the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. HIV-l-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing TCR or CAR transgenes are known in the art and have been previous described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6.327939, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 77: 1155, 2003; Frecha et al., Mol. Ther. 18.Y1AS, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).

Other vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi ci str onic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.

In certain embodiments, a vector is capable of delivering the transgene construct to a host cell (e.g., a hematopoietic progenitor cell or a human immune system cell). In specific embodiments, a vector is capable of delivering a transgene construct to human immune system cell, such as, for example, a CD4⁺ T cell, a CD8⁺ T cell, a CD4" CD8" double negative T cell, a yδ T cell, a natural killer cell, a dendritic cell, or any combination thereof. In further embodiments, a vector is capable of delivering a transgene construct to a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. In some embodiments, a vector that encodes a polynucleotide or transgene construct of the present disclosure may further comprise a polynucleotide that encodes a nuclease that can be used to perform a chromosomal knockout in a host cell (e.g., a CRISPR-Cas endonuclease or another endonuclease as disclosed herein) or that can be used to to deliver a therapeutic transgene or portion thereof to a host cell in a gene therapy replacement or gene repair therapy. Alternatively, a nuclease used for a chromosomal knockout or a gene replacement or gene repair therapy can be delivered to a host cell independent of a vector that encodes a polynucleotide or transgene construct of this disclosure.

Immunogenic Compositions

In other aspects, the present disclosure provides immunogenic compositions comprising or consisting of PBK or OIP5 peptide antigens disclosed herein. In certain embodiments, an immunogenic composition comprises: (i) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) a PBK antigen comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO: 10) (iii) a OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); and/or (iv) a OIP5 antigen comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5), or a variant of any one of SEQ ID NOs:3-5 having one, two, or three substitutions (i.e., one, two, or three different amino acids as compared to SEQ ID NO:3, 4, or 5, respectively).

In certain embodiments, the immunogenic composition is capable of eliciting an immune response in a subject against cancer cells, wherein, optionally, the cancer cells comprise glioblastoma cells, medulloblastoma cells, breast cancer cells, colorectal cancer cells, lung cancer cells, esophageal cancer cells, lymphoma cells, leukemia (e.g., acute myeloid leukemia) cells, melanoma cells, cholangiocarcinoma cells, hepatocellular carcinoma cells, or any combination thereof.

In some embodiments, an immunogenic composition is provided that comprises: (i) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10); (iii) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); (iv) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5); and/or (v) a variant of the isolated peptide or polypeptide of SEQ ID NO.:3, SEQ ID NO.: 10, SEQ ID NO.:4, or SEQ ID NO.: 5, wherein the variant comprises one, two, or three differences in amino acid sequence as compared to SEQ ID NO.:3, 10, 4, or 5, respectively, wherein the isolated peptide or polypeptide of any one of (i)-(vii) does not comprise an isolated full-length human PBK or OIP5.

In certain embodiments, (a) one or more copies of any one of (i)-(v) and/or (b) one or more of any of (i)-(v) is/are present in a fusion polypeptide, wherein the fusion polypeptide optionally further comprises the amino acid sequence of a self-cleaving peptide, and/or are present in a composition as isolated polypeptides each independently being of no more than about 250, no more than about 200, no more than about 150, no more than about 100, no more than about 50, no no more than about 25, no more than about 20, or no more than about 15 amino acids in length.

In certain embodiments, a composition comprises any one, two, or three of the amino acid sequences set forth in SEQ ID NOs:3-5 and 10; e.g., in a fusion polypeptide. An exemplary immunogenic fusion polypeptide can include two or more of the amino acid sequences set forth in SEQ ID NOs:3-5 and 10, in any order, and can include two or more copies of any one or more of the amino acid sequences set forth in SEQ ID NOs:3-5 and 10. In some embodiments, a self-cleaving peptide (e.g., P2A, T2A, E2A, F2A) is disposed between two PBK and/or OIP5 peptides of a fusion.

Also provided is an isolated polynucleotide encoding the immunogenic composition, wherein the polynucleotide is optionally contained in a vector. In some embodiments, the polynucleotide is codon-optimized for expression in a host cell, wherein the host cell is optionally a dendritic cell or a T cell.

Also provided is a host cell comprising the isolated polynucleotide, wherein the polynucleotide is heterologous to the host cell, and wherein the host cell is optionally an immune cell and is further optionally a professional antigen-presenting cell.

In certain embodiments, the host cell is a dendritic cell or a T cell.

In certain embodiments, the immunogenic composition further comprises a pharmaceutically acceptable adjuvant. An adjuvant is intended to enhance (or improve, augment) the immune response to the immunogenic peptides and fusion polypeptides comprising the peptide (i.e., increase the level of the specific immune response to the immunogenic peptide or fusion polypeptide and in a statistically, biologically, or clinically significant manner compared with the level of the specific immune response in the absence of administering the adjuvant). For administration in humans, a pharmaceutically acceptable adjuvant is one that has been approved or is approvable for human administration by pertinent regulatory bodies. Desired adjuvants augment the response to the immunogenic peptide or fusion polypeptide without causing conformational changes in the immunogen that might adversely affect the qualitative immune response. Suitable adjuvants include aluminum salts, such as alum (potassium aluminum sulfate), or other aluminum containing adjuvants such as aluminum hydroxide, aluminum phosphate, or aluminum sulfate. Other pharmaceutically suitable adjuvants include nontoxic lipid A-related adjuvants such as, by way of non-limiting example, nontoxic monophosphoryl lipid A (see, e.g., Persing et al., Trends Microbiol. 25 10:s32-s37 (2002)), for example, 3 De-O- acylated monophosphoryl lipid A (MPL) (see, e.g., United Kingdom Patent Application No. GB 2220211). Other useful adjuvants include QS21 and QuilA that comprise a triterpene glycoside or saponin isolated from the bark of the Quillaja saponaria Molina tree found in South America (see, e.g., Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell and 30 Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540). Other suitable adjuvants include oil in water emulsions, optionally in combination with immune stimulants, such as monophosphoryl lipid A (see, e.g., Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Other suitable adjuvants include polymeric or monomeric amino acids such as polyglutamic acid or polylysine, liposomes, and CpG (see, e.g., Klinman, 35 Int. Rev. Immunol. 25(3-4): 135-54 (2006); U.S. Patent No. 7,402,572; European Patent No. 772 619). Also provided are host cells comprising a heterologous polynucleotide that encodes an immunogenic PBK and/or OIP5 peptide or fusion polypeptide as provided herein. In certain embodiments, a host cell comprises an immune cell, such as a human immune cell.

In certain embodiments, a host cell comprises a dendritic cell or a T cell. In certain embodiments, (e.g. binding protein, polynucleotide, vector, host cell, pharmaceutical composition, or immunogenic composition) is administered to a subject who is HLA-A*02:01⁺.

In certain embodiments, composition of the present disclosure (e.g. binding protein, polynucleotide, vector, host cell, pharmaceutical composition, or immunogenic composition) is administered to a subject who is HLA-B*07:02⁺. Uses

In additional aspects, the present disclosure provides methods of eliciting an immune response against glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, neuroblastoma, hepatoblastoma, PNET, glioma, cranipharyngioma, choroid plexus tumors, schwannomas, meningiomas, Wilm’s tumor, germ cell tumors, or hepatocellular carcinoma, wherein the methods comprise administering to a human subject an immunogenic composition as disclosed herein.

Also provided are methods for expanding a population of T cells that bind to a peptide (e.g., a peptide comprised in a peptide:HLA complex) selected from: (i) a peptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) a peptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10); (iii) a peptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); and (iv) a peptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5), wherein the methods comprising contacting a sample comprising one or more T cells that bind to the peptide with a presently disclosed immunogenic composition, polynucleotide, host cell, and/or antigen-presenting cells that express or have been contacted with a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:3-5 and 10.

Also provided is a method for generating and/or isolating T cells, the method comprising contacting a sample comprising T cells, wherein the sample optionally comprises peripheral blood cells, with (a) a presently disclosed immunogenic composition; and/or (b) antigen-presenting cells (APCs) that express or have been contacted with

(i) a peptide or polypeptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) a peptide or polypeptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10); (iii) a peptide or polypeptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); and (iv) a peptide or polypeptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5); or (v) any combination of (i)-(iv); and optionally sorting T cells from other cells in the sample, thereby isolating and/or generating T cells. Also provided is a T cell isolated and/or generated by this method.

In still other aspects, the present disclosure provides methods for treating a disease or disorder associated with (e.g. that expresses or is believed or known to express or is confirmed to express) (i) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3) or RPSAAHIVEA (SEQ ID NO : 10 and/or (ii) a OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4) or KSLMKILSEV (SEQ ID NO:5) in a subject, wherein the methods comprise administering to the subject a composition (e.g., binding protein, polynucleotide, vector, host cell, or pharmaceutical composition) of the present disclosure, thereby treating the disease or condition. Also provided are presently disclosed binding proteins, polynucleotides, vectors, host cells, compositions, and immunogenic compositions for use in the treatment of a disease or disorder associated with a PBK or OIP5 antigen as disclosed herein and/or for use in the manufacture of a medicament for treating a disease or disorder associated with a PBK or OIP5 antigen as disclosed herein.

"Treat" or "treatment" or "ameliorate" refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). In general, an appropriate dose or treatment regimen comprising a modified immune cell of the present disclosure, and optionally an adjuvant, is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; prolonged survival; or any combination thereof.

A "therapeutically effective amount" or "effective amount", as used herein, refers to an amount of a composition sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner. When referring to an individual active ingredient or a cell expressing a single active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially or simultaneously. A combination may also be a cell expressing more than one active ingredient.

As used herein, "statistically significant" refers to a p value of 0.050 or less when calculated using the Students t-test and indicates that it is unlikely that a particular event or result being measured has arisen by chance.

The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refer to biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject and generally recognized as safe or not causing a serious adverse event.

In certain embodiments, a subject treated according to the presently disclosed methods is HLA-A*02:01⁺. In certain embodiments, a subject treated according to the presently disclosed methods is HLA-B*07:02⁺. In some embodiments, the disease or condition is a cancer. In certain embodiments, the cancer comprises a solid tumor or a hematological malignancy.

Exemplary cancers that can form tumors and can be targeted with the methods of this disclosure include sarcomas and carcinomas, including, for example, chondrosarcoma; fibrosarcoma (fibroblastic sarcoma); Dermatofibrosarcoma protuberans (DFSP); osteosarcoma; rhabdomyosarcoma; Ewing’s sarcoma; a gastrointestinal stromal tumor; Leiomyosarcoma; angiosarcoma (vascular sarcoma); Kaposi’s sarcoma; liposarcoma; pleomorphic sarcoma; synovial sarcoma; Squamous cell carcinoma; Adenocarcinoma; Adenosquamous carcinoma; anaplastic carcinoma; Large cell carcinoma; Small cell carcinoma; a breast carcinoma (e.g., Ductal Carcinoma in situ (non-invasive), Lobular carcinoma in situ (non-invasive), Invasive Ductal Carcinoma, Invasive lobular carcinoma, Non-invasive Carcinoma); a liver carcinoma (e.g., Hepatocellular Carcinoma, Cholangiocarcinomas or Bile Duct Cancer); a lung carcinoma (e.g., Adenocarcinoma, Squamous Cell Carcinoma (Epidermoid Carcinoma), Large-cell undifferentiated carcinoma, Bronchioalveolar carcinoma); an ovarian carcinoma (e.g., Surface epithelial-stromal tumor (Adenocarcinoma) or ovarian epithelial carcinoma (which includes serous tumor, endometrioid tumor and mucinous cystadenocarcinoma), Epidermoid (Squamous cell carcinoma), Embryonal carcinoma and choriocarcinoma ( germ cell tumors)); a kidney carcinoma (e.g., Renal adenocarcinoma, hypernephroma, Transitional cell carcinoma (renal pelvis), Squamous cell carcinoma, Bellini duct carcinoma, Clear cell adenocarcinoma, Transitional cell carcinoma, Carcinoid tumor of the renal pelvis); an adrenal carcinoma (e.g., Adrenocortical carcinoma), a carcinoma of the testis (e.g., Germ cell carcinoma (Seminoma, Choriocarcinoma, Embryonal carciroma, Teratocarcinoma), Serous carcinoma); Gastric carcinoma (e.g., Adenocarcinoma); an intestinal carcinoma (e.g., Adenocarcinoma of the duodenum); a colorectal carcinoma; or a skin carcinoma (e.g., Basal cell carcinoma, Squamous cell carcinoma), neuroblastoma, hepatoblastoma, Brain tumors subtypes (e.g., gliomas, PNETs, cranipharyngioma, choroid plexus tumors, schwannomas, meningiomas), Wilms tumor, Germ cell tumors.

In certain embodiments, methods of the present disclosure target a solid tumor formed by a cancer selected from an ovarian carcinoma, an ovarian epithelial carcinoma, a cervical adenocarcinoma or small cell carcinoma, a pancreatic carcinoma, a colorectal carcinoma (e.g., an adenocarcinoma or squamous cell carcinoma), a lung carcinoma, a breast ductal carcinoma, or an adenocarcinoma of the prostate.

The level of an immune response against a solid tumor (e.g., a CTL (cytotoxic T lymphocyte) immune response) may be determined by any one of numerous immunological methods described herein. The level of a CTL immune response may be determined prior to and following administration of any one of the herein described antigen-specific binding receptors expressed by, for example, a T cell. Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods (see, e.g., Henkart et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and references cited therein).

In certain embodiments, the hematological malignancy comprises a leukemia (e.g., an acute leukemia or a chronic leukemia). In specific embodiments, the leukemia comprises acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), mixed phenotype acute leukemia (MP AL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia, hairy cell leukemia, or chronic lymphocytic leukemia (CLL). In certain embodiments, the hematological malignancy comprises a lymphoma. In certain embodiments, the lymphoma comprises Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma (NHL), a central nervous system lymphoma, small lymphocytic lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, extraosseous plasmacytoma, extra-nodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B- cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt's lymphoma/leukemia, B-cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, and post-transplant lymphoproliferative disorder. In certain embodiments, the hematological malignancy comprises a myelodysplastic disorder, such as, for example, refractory cytopenia with unilineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts - thrombocytosis (RARS-t), refractory cytopenia with multinieage dysplasia (RCMD), refractory cytopenia with multinieage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), myelodysplasia unclassifiable, and refractory cytopenia of childhood. In further embodiments, the hematological malignancy comprises a myeloma.

In some embodiments, a cancer treatable by a method of the present disclosure comprises glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, or hepatocellular carcinoma, neuroblastoma, hepatoblastoma, Brain tumors subtypes (e.g., gliomas, PNETs, cranipharyngioma, choroid plexus tumors, schwannomas, meningiomas), Wilms tumor, Germ cell tumors.

Subjects that can be treated by the present invention are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes. In any of the aforementioned embodiments, the subject may be a human subject. The subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

A pediatric subject refers to an infant, juvenile, or adolescent subject. In some embodiments, a subject is 18 years old or younger, or 15 years old or younger, or 12 years old or younger, or 8 years old or younger, or 6 years old or younger, or 4 years old or younger, or 2 years old or younger. Compositions according to the present disclosure may be administered in a manner appropriate to the disease, condition, or disorder to be treated as determined by persons skilled in the medical art. In any of the above embodiments, an engineered immune cell or unit dose as described herein is administered intravenously, intraperitoneally, intratum orally, intracerebrally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid so as to encounter target cells (e.g., cancer cells). An appropriate dose, suitable duration, and frequency of administration of the compositions will be determined by such factors as a condition of the patient; size, type, and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.

The amount of cells in a composition or unit dose is at least one cell (for example, one engineered CD8⁺ T cell subpopulation; one engineered CD4⁺ T cell subpopulation) or is more typically greater than 10² cells, for example, up to 10⁶, up to 10⁷, up to 10⁸ cells, up to 10⁹ cells, or more than IO¹⁰ cells, such as 10¹¹ cells. In certain embodiments, the cells are administered in a range from about 10⁵ to about 10¹¹ cells/m², preferably in a range of about 10⁶ to about 10⁹ cells/m². The number of cells will depend upon the ultimate use for which the composition is intended as well the type of cells included therein. For example, cells modified to contain a fusion protein specific for a particular antigen will comprise a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For uses provided herein, cells are generally in a volume of a liter or less, 500 mis or less, 250 mis or less, or 100 mis or less. In embodiments, the density of the desired cells is typically greater than 10⁴ cells/ml and generally is greater than 10⁷ cells/ml, generally 10⁸ cells/ml or greater. The cells may be administered as a single infusion or in multiple infusions over a range of time. A clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, or 10¹¹ cells. In certain embodiments, a unit dose of the modifiedimmune cells can be co-administered with (e.g., simultaneously or contemporaneously) hematopoietic stem cells from an allogeneic donor (e.g., a donor that is HAl^H-negative, HLA-A2-negative, or both).

Also contemplated are pharmaceutical compositions (i.e., compositions) that comprise modified immune cells as disclosed herein and a pharmaceutically acceptable carrier, diluents, or excipient. Suitable excipients include water, saline, dextrose, glycerol, or the like and combinations thereof. In embodiments, compositions comprising fusion proteins or host cells as disclosed herein further comprise a suitable infusion media. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% dextrose in water, Ringer's lactate can be utilized. An infusion medium can be supplemented with human serum albumin or other human serum components.

Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's condition, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity).

An effective amount of a pharmaceutical composition refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein. An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount" may be used in reference to treatment, whereas "prophylactically effective amount" may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.

Generally, a therapeutically effective dose of an antibody or antigen binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1.75 g). For polynucleotides, vectors, host cells, and related compositions of the present disclosure, a therapeutically effective dose may be different than for an antibody or antigen-binding fragment.

In some embodiments, a vector is provided that comprises a DNA plasmid construct encoding a TCR-mimic antibody or antigen-binding fragment, or a portion thereof (e.g., so-called "DMAb"; see, e.g., Muthumani et al., J Infect Dis. 214(3):369- 378 (2016); Muthumani et al., Hum Vaccin Immunother 9:2253-2262 (2013)); Flingai et al., Sci Rep. 5: 12616 (2015); and Elliott et al., NPJ Vaccines 18 (2017), which antibodycoding DNA constructs and related methods of use, including administration of the same, are incorporated herein by reference). In certain embodiments, a DNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigen-binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide. In some embodiments, the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide comprised in a single plasmid. In other embodiments, the substituent components of the antibody or antigenbinding fragment are encoded by a polynucleotide comprised in two or more plasmids (e.g., a first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL). An exemplary expression vector is pVaxl, available from Invitrogen®. A DNA plasmid of the present disclosure can be delivered to a subject by, for example, electroporation (e.g., intramuscular electroporation), or with an appropriate formulation (e.g., hyaluronidase).

The pharmaceutical compositions described herein may be presented in unitdose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until infusion into the patient. In certain embodiments, a unit dose comprises an engineered immune cell as described herein at a dose of about 10⁷ cells/m² to about 10¹¹ cells/m². The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including, e.g., parenteral or intravenous administration or formulation.

If the subject composition is administered parenterally, the composition may also include sterile aqueous or oleaginous solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer’s solution, isotonic salt solution, 1,3 -butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water. Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of engineered immune cells or active compound calculated to produce the desired effect in association with an appropriate pharmaceutical carrier. In general, an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide a benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine.

For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.

As used herein, administration of a composition refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally. Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state. Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., engineered immune cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof).

In certain embodiments, a plurality of doses of a composition described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks.

Treatment or prevention methods of this disclosure may be administered to a subject as part of a treatment course or regimen, which may comprise additional treatments prior to, or after, administration of the instantly disclosed unit doses, cells, or compositions. For example, in certain embodiments, a subject receiving a unit dose of the composition is receiving or had previously received a hematopoietic cell transplant (HCT; including myeloablative and non-myeloablative HCT). Techniques and regimens for performing HCT are known in the art and can comprise transplantation of any suitable donor cell, such as a cell derived from umbilical cord blood, bone marrow, or peripheral blood, a hematopoietic stem cell, a mobilized stem cell, or a cell from amniotic fluid. Accordingly, in certain embodiments, a composition of the present disclosure can be administered with or shortly after hematopoietic stem cells in a modified HCT therapy.

In further embodiments, the subject had previously received lymphodepleting chemotherapy prior to receiving the composition or HCT. In certain embodiments, a lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, anti-thymocyte globulin, or a combination thereof.

In certain embodiments, the subject had previously received one or more of surgery; radiation therapy, or chemotherapy, which therapies include those described herein or otherwise known in the art. In certain embodiments, for example, chemotherapy comprises vincristine, cisplatin, cyclophosphamide, filgrastim, etoposide, thiotepa, or any combination thereof.

Methods according to this disclosure may further include administering one or more additional agents to treat the disease or disorder in a combination therapy. For example, in certain embodiments, a combination therapy comprises administering a composition as provided herein (e.g. binding protein, polynucleotide, vector, host cell, composition, or immunogenic composition) with (concurrently, simultaneously, or sequentially) an immune checkpoint inhibitor. In some embodiments, a combination therapy comprises administering a composition with an agonist of a stimulatory immune checkpoint agent. In further embodiments, a combination therapy comprises administering a composition with a secondary therapy, such as chemotherapeutic agent, a radiation therapy, a surgery, an antibody, or any combination thereof.

As used herein, the term "immune suppression agent" or "immunosuppression agent" refers to one or more cells, proteins, molecules, compounds or complexes providing inhibitory signals to assist in controlling or suppressing an immune response. For example, immune suppression agents include those molecules that partially or totally block immune stimulation; decrease, prevent or delay immune activation; or increase, activate, or up regulate immune suppression. Exemplary immunosuppression agents to target (e.g., with an immune checkpoint inhibitor) include PD-1, PD-L1, PD- L2, LAG3, CTLA4, B7-H3, B7-H4, CD244/2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-IRA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

An immune suppression agent inhibitor (also referred to as an immune checkpoint inhibitor) may be a compound, an antibody, an antibody fragment or fusion polypeptide (e.g., Fc fusion, such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme or RNAi molecule, or a low molecular weight organic molecule. In any of the embodiments disclosed herein, a method may comprise a composition as provided herein (e.g. binding protein, polynucleotide, vector, host cell, composition, or immunogenic composition) with one or more inhibitor of any one of the following immune suppression components, singly or in any combination.

In certain embodiments, a composition is used in combination with a PD-1 inhibitor, for example a PD-1 -specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MED 10680 (formerly AMP-514), AMP -224, BMS-936558 or any combination thereof. In further embodiments, a composition of the present disclosure is used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof. In certain embodiments, a composition of the present disclosure is used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof. In certain embodiments, a composition is used in combination with an inhibitor of CTLA4. In particular embodiments, a composition is used in combination with a CTLA4 specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof. In certain embodiments, a composition is used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. A B7-H4 antibody binding fragment may be a scFv or fusion protein thereof, as described in, for example, Dangaj et al., Cancer Res. 73:4820, 2013, as well as those described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO /201640724A1 and WO 2013/025779A1. In certain embodiments, a composition is used in combination with an inhibitor of CD244. In certain embodiments, a composition is used in combination with an inhibitor of BLTA, HVEM, CD 160, or any combination thereof. Anti CD- 160 antibodies are described in, for example, PCT Publication No. WO 2010/084158. In certain embodiments, a composition is used in combination with an inhibitor of TIM3. In certain embodiments, a composition is used in combination with an inhibitor of Gal9. In certain embodiments, a composition is used in combination with an inhibitor of adenosine signaling, such as a decoy adenosine receptor. In certain embodiments, a composition is used in combination with an inhibitor of A2aR. In certain embodiments, a composition is used in combination with an inhibitor of KIR, such as lirilumab (BMS- 986015). In certain embodiments, a compositionis used in combination with an inhibitor of an inhibitory cytokine (typically, a cytokine other than TGFP) or Treg development or activity. In certain embodiments, a composition is used in combination with an IDO inhibitor, such as levo- 1 -methyl tryptophan, epacadostat (INCB024360; Liu et al., Blood 775:3520-30, 2010), ebselen (Terentis et al. , Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (l-MT)-tira- pazamine, or any combination thereof. In certain embodiments, a composition is used in combination with an arginase inhibitor, such as N(omega)-Nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)- amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine (BEC), or any combination thereof. In certain embodiments, a composition is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.). In certain embodiments, a composition is used in combination with an inhibitor of TIGIT such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both. In certain embodiments, a composition is used in combination with an inhibitor of PVRIG, PVRL2, or both. Anti- PVRIG antibodies are described in, for example, PCT Publication No.

WO 2016/134333. Anti-PVRL2 antibodies are described in, for example, PCT Publication No. WO 2017/021526. In certain embodiments, a composition is used in combination with a LAIR1 inhibitor. In certain embodiments, a composition is used in combination with an inhibitor of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof. In certain embodiments, a composition is used in combination with an agent that increases the activity (i.e., is an agonist) of a stimulatory immune checkpoint molecule. For example an engineered immune cell can be used in combination with a CD137 (4-1BB) agonist (such as, for example, urelumab), a CD134 (OX-40) agonist (such as, for example, MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, a CD27 agonist (such as, for example, CDX-1127), a CD28 agonist (such as, for example, TGN1412, CD80, or CD86), a CD40 agonist (such as, for example, CP-870,893, rhuCD40L, or SGN-40), a CD122 agonist (such as, for example, IL-2) an agonist of GITR (such as, for example, humanized monoclonal antibodies described in PCT Patent Publication No. WO 2016/054638), an agonist of ICOS (CD278) (such as, for example, GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof). In any of the embodiments disclosed herein, a method may comprise administering a composition one or more agonist of a stimulatory immune checkpoint molecule, including any of the foregoing, singly or in any combination.

In certain embodiments, a combination therapy comprises a composition and a secondary therapy comprising one or more of: an antibody or antigen binding-fragment thereof that is specific for a cancer antigen expressed by the non-inflamed solid tumor, a radiation treatment, a surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

In certain embodiments, a combination therapy method comprises administering a composition and further administering a radiation treatment or a surgery. Radiation therapy is well-known in the art and includes X-ray therapies, such as gammairradiation, and radiopharmaceutical therapies. Surgeries and surgical techniques appropriate to treating a given cancer in a subject are well-known to those of ordinary skill in the art. In certain embodiments, a combination therapy method comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent. A chemotherapeutic agent includes, but is not limited to, an inhibitor of chromatin function, a topoisomerase inhibitor, a microtubule inhibiting drug, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), a DNA synthesis inhibitor, a DNA interactive agent (such as an intercalating agent), and a DNA repair inhibitor. Illustrative chemotherapeutic agents include, without limitation, the following groups: anti-metabolites/anti-cancer agents, such as pyrimidine analogs (5 -fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2- chlorodeoxyadenosine (cladribine)); antiproliferative/antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, tri ethylenethiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L- asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative/antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates -busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes — dacarbazinine (DTIC); antiproliferative/antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers, toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disruptors.

Cytokines can be used to manipulate host immune response towards anticancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting immune anticancer or antitumor response include, for example, IFN-a, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, singly or in any combination with a composition of this disclosure.

The present disclosure also provides the following non-limiting Embodiments: Embodiment 1. An isolated binding protein that is capable of binding to a PDZ-binding kinase (PBK) peptide antigen:HLA complex, wherein the PBK peptide antigen comprises or consists of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3), and wherein, optionally, the binding comprises specific binding. Embodiment 2. The binding protein of Embodiment 1, wherein the HLA comprises HLA-A*02:01

Embodiment 3. An isolated binding protein that is capable of binding to a

PDZ-binding kinase (PBK) peptide antigen:HLA complex, wherein the PBK peptide antigen comprises or consists of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10), and wherein, optionally, the binding comprises specific binding.

Embodiment 4. The binding protein of Embodiment 3, wherein the HLA comprises HLA-B*07:02.

Embodiment 5. The binding protein of any one of Embodiments 1-4, comprising an immunoglobulin superfamily variable domain.

Embodiment 6. The binding protein of any one of Embodiments 1-5, comprising a TCR a-chain variable domain ( Vα) and/or a TCR β-chain variable domain (VP).

Embodiment 7. The binding protein of any one of Embodiments 1-5, comprising a heavy chain variable domain (VH) and/or a light chain variable domain (VL) of a TCR-mimic antibody.

Embodiment 8. The binding protein of any one of Embodiments 1, 2, and 5-7, comprising: (i) the amino acid sequence of SEQ ID NO.:34, wherein SEQ ID NO.:34 is optionally a complementarity determining region (CDR3)a; and/or (ii) the amino acid sequence of SEQ ID NO.:37, wherein SEQ ID NO.:37 is optionally a CDR3p.

Embodiment 9. The binding protein of Embodiment 8, further comprising:

(iii) the amino acid sequence of SEQ ID NO.:32, wherein SEQ ID NO.:32 is optionally a CDRla; (iv) the amino acid sequence of SEQ ID NO.:33, wherein SEQ ID NO.:33 is optionally a CDR2a; (v) the amino acid sequence of SEQ ID NO.:35, wherein SEQ ID NO.:35 is optionally a CDR1P; and/or (vi) the amino acid sequence of SEQ ID NO.:36, wherein SEQ ID NO.:36 is optionally a CDR2p.

Embodiment 10. The binding protein of any one of Embodiments 1, 2, 5, 6,

8, and 9, comprising: (1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:32-34, respectively; and (2) a TCR VP comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:35-37, respectively.

Embodiment 11. The binding protein of any one of Embodiments 6 and 8-

10, comprising: (a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs.:56, 64, 66, and 67; and/or (b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs.:57 and 65.

Embodiment 12. The binding protein of any one of Embodiments 6 and 8-

11, comprising the framework amino acid sequence set forth in any one of SEQ ID NOs.: 19-31, or any combination of these.

Embodiment 13. The binding protein of any one of Embodiments 1, 2, 5, 6, and 8-12, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.: (i) 56 and 57, respectively; (ii) 56 and 65, respectively; (iii) 64 and 57, respectively; (iv) 64 and 65, respectively; (v) 66 and 57, respectively; (vi) 66 and 65, respectively; (vii) 67 and 57, respectively; or (viii) 67 and 65, respectively.

Embodiment 14. The binding protein of any one of Embodiments 1, 2, 5, 6, and 8-13, comprising the amino acid sequences set forth in SEQ ID NOs.: (i) 11 and 12, respectively; (ii) 13 and 12, respectively; (iii) 15 and 12, respectively; (iv) 16 and 12, respectively; (v) 11 and 14, respectively; (vi) 13 and 14, respectively; (vii) 15 and 14, respectively; or (viii) 16 and 14, respectively.

Embodiment 15. The binding protein of any one of Embodiments 3-7, comprising: (i) the amino acid sequence of SEQ ID NO.:40, wherein SEQ ID NO.:40 is optionally a complementarity determining region (CDR3)a; and/or (ii) the amino acid sequence of SEQ ID NO.:43, wherein SEQ ID NO.:43 is optionally a CDR3p. Embodiment 16. The binding protein of Embodiment 15, further comprising:

(iii) the amino acid sequence of SEQ ID NO.:38, wherein SEQ ID NO.:38 is optionally a CDRla; (iv) the amino acid sequence of SEQ ID NO.:39, wherein SEQ ID NO.:39 is optionally a CDR2a; (v) the amino acid sequence of SEQ ID NO.:41, wherein SEQ ID NO.:41 is optionally a CDR1P; and/or (vi) the amino acid sequence of SEQ ID NO.:42, wherein, optionally, SEQ ID NO.:42 is optionally a CDR2p.

Embodiment 17. The binding protein of any one of Embodiments 3-6, 15, and 16, comprising: (1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:38-40, respectively; and (2) a TCR VP comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:41-43, respectively.

Embodiment 18. The binding protein of any one of Embodiments 3-6 and 15-17, comprising: (a)a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:58; and/or (b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:59.

Embodiment 19. The binding protein of any one of Embodiments 3-6 and 15-17, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.:58 and 59, respectively.

Embodiment 20. The binding protein of any one of Embodiments 1, 2, 5, and 6, comprising:

(i) a CDR3P according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; (ii) a CDR3a according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3;

(iii) any one or more of the Vα, CDRa, VP, CDRβ, and/or framework amino acid sequences set forth in any one of SEQ ID NOs. : 11-16 or 19-31, respectively, or a variant thereof having at least 90% identity thereto, or a variant thereof comprising one, two, or three amino acid substitutions, wherein, optionally, the Vα has at least 90% identity to a mature form of the Vα amino acid sequence in SEQ ID NO.: 11, 13, 15, or 16 with the signal sequence removed, and/or the Vβ has at least 90% identity to a mature form of the Vβ amino acid sequence in SEQ ID NO.: 12 or 14, with the signal sequence removed;

(iv) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; and/or

(v) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3, provided that

(a) at least three or four of the CDRs have no mutations;

(b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and

(c) the binding protein retains its ability to bind to a peptide comprising a MMTLSIPHI (SEQ ID NO.:3):HLA complex.

Embodiment 21. The binding protein of Embodiment 20, further comprising a CDRip, a CDR2P, a CDRla, and/or a CDR2a according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3. § Embodiment 22. The binding protein any one of Embodiments 3-6, wherein the encoded binding protein comprises:

(i) a CDR3β according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5;

(ii) a CDR3a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5;

(iii) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; and/or

(iv) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5, provided that

(a) at least three or four of the CDRs have no mutations;

(b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and

(c) the encoded binding protein retains its ability to bind to a RPSAAHIVEA (SEQ ID NO.:10):HLA complex.

Embodiment 23. The binding protein of Embodiment 22, wherein the encoded binding protein further comprises a CDRip, a CDR2P, a CDRlα, and/or a CDR2a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5.

Embodiment 24. An isolated binding protein that is capable of binding to an OPA-interacting protein 5 (OIP5) peptide antigen:HLA complex, wherein the OIP5 peptide antigen comprises or consists of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5), wherein, optionally, the binding comprises specific binding.

Embodiment 25. The binding protein of Embodiment 24, wherein the HL A comprises HLA-A*02:01.

Embodiment 26. The binding protein of Embodiment 24 or 25, comprising an immunoglobulin superfamily variable domain.

Embodiment 27. The binding protein of any one of Embodiments 24-26, comprising a TCR a-chain variable domain ( Vα) and a TCR β-chain variable domain (VP). Embodiment 28. The binding protein of any one of Embodiments 24-26, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) of a TCR-mimic antibody.

Embodiment 29. The binding protein of any one of Embodiments 24-28, comprising: (i) the amino acid sequence of SEQ ID NO.:46, wherein SEQ ID NO.:46 is optionally a complementarity determining region (CDR3)a; and/or (ii) the amino acid sequence of SEQ ID NO.:49, wherein SEQ ID NO.:49 is optionally a CDR3p.

Embodiment 30. The binding protein of Embodiment 29, further comprising:

(iii) the amino acid sequence of SEQ ID NO.:44, wherein SEQ ID NO.:44 is optionally a CDRla; (iv) the amino acid sequence of SEQ ID NO.:45, wherein SEQ ID NO.:45 is optionally a CDR2a; (v) the amino acid sequence of SEQ ID NO.:47, wherein SEQ ID NO.:47 is optionally a CDRIP; and/or (vi) the amino acid sequence of SEQ ID NO.:48, wherein SEQ ID NO.:48 is optionally a CDR2p.

Embodiment 31. The binding protein of any one of Embodiments 24-27 and 29-30, comprising: (1) a TCR Vα comprising the CDRlα, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:44-46, respectively; and (2) a TCR VP comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:47-49, respectively.

Embodiment 32. The binding protein of Embodiment 29 or 30, comprising: a TCR Vα comprising

(1) the CDR3a amino acid sequence of SEQ ID NO. :46;

(2) the CDRla amino acid sequence of SEQ ID NO.:44 or 50;

(3) the CDR2a amino acid sequence of SEQ ID NO.:45 or 51; and a TCR Vβ comprising

(4) the CDR3P amino acid sequence of SEQ ID NO.:49;

(5) the CDRip amino acid sequence of SEQ ID NO.:47 or 53; and

(6) the CDR2 P amino acid sequence of SEQ ID NO. :48 or 54.

Embodiment 33. The binding protein of any one of Embodiments 24-27 and 29-32, comprising: (a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:60; and/or

(b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:61.

Embodiment 34. The binding protein of any one of Embodiments 24-27 and 29-33, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.:60 and 61, respectively.

Embodiment 35. The binding protein of any one of Embodiments 24-28, comprising:

(i) the amino acid sequence of SEQ ID NO.: 52, wherein SEQ ID NO.: 52 is optionally a complementarity determining region (CDR3)a; and/or

(ii) the amino acid sequence of SEQ ID NO.:55, wherein SEQ ID NO.:55 is optionally a CDR3p.

Embodiment 36. The binding protein of Embodiment 33, further comprising:

(iii) the amino acid sequence of SEQ ID NO.: 50, wherein SEQ ID NO.: 50 is optionally a CDRla;

(iv) the amino acid sequence of SEQ ID NO.:51, wherein SEQ ID NO.:51 is optionally a CDR2a;

(v) the amino acid sequence of SEQ ID NO.:53, wherein SEQ ID NO.:53 is optionally a CDR1P; and/or

(vi) the amino acid sequence of SEQ ID NO.: 54, wherein SEQ ID NO.: 54 is optionally a CDR2p.

Embodiment 37. The binding protein of any one of Embodiments 24-27 and 35-36, comprising:

(1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:50-52, respectively; and (2) a TCR Vβ comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:53-55, respectively.

Embodiment 38. The binding protein of Embodiment 35, comprising: a TCR Vα comprising

(1) the CDR3a amino acid sequence of SEQ ID NO.:52;

(2) the CDRla amino acid sequence of SEQ ID NO.:44 or 50;

(3) the CDR2a amino acid sequence of SEQ ID NO.:45 or 51; and a TCR Vβ comprising

(4) the CDR3P amino acid sequence of SEQ ID NO.:55;

(5) the CDRip amino acid sequence of SEQ ID NO.:47 or 53; and

(6) the CDR2 P amino acid sequence of SEQ ID NO. :48 or 54.

Embodiment 39. The binding protein of any one of Embodiments 24-27 and 35-38, comprising:

(a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:62; and/or

(b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:63.

Embodiment 40. The binding protein of any one of Embodiments 24-27 and 35-39, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.:62 and 63, respectively.

Embodiment 41. The binding protein of any one of Embodiments 1-40, further comprising a TCR a-chain constant domain (Ca), a TCR β-chain constant domain, (CP), or both, wherein, optionally, the Vβ and the Cβ together comprise a TCR β chain and/or the Vα and the Ca together comprise a TCR α chain.

Embodiment 42. The binding protein of Embodiment 41, wherein the Ca comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:68.

Embodiment 43. The binding protein of Embodiment 41 or 42, wherein the Cβ comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:69 or 70.

Embodiment 44. The binding protein of any one of Embodiments 41-43, wherein the Cβ and/or the Cα comprises one or more non-native amino acid at a position such that when the the Cβ and the Ca associate to form a dimer, a non-native disulfide bond is formed between the Cβ and the Ca, wherein, optionally, the non- native amino acid comprises a cysteine in the Cβ and/or a cysteine in the Ca, wherein, further optionally, the Cβ comprises a cysteine amino acid at amino acid position 57 and/or the Ca comprises a cysteine amino acid at amino acid position 48.

Embodiment 45. The binding protein of any one of Embodiments 1-44, wherein: (1) the binding protein comprises a TCR, a scTv, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), a TCR-mimic antibody or antigen-binding fragment thereof, or any combination thereof; and/or (2) wherein a substitution mutation in SEQ ID NO.:3 of any of the following amino acids of SEQ ID NO.:3 to alanine does not substantially reduce binding by the binding protein: the amino acid at position 1 of SEQ ID NO.:3, the amino acid at position 3 of SEQ ID NO.:3, the amino acid at position 4 of SEQ ID NO.:3, the amino acid at position 9 of SEQ ID NO.:3.

Embodiment 46. The binding protein of Embodiment 45, wherein binding protein comprises a TCR.

Embodiment 47. The binding protein of Embodiment 45, wherein the binding protein comprises a scTv.

Embodiment 48. The binding protein of Embodiment 45, wherein the binding protein comprises a scTCR.

Embodiment 49. The binding protein of Embodiment 45, wherein the binding protein comprises a CAR. Embodiment 50. An isolated polynucleotide encoding the binding protein of any one of Embodiments 1-49.

Embodiment 51. The polynucleotide of Embodiment 50, wherein the polynucleotide is codon-optimized for expression in a host cell, wherein, optionally, the host cell comprises an immune system cell, wherein, further optionally, the immune system cell comprises a T cell, a NK-T cell, or a NK cell.

Embodiment 52. The polynucleotide of Embodiment 51, further comprising:

(i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

Embodiment 53. The polynucleotide of Embodiment 52, comprising: (a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain; (b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor p chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).

Embodiment 54. The polynucleotide of Embodiment 52 or 53, further comprising a polynucleotide that encodes a self-cleaving peptide and is disposed between:

(1) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain; and/or

(2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain.

Embodiment 55. The polynucleotide of any one of Embodiments 52-54, comprising, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)- (pnBP); (ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnBP); (iii) (pnBP)-(pnSCPi)- (pnCD8a)-(pnSCP2)-(pnCD8P); (iv) (pnBP)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnCD8a); (v) (pnCD8a)-(pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8P); or (vi) (pnCD8P)-(pnSCPi)- (pnBP)-(pnSCP2)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnBP is the polynucleotide encoding a binding protein, and wherein pnSCPi and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptides are optionally the same or different.

Embodiment 56. The polynucleotide of any one of Embodiments 50-55, wherein the encoded binding protein comprises a TCRα chain and a TCRβ chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRα chain and the polynucleotide encoding a TCRβ chain, wherein, optionally, the polynucleotide comprises, operably linked in-frame: (i) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRP)-(pnSCP3)- (pnTCRa); (ii)(pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRP)-(pnSCP3)- (pnTCRa); (iii) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)- (pnTCRP); (iv) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)- (pnTCRP); (v) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)-(pnCD8a)-(pnSCP3)- (pnCD8p); (vi) (pnTCRp)-(pnSCPi)-(pnTCRa)-(pnSCP₂)-(pnCD8p)-(pnSCP3)- (pnCD8a); (vii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8a)-(pnSCP3)- (pnCD8P); or (viii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8P)-(pnSCP3)- (pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnTCRa is the polynucleotide encoding a TCR α chain, wherein pnTCRP is the polynucleotide encoding a TCR β chain, and wherein pnSCPi, pnSCP2, and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptides are further optionally the same or different.

Embodiment 57. The polynucleotide of any one of Embodiments 50-56, wherein the polynucleotide comprises DNA, RNA (optionally mRNA), or both. Embodiment 58. The polynucleotide of Embodiment 57, comprising DNA.

Embodiment 59. A vector comprising the polynucleotide of any one of

Embodiments 50-58.

Embodiment 60. The vector of Embodiment 59, wherein the vector comprises a viral vector.

Embodiment 61. The vector of Embodiment 60, wherein the viral vector comprises a lentiviral vector or a y-retroviral vector.

Embodiment 62. The vector of any one of Embodiments 59-61, wherein the vector is capable of delivering the polynucleotide to a host cell.

Embodiment 63. The vector of Embodiment 62, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

Embodiment 64. The vector of Embodiment 63, wherein the human immune system cell is a CD4⁺ T cell, a CD8⁺ T cell, a CD4'CD8‘ double negative T cell, a yδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof.

Embodiment 65. The vector of Embodiment 64, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

Embodiment 66. A host cell comprising the polynucleotide of any one of Embodiments 50-58 and/or the vector of any one of Embodiments 59-65, and/or express the binding protein of any one of Embodiments 1-49.

Embodiment 67. The host cell of Embodiment 66, wherein the modified cell comprises a hematopoietic progenitor cell and/or an immune cell, optionally a human immune cell.

Embodiment 68. The host cell of Embodiment 67, wherein the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof.

Embodiment 69. The host cell of Embodiment 68, wherein the immune cell comprises a CD4⁺ T cell, a CD8⁺ T cell, a CD4" CD8" double negative T cell, a yδ T cell, or any combination thereof, wherein, optionally, the immune cell comprises a CD4⁺ T cell and a CD8⁺ T cell, wherein, further optionally, the CD4⁺ T cell, the CD8⁺ T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor p chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

Embodiment 70. The host cell of Embodiment 68 or 69, wherein the immune cell comprises a CD8⁺ T cell and/or a CD4⁺ T cell.

Embodiment 71. The host cell of any one of Embodiments 66-70, wherein the modified cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TIGIT gene; a TCR component gene, a FasL gene, or any combination thereof.

Embodiment 72. The host cell of Embodiment 71, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an α2 macroglobulin gene, an α3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.

Embodiment 73. The host cell of Embodiment 71 or 72, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.

Embodiment 74. The host cell of any one of Embodiments 66-73, wherein the polynucleotide encoding the binding protein is heterologous to the host cell and is comprised in an endogenous TCR gene locus.

Embodiment 75. The host cell of any one of Embodiments 66-74, further comprising a heterologous polynucleotide encoding: (i) a safety switch protein; (ii) a selection marker; (iii) a CD8 co-receptor β-chain; (iv) a CD8 co-receptor a-chain; or (v) any combination thereof.

Embodiment 76. A composition, comprising: (i) the binding protein of any one of Embodiments 1-49; (ii) the polynucleotide of any one of Embodiments 50-58; (iii) the vector of any one of Embodiments 59-65; and/or (iv) the host cell of any one of Embodiments 66-75, optionally comprising CD4+ T cells, CD8+ T cells, or both, and a pharmaceutically acceptable carrier, excipient, or diluent.

Embodiment 77. The composition of Embodiment 76, comprising the host cell, wherein the host cell comprises an immune cell, optionally CD8+ T cells and/or CD4+ T cells, wherein, further optionally, the CD8+ T cells and CD4+ T cells are present in about a 1 :1 ratio, and/or the composition comprises substantially no naive T cells.

Embodiment 78. A method for treating a disease or disorder associated with expression of (1) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3), (2) a PBK antigen comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO. : 10), and/or (3) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5) in a subject, the method comprising administering to the subject an effective amount of: (i) the binding protein of any one of Embodiments 1-49; (ii) the polynucleotide of any one of Embodiments 50-58; (iii) the vector of any one of Embodiments 59-65; (iv) the host cell of any one of Embodiments 66-75; and/or (v) the composition of Embodiment 76 or 77.

Embodiment 79. The binding protein of any one of Embodiments 1-49, the polynucleotide of any one of Embodiments 50-58, the vector of any one of Embodiments 59-65, the host cell of any one of Embodiments 66-75, and/or the composition of Embodiment 76 or 77, for use in a method of treating a disease or disorder associated with expression of (1) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3), (2) a PBK antigen comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10), and/or (3) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5) in a subject.

Embodiment 80. The binding protein of any one of Embodiments 1-49, the polynucleotide of any one of Embodiments 50-58, the vector of any one of Embodiments 59-65, the host cell of any one of Embodiments 66-75, and/or the composition of Embodiment 76 or 77, for use in the manufacture of a medicament for treating a disease or disorder associated with expression of (1) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3), (2) a PBK antigen comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10), and/or

(3) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5) in a subject.

Embodiment 81. The method of Embodiment 78 or the binding protein, polynucleotide, vector, host cell, and/or composition for use of Embodiment 79 or 80, wherein the subject is HLA-A*02:01⁺ or HLA-B*07:02⁺.

Embodiment 82. The method of Embodiment 78 or 81 or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-81, wherein the disease or condition is a cancer.

Embodiment 83. The method of Embodiment 78, 81, or 82, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-82, wherein the cancer comprises a solid tumor or a hematological malignancy.

Embodiment 84. The method of Embodiment 78, 81, 82, or 83, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-83, wherein the cancer comprises glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, neuroblastoma, hepatoblastoma, PNET, glioma, cranipharyngioma, choroid plexus tumors, schwannomas, meningiomas, Wilm’s tumor, germ cell tumors, hepatocellular carcinoma, or any combination thereof.

Embodiment 85. The method of Embodiment 78, 81, 82, 83, or 84, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-84, wherein the subject is human.

Embodiment 86. The method of Embodiment 78, 81, 82, 83, 84, or 85, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-85, wherein the subject is a pediatric subject. Embodiment 87. The method of Embodiment 78, 81, 82, 83, 84, 85, or 86, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-86, wherein the subject has previously received one or more of: (i) surgery; (ii) radiation therapy; (iii) chemotherapy; or (iv) a hematopoietic stem cell transplant (HSC).

Embodiment 88. The method of Embodiment 78, 81, 82, 83, 84, 85, 86, or

87, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-87, wherein one or more of the modified immune cells comprised in the composition is autologous to the subject.

Embodiment 89. The method of Embodiment 78, 81, 82, 83, 84, 85, 86, 87, or 88, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-88, wherein the method further comprises administering an inhibitor of an immune checkpoint molecule to the subject.

Embodiment 90. The method of Embodiment 78, 81, 82, 83, 84, 85, 86, 87,

88, or 89, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of Embodiments 79-89, wherein the method comprises administering the binding protein, polynucleotide, vector, modified host cell, and/or composition to the subject intrathecally, intracerebroventricularly, into the cerebrospinal fluid (CSF), intracranially, intraventricularly, intravenously, locally, or systemically.

Embodiment 91. An immunogenic composition, comprising: (i) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10); (iii) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); (iv) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5); and/or (v) a variant of the isolated peptide or polypeptide of SEQ ID NO.:3, SEQ ID NO.: 10, SEQ ID NO.:4, or SEQ ID NO.:5, wherein the variant comprises one, two, or three differences in amino acid sequence as compared to SEQ ID NO.:3, 10, 4, or 5, respectively, wherein the isolated peptide or polypeptide of any one of (i)-(vii) does not comprise an isolated full-length human PBK or OIP5.

Embodiment 92. The immunogenic composition of Embodiment 91, wherein (a) one or more copies of any one of (i)-(v) and/or (b) one or more of any of (i)-(v) is/are present in a fusion polypeptide, wherein the fusion polypeptide optionally further comprises the amino acid sequence of a self-cleaving peptide.

Embodiment 93. The immunogenic composition of Embodiment 91 or 92, wherein the immunogenic composition is capable of eliciting an immune response in a subject against cancer cells, wherein, optionally, the cancer cells comprise glioblastoma cells, medulloblastoma cells, breast cancer cells, colorectal cancer cells, lung cancer cells, esophageal cancer cells, lymphoma cells, leukemia (e.g., acute myeloid leukemia) cells, melanoma cells, cholangiocarcinoma cells, hepatocellular carcinoma cells, or any combination thereof.

Embodiment 94. The immunogenic composition of any one of Embodiments 91-93, further comprising an adjuvant.

Embodiment 95. An isolated polynucleotide encoding the immunogenic composition of any one of Embodiments 91-93, wherein the polynucleotide is optionally contained in a vector.

Embodiment 96. The polynucleotide of Embodiment 95, wherein the polynucleotide is codon-optimized for expression in a host cell, wherein the host cell is optionally a dendritic cell or a T cell.

Embodiment 97. A host cell comprising the isolated polynucleotide of Embodiment 95 or 96, wherein the polynucleotide is heterologous to the host cell, and wherein the host cell is optionally an immune cell and is further optionally a professional antigen-presenting cell.

Embodiment 98. The host cell of Embodiment 97, wherein the host cell is a dendritic cell or a T cell.

Embodiment 99. A method of eliciting an immune response in a subject against glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, and/or hepatocellular carcinoma, the method comprising administering to the subject the binding protein of any one of Embodiments 1-49, the polynucleotide of any one of Embodiments 50-58, the vector of any one of Embodiments 59-65, the host cell of any one of Embodiments 66-75, the composition of Embodiment 76 or 77, the immunogenic composition of any one of Embodiments 91-94, and/or the host cell of Embodiment 98 or 99.

Embodiment 100. A method for expanding a population of T cells that bind to a peptide (e.g., a peptide comprised in a peptide:HLA complex) selected from: (i) a peptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) a peptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO. : 10); (iii) a peptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); and (iv) a peptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5), the method comprising contacting a sample comprising one or more T cells that bind to the peptide with the immunogenic composition of any one of Embodiments 91-94, the polynucleotide of Embodiment 95 or 96, the host cell of Embodiment 97 or 98, and/or antigen-presenting cells that express or have been contacted with a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:3-5 and 10.

Embodiment 101. A method for generating and/or isolating T cells, the method comprising contacting a sample comprising T cells, wherein the sample optionally comprises peripheral blood cells, with: (a) an immunogenic composition of any one of Embodiments 91-94; and/or (b) antigen-presenting cells (APCs) that express or have been contacted with: (i) a peptide or polypeptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) a peptide or polypeptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10); (iii) a peptide or polypeptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); and (iv) a peptide or polypeptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5); or (v) any combination of (i)-(iv); and optionally sorting T cells from other cells in the sample, thereby isolating and/or generating T cells.

Embodiment 102. A T cell isolated and/or generated by the method of Embodiment 101.

The present disclosure also provides the following non-limiting Additional Embodiments:

Additional Embodiment 1. A modified immune cell, comprising a heterologous polynucleotide encoding a binding protein that includes a T cell receptor (TCR) a-chain variable ( Vα) domain and a TCR β-chain variable (VP) domain, wherein the encoded binding protein is capable of specifically binding to a PDZ-binding kinase (PBK) antigen:HLA complex, wherein the PBK antigen comprises or consists of the amino acid sequence MMTLSIPHI (SEQ ID NO:3).

Additional Embodiment 2. The modified immune cell of Additional Embodiment 1, wherein the encoded binding protein comprises: (i) a CDR3P according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; (ii) a CDR3a according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3;

(iii) any one or more of the Vα, CDRa, VP, CDRP, and/or framework amino acid sequences set forth in any one of SEQ ID NOs.: 11-16 or 19-31, respectively, or a variant thereof having at least 90% identity thereto, or a variant thereof comprising one, two, or three amino acid substitutions, wherein, optionally, the Vα has at least 90% identity to a mature form of the Vα amino acid sequence in SEQ ID NO.: 11, 13, 15, or 16 with the signal sequence removed, and/or the Vβ has at least 90% identity to a mature form of the Vβ amino acid sequence in SEQ ID NO.: 12 or 14, with the signal sequence removed;

(iv) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; and/or (v) a V dβomain having at least 90% amino acid identity to the Vβ domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3, provided that (a) at least three or four of the CDRs have no mutations; (b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and (c) the encoded binding protein retains its ability to bind to a peptide comprising a MMTLSIPHLHLA complex.

Additional Embodiment 3. The modified immune cell of Additional Embodiment 1 or 2, wherein the encoded binding protein further comprises a CDRip, a CDR2P, a CDRla, and/or a CDR2a according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3.

Additional Embodiment 4. A modified immune cell, comprising a heterologous polynucleotide encoding a binding protein that includes a T cell receptor (TCR) α chain variable ( Vα) domain and a TCR β-chain variable (VP) domain, wherein the encoded binding protein is capable of specifically binding to an OPA-interacting protein 5 (OIP5) antigen:HLA complex, wherein the OIP5 antigen comprises or consists of the amino acid sequence SLMKILSEV (SEQ ID NO:4) or KSLMKILSEV (SEQ ID NO:5).

Additional Embodiment 5. The modified immune cell of Additional Embodiment 4, wherein the encoded binding protein comprises: (i) a CDR3P according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; (ii) a CDR3a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; (iii) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; and/or (iv) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5, provided that (a) at least three or four of the CDRs have no mutations; (b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and (c) the encoded binding protein retains its ability to bind to a SLMKILSEV:HLA complex and/or a KSLMKILSEV:HLA complex.

Additional Embodiment 6. The modified immune cell of Additional Embodiment 4 or 5, wherein the encoded binding protein further comprises a CDRip, a CDR2β, a CDRl,, and/or a CDR2a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5.

Additional Embodiment 7. The modified immune cell of any one of Additional Embodiments 1-6, wherein the HLA comprises HLA-A*02:01 or HLA- B*07:02.

Additional Embodiment 8. The modified immune cell of any one of Additional Embodiments 1-7, wherein the encoded binding protein comprises a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or any combination thereof.

Additional Embodiment 9. The modified immune cell of Additional Embodiment 8, wherein the encoded binding protein comprises a TCR.

Additional Embodiment 10. The modified immune cell of Additional Embodiment 8, wherein the encoded binding protein comprises a scTCR.

Additional Embodiment 11. The modified immune cell of Additional Embodiment 8, wherein the encoded binding protein comprises a CAR.

Additional Embodiment 12. The modified immune cell of any one of Additional Embodiments 1-11, further comprising a polynucleotide that encodes a TCR β-chain constant domain (CP), a polynucleotide that encodes a TCR a-chain constant domain (Ca), or both.

Additional Embodiment 13. The modified immune cell of Additional Embodiment 12, comprising a polynucleotide that encodes a TCR Cβ and a polynucleotide that encodes a TCR Ca, wherein the encoded TCR Cβ comprises a cysteine amino acid at amino acid position 57, and wherein the encoded TCR Ca comprises a cysteine amino acid at amino acid position 48.

Additional Embodiment 14. The modified immune cell of any one of Additional Embodiments 1-13, wherein the immune cell comprises a T cell, aNK cell, a NK-T cell, or any combination thereof. Additional Embodiment 15. The modified immune cell of Additional

Embodiment 14, wherein the immune cell comprises a CD8⁺ T cell and/or a CD4⁺ T cell.

Additional Embodiment 16. The modified immune cell of any one of Additional Embodiments 1-15, wherein the heterologous polynucleotide encoding the binding protein is codon optimized.

Additional Embodiment 17. The modified immune cell of any one of Additional Embodiments 1-16, wherein the immune cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TCR component gene; or any combination thereof.

Additional Embodiment 18. The modified immune cell of Additional Embodiment 17, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene; an α2 macroglobulin gene; an α3 macroglobulin gene; a pi microglobulin gene; or a P2 microglobulin gene; or any combination thereof.

Additional Embodiment 19. The modified immune cell of Additional Embodiment 17 or 18, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene; a TCR P variable region gene; a TCR constant region gene; or any combination thereof.

Additional Embodiment 20. The modified immune cell of any one of Additional Embodiments 1-19, further comprising a heterologous polynucleotide encoding: (i) a safety switch protein; (ii) a selection marker; (iii) a CD8 co-receptor P- chain; (iv) a CD8 co-receptor a-chain; or (v) any combination thereof.

Additional Embodiment 21. A composition, comprising a modified immune cell of any one of Additional Embodiments 1-20 and a pharmaceutically acceptable carrier, diluent, or excipient.

Additional Embodiment 22. A method for treating a disease or disorder associated with (i) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3) or (ii) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4) or KSLMKILSEV (SEQ ID NO:5) in a subject, the method comprising administering to the subject an effective amount of a composition of Additional Embodiment 21, provided that the composition comprises a modified immune cell specific for the antigen, thereby treating the disease or condition.

Additional Embodiment 23. The method of Additional Embodiment 22, wherein the subject is HLA-A*02:01⁺ or HLA0B*07:02⁺.

Additional Embodiment 24. The method of Additional Embodiment 22 or 23, wherein the disease or condition is a cancer.

Additional Embodiment 25. The method of Additional Embodiment 24, wherein the cancer comprises a solid tumor or a hematological malignancy.

Additional Embodiment 26. The method of Additional Embodiment 24 or 25, wherein the cancer comprises glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, neuroblastoma, hepatoblastoma, PNET, glioma, cranipharyngioma, choroid plexus tumors, schwannomas, meningiomas, Wilm’s tumor, germ cell tumors, and/or hepatocellular carcinoma.

Additional Embodiment 27. The method of any one of Additional Embodiments 22-26, wherein the subject is human.

Additional Embodiment 28. The method of Additional Embodiment 27, wherein the subject is a pediatric subject.

Additional Embodiment 29. The method of any one of Additional Embodiments 22-28, wherein the subject has previously received one or more of: (i) surgery; (ii) radiation therapy; (iii) chemotherapy; or (iv) a hematopoietic stem cell transplant (HSC).

Additional Embodiment 30. The method of any one of Additional Embodiments 22-29, wherein one or more of the modified immune cells comprised in the composition is autologous to the subject.

Additional Embodiment 31. The method of any one of Additional Embodiments 22-30, further comprising administering an inhibitor of an immune checkpoint molecule to the subject. Additional Embodiment 32. An isolated polynucleotide encoding a binding protein that includes a T cell receptor (TCR) α chain variable ( Vα) domain and a TCR β-chain variable (Vβ) domain, wherein the encoded binding protein is capable of specifically binding to a PDZ-binding kinase (PBK) antigen:HLA complex, wherein the PBK antigen comprises or consists of the amino acid sequence MMTLSIPHI (SEQ ID NO:3), wherein the polynucleotide is codon optimized for expression in a host cell.

Additional Embodiment 33. The isolated polynucleotide of Additional Embodiment 32, wherein the encoded binding protein comprises: (i) a CDR3P according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; (ii) a CDR3α according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; (iii)any one or more of the Vα, CDRa, VP, CDRP, and/or framework amino acid sequences set forth in any one of SEQ ID NOs. : 11 - 16 or 19-31 , respectively, or a variant thereof having at least 90% identity thereto, or a variant thereof comprising one, two, or three amino acid substitutions, wherein, optionally, the Vα has at least 90% identity to a mature form of the Vα amino acid sequence in SEQ ID NO.: 11, 13, 15, or 16 with the signal sequence removed, and/or the Vβ has at least 90% identity to a mature form of the Vβ amino acid sequence in SEQ ID NO.: 12 or 14, with the signal sequence removed; (iv) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; and/or (v) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3, provided that

(a) at least three or four of the CDRs have no mutations; (b)the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and (c) the encoded binding protein retains its ability to bind to a MMTLSIPHLHLA complex.

Additional Embodiment 34. The isolated polynucleotide of Additional Embodiment 32 or 33, wherein the encoded binding protein further comprises a CDRip, a CDR2β, a CDRla, and/or a CDR2a according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3.

Additional Embodiment 35. An isolated polynucleotide encoding a binding protein that includes a T cell receptor (TCR) α chain variable ( Vα) domain and a TCR β-chain variable (VP) domain, wherein the encoded binding protein is capable of specifically binding to an OPA-interacting protein 5 (OIP5) antigen:HLA complex, wherein the OIP5 antigen comprises or consists of the amino acid sequence SLMKILSEV (SEQ ID NO:4) or KSLMKILSEV (SEQ ID NO:5), wherein the polynucleotide is codon optimized for expression in a host cell.

Additional Embodiment 36. The isolated polynucleotide of Additional Embodiment 35, wherein the encoded binding protein comprises: (i) a CDR3P according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; (ii) a CDR3a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; (iii) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; and/or (iv) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5, provided that (a) at least three or four of the CDRs have no mutations; (b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and (c) the encoded binding protein retains its ability to bind to a SLMKILSEV:HLA complex and/or a KSLMKILSEV: HL A complex.

Additional Embodiment 37. The isolated polynucleotide of Additional Embodiment 35 or 36, wherein the encoded binding protein further comprises a CDRip, a CDR2P, a CDRla, and/or a CDR2a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5. Additional Embodiment 38. The isolated polynucleotide of any one of Additional Embodiments 32-37, wherein the HLA comprises HLA A*02:01 or HLA- B*07:02.

Additional Embodiment 39. The isolated polynucleotide of any one of Additional Embodiments 32-38, wherein the encoded binding protein comprises a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or any combination thereof.

Additional Embodiment 40. The isolated polynucleotide of Additional Embodiment 39, wherein the encoded binding protein comprises a TCR.

Additional Embodiment 41. The isolated polynucleotide of Additional Embodiment 39, wherein the encoded binding protein comprises a scTCR.

Additional Embodiment 42. The isolated polynucleotide of Additional Embodiment 39, wherein the encoded binding protein comprises a CAR.

Additional Embodiment 43. The isolated polynucleotide of any one of Additional Embodiments 32-42, further comprising a polynucleotide that encodes a TCR β-chain constant domain (CP), a polynucleotide that encodes a TCR a-chain constant domain (Ca), or both.

Additional Embodiment 44. The isolated polynucleotide of Additional Embodiment 43, comprising a polynucleotide that encodes a TCR Cβ and a polynucleotide that encodes a TCR Ca, wherein the encoded TCR Cβ comprises a cysteine amino acid at amino acid position 57, and wherein the encoded TCR Ca comprises a cysteine amino acid at amino acid position 48.

Additional Embodiment 45. The isolated polynucleotide of any one of Additional Embodiments 32-44, wherein the polynucleotide is codon-optimized for expression in an immune cell.

Additional Embodiment 46. The isolated polynucleotide of Additional Embodiment 45, wherein the immune cell is a T cell, a NK cell, or a NK-T cell.

Additional Embodiment 47. The isolated polynucleotide of Additional Embodiment 46, wherein the immune cell comprises a CD8⁺ T cell and/or a CD4⁺ T cell. Additional Embodiment 48. The isolated polynucleotide of any one of Additional Embodiments 32-47, comprising a polynucleotide encoding a self-cleaving peptide disposed between the Vβ-encoding polynucleotide and the Vα-encoding polynucleotide, or disposed between the TCR β chain-encoding polynucleotide and the TCR α chain-encoding polynucleotide.

Additional Embodiment 49. A vector comprising the isolated polynucleotide of any one of Additional Embodiments 32-48.

Additional Embodiment 50. An immunogenic composition, comprising: (i) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); (iii) an OIP5 antigen comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO: 5); (iv) a variant peptide of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, wherein the variant comprises one, two, or three differences in amino acid sequence as compared to SEQ ID NO:3, 4, or 5, respectively.

Additional Embodiment 51. The immunogenic composition of Additional Embodiment 50, wherein (a) two or more copies of any one of (i)-(vi) and/or (b) two or more of any of (i)-(vi) are present in a fusion polypeptide, wherein the fusion polypeptide optionally further comprises an amino acid sequence of a self-cleaving peptide.

Additional Embodiment 52. The immunogenic composition of Additional Embodiment 50 or 51, wherein the immunogenic composition is capable of eliciting an immune response against glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer, esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, and/or hepatocellular carcinoma.

Additional Embodiment 53. The immunogenic composition of any one of Additional Embodiments 50-52, further comprising an adjuvant.

Additional Embodiment 54. An isolated polynucleotide encoding the immunogenic composition of any one of Additional Embodiments 50-52, wherein the polynucleotide is optionally contained in a vector. Additional Embodiment 55. The isolated polynucleotide of Additional Embodiment 54, wherein the polynucleotide is codon-optimized for expression in a host cell, wherein the host cell is optionally a dendritic cell or a T cell.

Additional Embodiment 56. A host cell comprising the isolated polynucleotide of Additional Embodiment 54 or 55, wherein the host cell is optionally an immune cell and is further optionally a professional antigen-presenting cell.

Additional Embodiment 57. The host cell of Additional Embodiment 56, wherein the host cell is a dendritic cell or a T cell.

Additional Embodiment 58. A method of eliciting an immune response in a subject against glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, and/or hepatocellular carcinoma, the method comprising administering to the subject the modified immune cell of any one of Additional Embodiments 1-20, the composition of Additional Embodiment 21, the polynucleotide of any one of Additional Embodiments 32-48, the immunogenic composition of any one of Additional Embodiments 50-53, and/or the host cell of Additional Embodiment 56 or 57.

Additional Embodiment 59. A method for expanding a population of T cells that specifically bind to an antigen selected from: (i) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) a OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); and/or (iii) a OIP5 antigen comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5), the method comprising contacting a sample comprising one or more T cells that specifically bind to the antigen with the immunogenic composition of any one of Additional Embodiments 50-53.

Additional Embodiment 60. A method for generating and/or isolating T cells, the method comprising contacting peripheral blood cells with: (a) an immunogenic composition of any one of Additional Embodiments 50-52; and/or (b) antigen- presenting cells (APCs) that have been pulsed with (i) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3); (ii) a OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID

NO:4); (iii) a OIP5 antigen comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO: 5); or

(iv) any combination of (i)-(iii); and optionally sorting T cells from the peripheral blood cells, thereby isolating and/or generating T cells.

SEQUENCES

SEQ ID NO:1 (human PBK, isoform 1 amino acid sequence)

MEGISNFKTP SKLSEKKKSV LCSTPTINIP ASPFMQKLGF GTGVNVYLMK RSPRGLSHSP WAVKKINPIC NDHYRSVYQK RLMDEAKILK SLHHPNIVGY RAFTEANDGS LCLAMEYGGE KSLNDLIEER YKASQDPFPA AIILKVALNM ARGLKYLHQE KKLLHGDIKS SNVVIKGDFE TIKICDVGVS LPLDENMTVT DPEACYIGTE PWKPKEAVEE NGVITDKADI FAFGLTLWEM MTLSIPHINL SNDDDDEDKT FDESDFDDEA YYAALGTRPP INMEELDESY QKVIELFSVC

TNEDPKDRPS A AHIVEALET DV

SEQ ID NO:2 (human OIP5, isoform 1 amino acid sequence)

MAAQPLRHRS RCATPPRGDF CGGTERAIDQ ASFTTSMEWD TQVVKGSSPL GPAGLGAEEP AAGPQLPSWL QPERCAVFQC AQCHAVLADS VHLAWDLSRS LGAVVFSRVT NNVVLEAPFL VGIEGSLKGS TYNLLFCGSC GIPVGFHLYS THAALAALRG HFCLSSDKMV CYLLKTKAIV NASEMDIQNV PLSEKIAELK EKIVLTHNRL KSLMKILSEV TPDQSKPEN

SEQ ID NO:3 (PBK peptide)

MMTLSIPHI

SEQ ID NO:4 (OIP5 peptide)

SLMKILSEV

SEQ ID NO:5 (OIP5 peptide)

KSLMKILSEV

SEQ ID NO:6 (PBK peptide)

SLPLDENMTV

SEQ ID NO:7 (hTCR_Calpha-R) CAGCCGCAGCGTCATGAGCAGATTA

SEQ ID NO:8 (hTCR_Cbl-R)

CCACTTCCAGGGCTGCCTTCAGAAATC

SEQ ID NO:9 (hTCR_Cb2-R)

TGGGATGGTTTTGGAGCTAGCCTCTGG

SEQ ID NO: 10 (PBK peptide )

RPSAAHIVEA

SEQ ID NO:11 (TCR 9.3 native Vα with signal sequence) (signal sequence underlined, CDRs shown in bold)

MVKIROFLLAILWLQLSCVSAAKNEVEOSPONLTAOEGEFITINCSYSVGISALH

WLQQHPGGGIVSLFMLSSGKKKHGRLIATINIQEKHSSLHITASHPRDSAVYICA

VTKWDSNYQLIWGAGTKLIIKP

SEQ ID NO: 12 (TCR 9.3, native Vβ with signal sequence) (signal sequence underlined, CDRs shown in bold)

MGTSLLCWMALCLLGADHADTGVSONPRHKITKRGONVTFRCDPISEHNRLY

WYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDS

AMYLCASTPLAGALISSYEQYFGPGTRLTVT

SEQ ID NO: 13 (TCR 9.3_ Vα_variant with signal sequence) (signal sequence underlined, CDRs shown in bold)

MVKIROFLLAILWLQLSCVSAAKNEVEOSPONLTAOEGEFITINCSYSVGISALH

WLQQHPGGGIVSLFMLSSGKKKHGRLIATINIQEKHSSLHITASHLRDSAVYICA

VTKWDSNYQLIWGAGTKLIIKP

SEQ ID NO: 14: (TCR 9.3_Vp_variant with signal sequence) (signal sequence underlined, CDRs shown in bold)

MGTSLLCWMALCLLGADHADTGVSONPRYKITKRGONVTFRCDPISEHNRLY

WYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDS

AMYLCASTPLAGALISSYEQYFGPGTRLTVT

SEQ ID NO: 15: (TCR 9.3_ Vα_variant with signal sequence) (signal sequence underlined, CDRs shown in bold) MVKIRQFLLAILWLOLSCVSAAKNEVEOSPONLTAOEGEFVTINCTYSVGISAL

HWLQQHPGGGMVSLFMLSSGKKKHGRLIATINIQEKHSSLHITASHLRDSAVYI

CAVTKWDSNYQLIWGAGTKLIIKP

SEQ ID NO: 16: (TCR 9.3 Vα variant with signal sequence) (signal sequence underlined, CDRs shown in bold)

MVKIRQFLLAILWLOLSCVSAAKNEVTOSOONLTAOEGEFVTINCTYSVGISAF

HWLQQHPGGGMVSLFDLSSGKAKHGRLIATINIQEKSSSLHITASHLRDSAVYI

CAVTKWDSNYQLIWGAGTKLIIKP

SEQ ID NO: 17 (TCR 9.3 nt_non-CO_α) atggtgaagatccggcaatttttgttggctattttgtggcttcagctaagctgtgtaagtgccgccaaaaatgaagtggagcaga gtcctcagaacctgactgcccaggaaggagaatttatcacaatcaactgcagttactcggtaggaataagtgccttacactggc tgcaacagcatccaggaggaggcattgtttccttgtttatgctgagctcagggaagaagaagcatggaagattaattgccacaa taaacatacaggaaaagcacagctccctgcacatcacagcctcccatcccagagactctgccgtctacatctgtgctgtcacta agtgggatagcaactatcagttaatctggggcgctgggaccaagctaattataaagccag

SEQ ID NO: 18 (TCR 9.3 nt_non-CO_β ) atgggcaccagcctcctctgctggatggccctgtgtctcctgggggcagatcacgcagatactggagtctcccagaacccca gacacaagatcacaaagaggggacagaatgtaactttcaggtgtgatccaatttctgaacacaaccgcctttattggtaccgac agaccctggggcagggcccagagtttctgacttacttccagaatgaagctcaactagaaaaatcaaggctgctcagtgatcgg ttctctgcagagaggcctaagggatctttctccaccttggagatccagcgcacagagcagggggactcggccatgtatctctgt gccagcaccccactagcgggggccttaattagctcctacgagcagtacttcgggccgggcaccaggctcacggtcacag

SEQ ID NO: 19 (TCR 9.3 Vα_framework 1)

KNEVEQSPQNLTAQEGEFITINCSYS

SEQ ID NO:20 (TCR 9.3 Vα_ framework 2)

LHWLQQHPGGGIVSLFM

SEQ ID NO:21 (TCR 9.3 Vα framework 3)

KKHGRLIATINIQEKHSSLHITASHPRDSAVYI

SEQ ID NO:22 (TCR 9.3 Vp framework 1)

DTGVSQNPRHKITKRGQNVTFRCDPI

SEQ ID NO:23 (TCR 9.3 Vp framework 2)

LYWYRQTLGQGPEFLTY

SEQ ID NO:24 (TCR 9.3 Vp framework 3) LEKSRLLSDRF S AERPKGSF STLEIQRTEQGDS AMYL

SEQ ID NO:25 (TCR 9.3_ Vα_framework_3_variant_l)

KKHGRLIATINIQEKHSSLHITASHLRDSAVYI

SEQ ID NO:26 (TCR 9.3 Vp_framework 1 variant_l)

DTGVSQNPRYKITKRGQNVTFRCDPI

SEQ ID NO:27 (TCR 9.3_ Vα_framework_l_variant_l)

KNEVEQSPQNLTAQEGEFVTINCTYS

SEQ ID NO:28 (TCR 9.3_ Vα_framework_2 variant l)

LHWLQQHPGGGMVSLFM

SEQ ID NO:29 (TCR 9.3_ Vα_framework_l_variant_2)

KNEVTQSQQNLTAQEGEFVTINCTYS

SEQ ID NO:30 (TCR 9.3_ Vα_framework_2 _variant_2)

FHWLQQHPGGGMVSLFD

SEQ ID NO:31 (TCR 9.3_ Vα_framework_3_variant_2)

AKHGRLIATINIQEKSSSLHITASHLRDSAVYI

SEQ ID NO:32 (TCR 9.3 CDRla)

VGISA

SEQ ID NO:33 (TCR 9.3 CDR2a)

LSSGK

SEQ ID NO:34 (TCR 9.3 CDR3a)

C AVTKWD SNYQLIW

SEQ ID NO:35 (TCR CDRip)

SEHNR

SEQ ID NO:36 (TCR 9.3 CDR2P)

FQNEAQ

SEQ ID NO:37 (TCR 9.3 CDR3P)

C ASTPLAGALIS S YEQYF

SEQ ID NO:38 (TCR 15.2 CDRla)

TSENNYY

SEQ ID NO:39 (TCR 15.2 CDR2a)

QEAYKQQN SEQ ID NO:40 (TCR 15.2 CDR3a)

CAFRVGISGTSYGKLTF

SEQ ID NO:41 (TCR 15.2 CDRip)

DFQATT

SEQ ID NO:42 (TCR 15.2 CDR2P)

SNEGSKA

SEQ ID NO:43 (TCR 15.2 CDR3P)

CSARVAPTGELFF

SEQ ID NO:44 (TCR 4.2 CDRla)

NSMFDY

SEQ ID NO: 45 (TCR 4.2 CDR2a)

ISSIKDK

SEQ ID NO: 46 (TCR 4.2 CDR3a)

CAASGADYKLSF

SEQ ID NO: 47 (TCR 4.2 CDRip)

DFQATT

SEQ ID NO:48 (TCR 4.2 CDR2P)

SNEGSKA

SEQ ID NO: 49 (TCR 4.2 CDR3P)

CSAGRIGDGETQYF

SEQ ID NO:50 (TCR 28.1 CDRla)

NSMFDY

SEQ ID NO:51 (TCR 28.1 CDR2a)

ISSIKDK

SEQ ID NO: 52 (TCR 28.1 CDR3a)

CAASPLGNQFYF

SEQ ID NO:53 (TCR 28.1 CDRip)

MNHEY

SEQ ID NO:54 (TCR 28.1 CDR2P)

SVGEGT

SEQ ID NO: 55 (TCR 28.1 CDR3P) CAS SPLVRS YNEQFF

SEQ ID NO:56 (TCR 9.3 Vα) (CDRs shown in bold)

KNEVEQSPQNLTAQEGEFITINCSYSVGISALHWLQQHPGGGIVSLFMLSSGKK

KHGRLIATINIQEKHSSLHITASHPRDSAVYICAVTKWDSNYQLIWGAGTKLIIK P

SEQ ID NO:57 (TCR 9.3 VP) (CDRs shown in bold)

DTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEA

QLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASTPLAGALISSYEQ YFGPGTRLTVT

SEQ ID NO:58 (TCR 15.2 Vα) (CDRs shown in bold)

AQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVIRQEA

YKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFRVGISGTSYGK LTFGQGTILTVHP

SEQ ID NO:59 (TCR 15.2 VP) (CDRs shown in bold)

GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNE

GSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARVAPTGELFF GEGSRLTVL

SEQ ID NQ:60 (TCR 4.2 Vα) (CDRs shown in bold)

DQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKD

KNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGADYKLSFGAGTTVT VRA

SEQ ID NO:61 (TCR 4.2 VP) (CDRs shown in bold)

GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNE

GSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSAGRIGDGETQY FGPGTRLLVL

SEQ ID NO:62 (TCR 28.1 Vα) (CDRs shown in bold)

DQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKD

KNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASPLGNQFYFGTGTSLT VIP

SEQ ID NO:63 (TCR 28.1 VP) (CDRs shown in bold) NAGVTQTPKFRVLKTGQSMTLLCAQDMNHEYMYWYRQDPGMGLRLIHYSV GEGTTAKGEVPDGYNVSRLKKQNFLLGLESAAPSQTSVYFCASSPLVRSYNEQ FFGPGTRLTVL

SEQ ID NO:64 (TCR 9.3 Vα_vl) (CDRs shown in bold)

KNEVEQSPQNLTAQEGEFITINCSYSVGISALHWLQQHPGGGIVSLFMLSSGKK KHGRLIATINIQEKHSSLHITASHLRDSAVYICAVTKWDSNYQLIWGAGTKLII KP

SEQ ID NO:65 (TCR 9.3 Vp_vl) (CDRs shown in bold)

DTGVSQNPRYKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEA QLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASTPLAGALISSYEQ YFGPGTRLTVT

SEQ ID NO:66 (TCR 9.3 Vα_v2) (CDRs shown in bold)

KNEVEQSPQNLTAQEGEFVTINCTYSVGISALHWLQQHPGGGMVSLFMLSSGK KKHGRLIATINIQEKHSSLHITASHLRDSAVYICAVTKWDSNYQLIWGAGTKLI IKP

SEQ ID NO:67 (TCR 9.3 Vα_v3)

KNEVTQSQQNLTAQEGEFVTINCTYSVGISAFHWLQQHPGGGMVSLFDLSSGK AKHGRLIATINIQEKSSSLHITASHLRDSAVYICAVTKWDSNYQLIWGAGTKLII KP

SEQ ID NO.:68 (Cal)

IQNPDPAVYQ LRDSKSSDKS VCLFTDFDSQ TNVSQSKDSD VYITDKTVLD

MRSMDFKSNS AVAWSNKSDF ACANAFNNSI IPEDTFFPSP ESSCDVKLVE

KSFETDTNLN FQNLSVIGFR ILLLKVAGFN LLMTLRLWSS

SEQ ID NO: 69 (Cp2)

DLKNVFPPKV AVFEPSEAEI SHTQKATLVC LATGFYPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSESYQQG VLSATILYEI LLGKATLYAV LVSALVLMAM VKRKDSRG

SEQ ID NQ:70 (Cpi)

DLNKVFPPEV AVFEPSEAEI SHTQKATLVC LATGFFPDHV ELSWWVNGKE VHSGVSTDPQ PLKEQPALND SRYCLSSRLR VSATFWQNPR NHFRCQVQFY GLSENDEWTQ DRAKPVTQIV SAEAWGRADC GFTSVSYQQG VLSATILYEI

LLGKATLYAV LVS ALVLM AM VKRKDF

SEQ ID NO:71 (signal peptide)

MVKIRQFLLAILWLQLSCVSAA

SEQ ID NO:72 (signal peptide)

MGTSLLCWMALCLLGADHA

SEQ ID NO:73 (signal peptide)

MTRVSLLWAVVVSTCLESGM

SEQ ID NO:74 (signal peptide)

MLLLLLLLGPGISLLLPGSLAGSGL

SEQ ID NO:75 (signal peptide)

MAMLLGASVLILWLQPDWVNSQQKND

SEQ ID NO:76 (signal peptide)

MLLLLLLLGPGISLLLPGSLAGSGL

SEQ ID NO:77 (signal peptide)

MAMLLGASVLILWLQPDWVNSQQKND

SEQ ID NO:78 (signal peptide)

MSLGLLCCGAF SLLWAGP V

SEQ ID NO:79 (RQR peptide tag)

ACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSGGGGSACPYSNPSLCSGGGGS

SEQ ID NO: 80 (CD8 co-receptor α chain, without signal sequence)

SQFRVSPLDRTWNLGETVELKCQVLLSNPTSGCSWLFQPRGAAASPTFLLYLSQ

NKPKAAEGLDTQRFSGKRLGDTFVLTLSDFRRENEGYYFCSALSNSIMYFSHFV PVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI WAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPVVKSGDKPSLSARYV

SEQ ID NO: 81 (CD8 co-receptor β chain isoform 1, without signal sequence)

LQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWD SAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGT QLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLL VSLGVAIHLCCRRRRARLRFMKQFYK

SEQ ID NO: 82 (CD8 co-receptor β chain isoform 2, without signal sequence) LQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWD SAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGT QLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLL

VSLGVAIHLCCRRRRARLRFMKQLRLHPLEKCSRMDY

SEQ ID NO: 83 (CD8 co-receptor β chain isoform 3, without signal sequence)

LQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWD

SAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGT QLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLL VSLGVAIHLCCRRRRARLRFMKQKFNIVCLKISGFTTCCCFQILQISREYGFGVL

LQKDIGQ

SEQ ID NO: 84 (CD8 co-receptor β chain isoform 4, without signal sequence)

LQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWD

SAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGT QLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLL VSLGVAIHLCCRRRRARLRFMKQKFNIVCLKISGFTTCCCFQILQISREYGFGVL

LQKDIGQ

SEQ ID NO: 85 (CD8 co-receptor β chain isoform 5, without signal sequence)

LQQTPAYIKVQTNKMVMLSCEAKISLSNMRIYWLRQRQAPSSDSHHEFLALWD

SAKGTIHGEEVEQEKIAVFRDASRFILNLTSVKPEDSGIYFCMIVGSPELTFGKGT QLSVVDFLPTTAQPTKKSTLKKRVCRLPRPETQKGPLCSPITLGLLVAGVLVLL VSLGVAIHLCCRRRRARLRFMKQPQGEGISGTFVPQCLHGYYSNTTTSQKLLNP

WILKT

EXAMPLES

EXAMPLE 1

ANTIGEN STUDY USING PATIENT-DERIVED TUMOR XENOGRAFT LIBRARY

Pediatric brain and spinal cord tumors (PBT) account for 1 in 4 childhood cancers. Approximately 20% pediatric patients with glioblastoma survive to 5 years, and 5-year survival rates for patients with medulloblastoma, Atypical Teratoid Rhabdoid Tumor (ATRT), or Diffuse Intrinsic Pontine Glioma (DIPG) are extremely low. Current therapies for PBT include radiation and chemotherapy, but these are not curative for many and can be accompanied by toxicities. Recurrent disease that persists beyond radiation and chemotherapy is even more difficult to treat, with very poor prognoses (see, e.g., Ramaswamy et al., Lancet Oncol. 74(12):1200-1207 (2013); Kline etal., J. Neurooncol. 137(1): 103-110 (2018)).

To identify potential targets for immunotherapy of PBT, a bank of pediatric brain tumor derived mouse xenograft cell lines was used (Brabetz et al., Nature Med. 24: 1752-1761 (2018)). Briefly, the mixed pediatric cohort (33 patients) comprised 55 samples with exome sequencing and/or low coverage genomic sequencing. Eighteen (18) patients provided 2 or more samples. Fifteen (15) primary tumors were included. The sample database was queried for cancer/testis (CT) antigen expression based on reported reference levels. Efforts focused on identifying cancer-specific mutations, splice variants, and/or overexpressed genes that were present in more than one patient- derived orthotopic xenograft (PDOX) tumor sample. Antigen expression and accessibility by immunotherapy (e.g., T cells), the tumor microenvironment, and potential location liabilities (e.g., brain inflammation, neurotoxicity, and the blood-brain barrier (BBB)) were other factors considered.

Cancer/testis antigens PBK (PDZ-binding kinase; see, e.g., UniProtKB Q96KB5) and OIP5 (OPA-interacting protein 5; see, e.g., UniProtKB 043482) were identified from the screen as potential targets. PBK was characterized in Drosophila as a mitotic kinase, and in humans is normally expressed in testis and fetal tissues. PBK is highly expressed in non-small cell lung cancer, bladder cancer, lymphomas, cervical cancer, gliomas, pancreatic cancer. OIP5 is normally expressed in testis, but is highly expressed in breast cancer, colorectal cancer, glioblastoma, lung cancer, and esophageal cancer. EXAMPLE 2

ANTIGEN VALIDATION AND IDENTIFICATION OF ANTIGEN-SPECIFIC T CELLS

Briefly, PBK and OIP5 amino acid sequences were evaluated in silico to predict HLA binding using NetMHCPan, IEDB, and Syfpeithi algorithms. Candidate peptides were then tested for binding to HLA and presentation by relevant antigen-presenting cells (APCs; dendritic cells (DCs) were used).

PBMCs from each of three donors were used to source CD14⁺ monocytes, which differentiated into monocyte-derived DCs (Day -4). At Day -1/Day 0, CD8⁺ T cells were isolated. Concurrently, DCs were pulsed with pool of control or experimental peptides, and T cells were cultured with the pulsed DCs at a 10:1 ratio. An initial 4-hour splitwell labeled chromium (⁵¹Cr) release assay (CRA) was performed, and responsive T cells were rapidly expanded using a standard protocol (see, e.g., Dossa et al., Blood 131(1): 108-120 (2018)). T cells were then re-stimulated using pulsed autologous PBMCs. A confirmatory CRA was performed (representative data shown in Figures 1 A-1C), followed by CRAs against (i) sub-pools of the peptides and then (ii) individual peptides. Initial expansions of each polyclonal line of reactive T cells were cloned by limiting dilution, followed by a second round of rapid expansion. Next, reactive T cell clones were tested against pulsed DCs in a peptide titration CRA. Finally, T cell clones were tested against clinical samples, as described further herein.

For T cell identification CRAs, three conditions were tested: (1) no peptide control; (2) control peptides (HA-1, WT1, PRAME (2 peptides)); and (3) experimental peptides. For all CRAs except peptide titrations, APCs were pulsed with 1 pg/ml peptide and loaded with chromium for 4-16 hrs. Following 4 hours incubation with T cells in split-well plates, supernatant (30 pL) was harvested, allowed to dry on Luma plates, and read the following day.

T cell identification CRA data are provided in Figures ID and IE (initial splitwell CRA) and IF (confirmatory CRA following rapid expansion). As seen in Figure IE, ten (10) T cell lines had specific killing activity against candidate peptide-pulsed APCs (6 PBK-specific; 4 OIP5-specific), and nine (9) were specific for control peptides (these responses were later confirmed to be HA-1 specific). Killing activity of polyclonal T cell lines 8, 9, 13, 16, 21, 24 and 39 from Donor 1, lines 34 and 40 from Donor 2, and lines 4, 11, and 16 from Donor 3 against PBK peptides (pooled or individual peptides) is shown in Figures 2A and 2B. HLA-typing and predicted binding of two peptides, #210 (SLPLDENMTV; SEQ ID NO.:6) and #212 (MMTLSIPHI; SEQ ID NO.:3) is shown at the bottom of Figure 2A. Novel peptide #212 elicited the strongest responses and was selected for a titration CRA against T cell clones from each of lines 8, 9, 16, 21, and 24 from Donor 1. Results are shown in Figure 3. Clones 8.1, 8.2, 8.4, 8.5, 9.3, 9.5, and 21.1 were selected for further studies.

Killing activity of polyclonal T cell lines 6, 15, 17, and 18 from Donor 1 against OIP5 peptides (pooled or individual peptides) is shown in Figure 4. HLA-typing and predicted binding of two novel peptides, #216 (SLMKILSEV; SEQ ID NO.:4) and #223 (KSLMKILSEV; SEQ ID NO.:5) is shown at the bottom of the figure. Both peptides were used in titration CRAs against T cell clones from each of lines 6, 15, 17, and 18. Results are shown in Figures 5A and 5B. Clones 6.1, 15.1, 15.2, and 17.5 were selected for further studies. Clone 15.2 was found to encode another TCR, reactive against the PBK antigen RPSAAHIVEA (SEQ ID NO.:2).

EXAMPLE 3

T CELL ACTIVITY AGAINST PATIENT-DERIVED TUMOR XENOGRAFT CELL LINES

A PDOX cell line (PBT-05) was derived from a HLA-A*02:01⁺ patient with high-grade glioma. This patient’s clinical annotations are shown in Figure 6A. Tumor cells from this line are resistant to most standard therapies and are extremely difficult to kill. To confirm that PBT-05 cells were workable model for studying immunotherapy targeting HLA-A2 -binding PBK and OIP5 antigens, MHC Class I expression was examined. As shown in Figure 7, PBT-05 cells have low MHC-I expression.

The selected T cell clones were tested for killing activity against (i) donor- derived lymphoblastoid cells (LCL) with or without the corresponding PBK or OIP5 peptide(s) (Figures 8A, 8C (PBK), and 9A (OIP5)) and (ii) PBT-05 tumor cells (alone, with peptide, with IFN-y, or with peptide + IFN-y; Figures 8B and 9B). All experiments were perfomed at a 20: 1 effector :target (E:T) ratio. These data show that PBK and OIP5-specific T cell clones of the present disclosure have no or low killing activity against self-cells in the absence of antigen, but robustly kill self cells in the presence of antigen. The T cells also killed PBT-05 cells in the presence of antigen, and this effect was pronounced when IFN-y was added. Moreover, the T cells killed PBT-05 cells without addition of exogenous antigen, indicating that PBT-05 cells present the relevant peptides for recognition by T cell clones.

T cell clone 9.3 was also tested for killing activity against HLA-A*02:01⁺ PBT cell lines PBT-05, GBM-511, ATRT-310, and ATRT-311 in the presence or absence of exogenous IFN-y, without the addition of exogenous antigen (Figure 8D). These data show that clone 9.3 recognizes endogenously processed and presented peptide from these cell lines. Further, clone 9.3 recognized and killed GBM-511 cells treated with IFN-y in a 24-hour killing assay using the Incucyte® bioimaging platform (Essen Bioscience) (Figure 10).

EXAMPLE 4

TCR SEQUENCING, CLONING, AND HETEROLOGOUS EXPRESSION OF TCRS

TCR V, D, and J genes from the β chains of the high-avidity T cell clones were sequenced. To generate transgenic TCRs, the TCR α chains of the clones were also sequenced. For both β and α chains, sequencing was performed by next-generation sequencing (Adaptive Biotechnologies) of approximately 6xl0⁴ T cells and rapid amplification of complementary DNA ends (RACE) polymerase chain reaction (PCR). RNA was extracted from each PBK or OIP5/HLA-A*02:01-specific T cell clone. Survey-level sequencing of the variable V-J or V-D-J regions of the TRA and TRB genes (Adaptive Biotechnologies, Seattle, WA) was performed on genomic DNA extracted from approximately 6xl0⁴ T cells from PBK or OIP5-specific T cell clones.

Full TCR regions were identified using 5' first-strand complementary DNA (cDNA) amplification and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) using a SMART er RACE cDNA Amplification Kit (Clontech Laboratories). Briefly, cDNA was synthesized from RNA using 5' CDS Primer A, SMARTer IIA oligo, and SMARTScribe Reverse Transcriptase. The cDNA was then used to perform a RACE-PCR reaction using Phusion High-Fidelity DNA Polymerase and gene-specific primers for the TCR a (hTCR_Calpha-R 5'- CAGCCGCAGCGTCATGAGCAGATTA-3' (SEQ ID NO.:7)) or TCR p chain (hTCR_Cbl-R 5'- CCACTTCCAGGGCTGCCTTCAGAAATC-3' (SEQ ID NO : 8) and hTCR_Cb2-R 5'- TGGGATGGTTTTGGAGCTAGCCTCTGG-3 ' (SEQ ID NO.:9)). RACE-PCR products are purified and sequenced to identify TCR a and P chains. TCR variable, diversity, and joining regions were defined using IMGT/V- QUEST software.

TCRs were constructed by pairing the TRA and TRB sequences encoding the dominant chains in each PBK or OIP5/HLA-A*02:01-specific T cell clone. TRA and TRB sequences were confirmed by PCR using a forward primer from the 5’ end of the appropriate V region and reverse primers from the TRA or TRB constant region(s), followed by Sanger sequencing.

Amino acid sequences of certain TCR V-regions (without leader peptides) are shown in Table 5. CDRs (IMGT junction definition) are shown in bold.

Table 5 V-region amino acid sequences and antigen-specificities of certain TCRs

Complementary cysteine residues at positions 48 (Thr to Cys) and 57 (Ser to Cys) are incorporated into the TRA and TRB constant domains to increase exogenous TCR pairing and decrease mispairing with endogenous TCR (see, e.g., Dossa et al., Blood 131: 108-120 (2018)).

To ensure coordinated gene expression, the TCR chains were separated by codon-diversified 2A elements from the porcine teschovirus (P2A). Transgenes were codon-optimized to enhance expression, synthesized by Gene Art (Life Technologies), and cloned into the pRRLSIN.cPPT.MSCV.WPRE LV by restriction digestion and ligation. Briefly, LentiX-293T cells (Clontech) were transfected with the LV backbone plasmids along with PAX2 and VSVg packaging plasmids using the CalPhos transfection system (Clontech) per the manufacturer’s protocol. Virus particles were harvested after 48 hours and filtered through a 0.45 pm filter prior to use. T cells immunomagnetically purified from normal donor PBMC were activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher Scientific) in 50 lU/mL IL- 2 for 24 hours. T cells are then transduced with LV supernatant. Lentiviral vectors were produced and cells were transduced.

Four days after transduction, T cells were stained with peptide/HLA-A*02:01 pHLA tetramer and anti-CD8 mAb. Peptide tetramer⁺ CD8⁺ T cells were sorted to >95% purity and expanded using OKT3, IL-2 and feeder cells. After 10 days, T cells were evaluated by flow cytometry and functional assays, including examination of heterologous TCR expression, expansion following stimulation, expression of activation and senescence markers, and specific killing of APCs, tumor cell lines, and pediatric tumor samples in vitro.

EXAMPLE 5 ANTIGEN SAFETY VALIDATION

To identify the specific epitopes for each TCR, variants of the PBK and OIP5 peptide antigens were generated in which one of each of the amino acid residues is substituted with an alanine. The core epitopes were evaluated against a database of known protein motifs from humans and other organisms to determine the potential of the selected TCRs for non-cancer-specific reactivity. T cells expressing transferred TCRs are also tested for reactivity against normal human tissues and iPSc-derived normal cell lines. EXAMPLE 6

FRAMEWORK ENGINEERING IN TCR V-REGIONS

Engineering in TCR variable domain framework regions was performed. Clone 9.3 native and variant sequences are shown in Table 6. The CDRs (IMGT junction) are shown in bold.

Table 6.

Constructs encoding native clone 9.3 V-regions and the above variants (each combination of WT with WT, WT with variant, and variant with variant) were cloned, and lentivirus encoding the constructs was made and titered. Constructs included a RQR8 peptide tag (Philip et al. Blood 724(8): 1277-1278 (2014)) for sorting. Primary CD8+ T cells were transduced with lentivirus and sorted.

In another experiment, the TCR from clone 9.3 and sequence variants are transduced into CD8+ T cells using CRISPR, localized to an endogenous TCR locus to disrupt expression of endogenous TCR.

Expression of heterologous TCRs is measured, and TCRs with favorable expression are tested further using peptide antigen titrations, CRAs, and long-term killing assays are performed.

EXAMPLE 7

MURINE XENOGRAFT MODEL OF PEDIATRIC BRAIN CANCER

In vivo studies are performed using a mouse xenograft model of pediatric brain cancer expressing PBK and/or OIP5. A PBT cell line (Brabetz et al. Nat. Med.

24(11): 1752-1761 (2018); research.fredhutch.org/olson/en/btrl.html) is injected into a suitable mouse model and allowed to become established (~4 to ~8 weeks). In one study, tumor cells are administered into the flank (Figure 24).

In another study, tumor cells are administered to generate an orthotopic model (Figure 24). Pretreatment includes 250 cGy XRT with or without IFN-y. T cells expressing the TCR from Clone 9.3 (or a variant thereof comprising sequence engineering the variable domains), are administered intravenously (Day 0) in combination with IL-2 (i.p. injection), and optionally in further combination with chemotherapy and/or radiation in accordance with standard-of-care therapy. 10 mice are used per group (vehicle; IFN-y only; T cells only (approx. 10 million T cells per mouse); IFN-y plus T cells); IFN-y is administered via i.p. injection.

Bleeds are conducted on Days 1, 7, and 14. The T cells are assayed for persistence and function beginning on day three (Day +3) after administration of T cells, including testing of the immunohistochemistry of the tumor and FACS (fluorescence-activated cell sorting) analysis of tumor infiltrating lymphocytes. Beginning at Day 7, weekly monitoring of parameters including tumor burden and overall survival, number and phenotype of tumor infiltrating lymphocytes, T cell survival over time, and gene expression in tumor cells is performed.

Another study using the orthotopic model involves intrathecal administration of T cells. This study involves pretreatment (D -1) with IFN-y or no pretreatment, but not radiation. On Day 0, approx. T cells are administered intrathecally. 10 mice are used per group (vehicle; IFN-y only; T cells only (approx. 5 million T cells per mouse); IFN- y plus T cells). Bleeds are conducted on Day 1 and Day 7

Transferred cells with additional modifications (e.g., knockdown or knockouts of immune checkpoint molecules; knockout of endogenous TCR; transgenic expression of cytokines such as IL-12 or IFN-y) are also generated.

Tested therapies include concurrent treatment of HLA-A*02:01-restricted T cells with drugs that increase expression of MHC Class I in cancer cells (see, e.g., Wan et al., PLoS One 7(3):e32542 (2012)) and concurrent treatment with both PBK-specific and OlP-specific T cells.

EXAMPLE 8

KILLING ACTIVITY OF T CELLS AGAINST OTHER CANCER CELL LINES

PBK- and OIP5-specific T cells are tested for functionality against other antigen-expressing cancer cell lines, including glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, and hepatocellular carcinoma.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 63/083,076, filed on September 24, 2020, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS What is claimed is:

1. An isolated binding protein that is capable of binding to a PDZ-binding kinase (PBK) peptide antigen:HLA complex, wherein the PBK peptide antigen comprises or consists of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3), and wherein, optionally, the binding comprises specific binding.

2. The binding protein of claim 1, wherein the HLA comprises HLA- A*02:01.

3. An isolated binding protein that is capable of binding to a PDZ-binding kinase (PBK) peptide antigen:HLA complex, wherein the PBK peptide antigen comprises or consists of the amino acid sequence RPSAAHIVEA (SEQ ID NO.: 10), and wherein, optionally, the binding comprises specific binding.

4. The binding protein of claim 3, wherein the HLA comprises HLA- B*07:02.

5. The binding protein of any one of claims 1-4, comprising an immunoglobulin superfamily variable domain.

6. The binding protein of any one of claims 1-5, comprising a TCR a-chain variable domain ( Vα) and/or a TCR β-chain variable domain (VP).

7. The binding protein of any one of claims 1-5, comprising a heavy chain variable domain (VH) and/or a light chain variable domain (VL) of a TCR-mimic antibody.

8. The binding protein of any one of claims 1, 2, and 5-7, comprising: (i) the amino acid sequence of SEQ ID NO.:34, wherein SEQ ID NO.:34 is optionally a complementarity determining region (CDR3)a; and/or

(ii) the amino acid sequence of SEQ ID NO.:37, wherein SEQ ID NO.:37 is optionally a CDR3p.

9. The binding protein of claim 8, further comprising:

(iii) the amino acid sequence of SEQ ID NO.:32, wherein SEQ ID NO.:32 is optionally a CDRlα;

(iv) the amino acid sequence of SEQ ID NO.:33, wherein SEQ ID NO.:33 is optionally a CDR2a;

(v) the amino acid sequence of SEQ ID NO.:35, wherein SEQ ID NO.:35 is optionally a CDR1P; and/or

(vi) the amino acid sequence of SEQ ID NO.:36, wherein SEQ ID NO.:36 is optionally a CDR2p.

10. The binding protein of any one of claims 1, 2, 5, 6, 8, and 9, comprising:

(1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:32-34, respectively; and

(2) a TCR Vβ comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:35-37, respectively.

11. The binding protein of any one of claims 6 and 8-10, comprising:

(a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs.:56, 64, 66, and 67; and/or

(b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in any one of SEQ ID NOs.:57 and 65.

12. The binding protein of any one of claims 6 and 8-11, comprising the framework amino acid sequence set forth in any one of SEQ ID NOs. : 19-31, or any combination of these.

13. The binding protein of any one of claims 1, 2, 5, 6, and 8-12, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.:

(i) 56 and 57, respectively;

(ii) 56 and 65, respectively;

(iii) 64 and 57, respectively;

(iv) 64 and 65, respectively;

(v) 66 and 57, respectively;

(vi) 66 and 65, respectively;

(vii) 67 and 57, respectively; or

(viii) 67 and 65, respectively.

14. The binding protein of any one of claims 1, 2, 5, 6, and 8-13, comprising the amino acid sequences set forth in SEQ ID NOs.:

(i) 11 and 12, respectively;

(ii) 13 and 12, respectively;

(iii) 15 and 12, respectively;

(iv) 16 and 12, respectively;

(v) 11 and 14, respectively;

(vi) 13 and 14, respectively;

(vii) 15 and 14, respectively; or

(viii) 16 and 14, respectively.

15. The binding protein of any one of claims 3-7, comprising: (i) the amino acid sequence of SEQ ID NO.:40, wherein SEQ ID NO.:40 is optionally a complementarity determining region (CDR3)a; and/or

(ii) the amino acid sequence of SEQ ID NO.:43, wherein SEQ ID NO.:43 is optionally a CDR3p.

16. The binding protein of claim 15, further comprising:

(iii) the amino acid sequence of SEQ ID NO.:38, wherein SEQ ID NO.:38 is optionally a CDRlα;

(iv) the amino acid sequence of SEQ ID NO.:39, wherein SEQ ID NO.:39 is optionally a CDR2a;

(v) the amino acid sequence of SEQ ID NO.:41, wherein SEQ ID NO.:41 is optionally a CDR1P; and/or

(vi) the amino acid sequence of SEQ ID NO.:42, wherein, optionally, SEQ ID NO.:42 is optionally a CDR2p.

17. The binding protein of any one of claims 3-6, 15, and 16, comprising:

(1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:38-40, respectively; and

(2) a TCR Vβ comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:41-43, respectively.

18. The binding protein of any one of claims 3-6 and 15-17, comprising:

(a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:58; and/or

(b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:59.

19. The binding protein of any one of claims 3-6 and 15-17, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.:58 and 59, respectively.

20. The binding protein of any one of claims 1, 2, 5, and 6, comprising:

(i) a CDR3β according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3;

(ii) a CDR3a according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3;

(iii) any one or more of the Vα, CDRa, VP, CDRP, and/or framework amino acid sequences set forth in any one of SEQ ID NOs. : 11-16 or 19-31, respectively, or a variant thereof having at least 90% identity thereto, or a variant thereof comprising one, two, or three amino acid substitutions, wherein, optionally, the Vα has at least 90% identity to a mature form of the Vα amino acid sequence in SEQ ID NO.: 11, 13, 15, or 16 with the signal sequence removed, and/or the Vβ has at least 90% identity to a mature form of the Vβ amino acid sequence in SEQ ID NO.: 12 or 14, with the signal sequence removed;

(iv) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3; and/or

(v) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3, provided that

(a) at least three or four of the CDRs have no mutations; (b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and

(c) the binding protein retains its ability to bind to a peptide comprising a MMTLSIPHI (SEQ ID NO.:3):HLA complex.

21. The binding protein of claim 20, further comprising a CDRip, a CDR2P, a CDRla, and/or a CDR2a according to Clone 8.1, Clone 8.2, Clone 8.4, Clone 8.5, Clone 9.3, Clone 9.5, or Clone 21.1 from Donor 1, according to a T cell from Line 34 or 40 from Donor 2, or according to a T cell from line 4, 11, or 16 from Donor 3.

22. The binding protein of claim any one of claims 3-6, wherein the encoded binding protein comprises:

(i) a CDR3P according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5;

(ii) a CDR3a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5;

(iii) a Vα domain having at least 90% amino acid identity to the Vα domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5; and/or

(iv) a Vβ domain having at least 90% amino acid identity to the Vβ domain of Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5, provided that

(a) at least three or four of the CDRs have no mutations;

(b) the CDRs that do have mutations have only up to two amino acid substitutions, up to a contiguous five amino acid deletion, or a combination thereof; and

(c) the encoded binding protein retains its ability to bind to a RPSAAHIVEA (SEQ ID NO.:10):HLA complex.

23. The binding protein of claim 22, wherein the encoded binding protein further comprises a CDRip, a CDR2P, a CDRla, and/or a CDR2a according to Clone 6.1, Clone 15.1, Clone 15.2, or Clone 17.5.

24. An isolated binding protein that is capable of binding to an OPA- interacting protein 5 (OIP5) peptide antigen:HLA complex, wherein the OIP5 peptide antigen comprises or consists of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5), wherein, optionally, the binding comprises specific binding.

25. The binding protein of claim 24, wherein the HLA comprises HLA- A*02:01.

26. The binding protein of claim 24 or 25, comprising an immunoglobulin superfamily variable domain.

27. The binding protein of any one of claims 24-26, comprising a TCR a- chain variable domain ( Vα) and a TCR β-chain variable domain (VP).

28. The binding protein of any one of claims 24-26, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) of a TCR-mimic antibody.

29. The binding protein of any one of claims 24-28, comprising:

(i) the amino acid sequence of SEQ ID NO.:46, wherein SEQ ID NO.:46 is optionally a complementarity determining region (CDR3)a; and/or

(ii) the amino acid sequence of SEQ ID NO.:49, wherein SEQ ID NO.:49 is optionally a CDR3p.

30. The binding protein of claim 29, further comprising:

(iii) the amino acid sequence of SEQ ID NO.:44, wherein SEQ ID NO.:44 is optionally a CDRlα;

(iv) the amino acid sequence of SEQ ID NO.:45, wherein SEQ ID NO.:45 is optionally a CDR2a; (v) the amino acid sequence of SEQ ID NO.:47, wherein SEQ ID NO.:47 is optionally a CDRIP; and/or

(vi) the amino acid sequence of SEQ ID NO.:48, wherein SEQ ID NO.:48 is optionally a CDR2p.

31. The binding protein of any one of claims 24-27 and 29-30, comprising:

(1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:44-46, respectively; and

(2) a TCR Vβ comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:47-49, respectively.

32. The binding protein of claim 29 or 30, comprising: a TCR Vα comprising

(1) the CDR3a amino acid sequence of SEQ ID NO. :46;

(2) the CDRla amino acid sequence of SEQ ID NO.:44 or 50;

(3) the CDR2a amino acid sequence of SEQ ID NO.:45 or 51; and a TCR Vβ comprising

(4) the CDR3P amino acid sequence of SEQ ID NO.:49;

(5) the CDRip amino acid sequence of SEQ ID NO.:47 or 53; and

(6) the CDR2 P amino acid sequence of SEQ ID NO. :48 or 54.

33. The binding protein of any one of claims 24-27 and 29-32, comprising:

(a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:60; and/or

(b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:61.

34. The binding protein of any one of claims 24-27 and 29-33, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.:60 and 61, respectively.

35. The binding protein of any one of claims 24-28, comprising:

(i) the amino acid sequence of SEQ ID NO.: 52, wherein SEQ ID NO.: 52 is optionally a complementarity determining region (CDR3)a; and/or

(ii) the amino acid sequence of SEQ ID NO.:55, wherein SEQ ID NO.:55 is optionally a CDR3β.

36. The binding protein of claim 33, further comprising:

(iii) the amino acid sequence of SEQ ID NO.: 50, wherein SEQ ID NO.: 50 is optionally a CDRlα;

(iv) the amino acid sequence of SEQ ID NO.:51, wherein SEQ ID NO.:51 is optionally a CDR2a;

(v) the amino acid sequence of SEQ ID NO.:53, wherein SEQ ID NO.:53 is optionally a CDR1β; and/or

(vi) the amino acid sequence of SEQ ID NO.: 54, wherein SEQ ID NO.: 54 is optionally a CDR2β.

37. The binding protein of any one of claims 24-27 and 35-36, comprising:

(1) a TCR Vα comprising the CDRla, CDR2a, and CDR3a amino acid sequences set forth in SEQ ID NOs.:50-52, respectively; and

(2) a TCR Vβ comprising the CDRip, CDR2P, and CDR3P amino acid sequences set forth in SEQ ID NOs.:53-55, respectively.

38. The binding protein of claim 35, comprising: a TCR Vα comprising

(1) the CDR3a amino acid sequence of SEQ ID NO.:52;

(2) the CDRla amino acid sequence of SEQ ID NO.:44 or 50;

(3) the CDR2a amino acid sequence of SEQ ID NO.:45 or 51; and a TCR Vβ comprising

(4) the CDR3β amino acid sequence of SEQ ID NO.:55;

(5) the CDR1β amino acid sequence of SEQ ID NO.:47 or 53; and

(6) the CDR2 β amino acid sequence of SEQ ID NO. :48 or 54.

39. The binding protein of any one of claims 24-27 and 35-38, comprising:

(a) a TCR Vα amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:62; and/or

(b) a TCR Vβ amino acid sequence having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:63.

40. The binding protein of any one of claims 24-27 and 35-39, comprising the TCR Vα and TCR Vβ amino acid sequences set forth in SEQ ID NOs.:62 and 63, respectively.

41. The binding protein of any one of claims 1-40, further comprising a TCR a-chain constant domain (Ca), a TCR β-chain constant domain, (CP), or both, wherein, optionally, the Vβ and the Cβ together comprise a TCR β chain and/or the Vα and the Ca together comprise a TCR α chain.

42. The binding protein of claim 41, wherein the Ca comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:68.

43. The binding protein of claim 41 or 42, wherein the Cβ comprises or consists of an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO.:69 or 70.

44. The binding protein of any one of claims 41-43, wherein the Cβ and/or the Cα comprises one or more non-native amino acid at a position such that when the the Cβ and the Ca associate to form a dimer, a non-native disulfide bond is formed between the Cβ and the Ca, wherein, optionally, the non-native amino acid comprises a cysteine in the Cβ and/or a cysteine in the Ca, wherein, further optionally, the CP comprises a cysteine amino acid at amino acid position 57 and/or the Ca comprises a cysteine amino acid at amino acid position 48.

45. The binding protein of any one of claims 1-44, wherein:

(1) the binding protein comprises a TCR, a scTv, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), a TCR-mimic antibody or antigen-binding fragment thereof, or any combination thereof; and/or

(2) wherein a substitution mutation in SEQ ID NO.:3 of any of the following amino acids of SEQ ID NO.:3 to alanine does not substantially reduce binding by the binding protein: the amino acid at position 1 of SEQ ID NO.:3, the amino acid at position 3 of SEQ ID NO.:3, the amino acid at position 4 of SEQ ID NO.:3, the amino acid at position 9 of SEQ ID NO.:3.

46. The binding protein of claim 45, wherein binding protein comprises a TCR.

47. The binding protein of claim 45, wherein the binding protein comprises a scTv.

48. The binding protein of claim 45, wherein the binding protein comprises a scTCR.

49. The binding protein of claim 45, wherein the binding protein comprises a CAR.

50. An isolated polynucleotide encoding the binding protein of any one of claims 1-49.

51. The polynucleotide of claim 50, wherein the polynucleotide is codon- optimized for expression in a host cell, wherein, optionally, the host cell comprises an immune system cell, wherein, further optionally, the immune system cell comprises a T cell, a NK-T cell, or a NK cell.

52. The polynucleotide of claim 51, further comprising:

(i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain;

(ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or comprises a CD 8 co-receptor β chain; or

(iii) a polynucleotide of (i) and a polynucleotide of (ii).

53. The polynucleotide of claim 52, comprising:

(a) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain;

(b) the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain; and

(c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).

54. The polynucleotide of claim 52 or 53, further comprising a polynucleotide that encodes a self-cleaving peptide and is disposed between: (1) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor a chain; and/or

(2) the polynucleotide encoding a binding protein and the polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor P chain.

55. The polynucleotide of any one of claims 52-54, comprising, operably linked in-frame:

(i) (pnCD8a)-(pnSCPi)-(pnCD8β)-(pnSCP2)-(pnBP);

(ii) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnBP);

(iii) (pnBP)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnCD8P);

(iv) (pnBP)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnCD8a);

(v) (pnCD8a)-(pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8P); or

(vi) (pnCD8P)-(pnSCPi)-(pnBP)-(pnSCP2)-(pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnBP is the polynucleotide encoding a binding protein, and wherein pnSCPi and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded selfcleaving peptides are optionally the same or different.

56. The polynucleotide of any one of claims 50-55, wherein the encoded binding protein comprises a TCRα chain and a TCRβ chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding a TCRα chain and the polynucleotide encoding a TCRβ chain, wherein, optionally, the polynucleotde comprises, operably linked in-frame:

(i) (pnCD8a)-(pnSCPi)-(pnCD8p)-(pnSCP₂)-(pnTCRp)-(pnSCP₃)- (pnTCRa); (ii) (pnCD8p)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRβ)-(pnSCP₃)- (pnTCRa);

(iii) (pnCD8a)-(pnSCPi)-(pnCD8P)-(pnSCP2)-(pnTCRa)-(pnSCP3)- (pnTCRp);

(iv) (pnCD8P)-(pnSCPi)-(pnCD8a)-(pnSCP2)-(pnTCRa)-(pnSCP3)- (pnTCRp);

(v) (pnTCRP)-(pnSCPi)-(pnTCRa)-(pnSCP2)-(pnCD8a)-(pnSCP3)- (pnCD8p);

(vi) (pnTCRp)-(pnSCPi)-(pnTCRa)-(pnSCP₂)-(pnCD8p)-(pnSCP3)- (pnCD8α);

(vii) (pnTCRa)-(pnSCPi)-(pnTCRP)-(pnSCP2)-(pnCD8a)-(pnSCP3)- (pnCD8P); or

(viii) (pnTCRa)-(pnSCPi)-(pnTCRp)-(pnSCP₂)-(pnCD8p)-(pnSCP3)- (pnCD8a), wherein pnCD8a is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnCD8p is the polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein pnTCRa is the polynucleotide encoding a TCR α chain, wherein pnTCRP is the polynucleotide encoding a TCR β chain, and wherein pnSCPi, pnSCP2, and pnSCP₃ are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and/or the encoded self-cleaving peptides are optionally the same or different.

57. The polynucleotide of any one of claims 50-56, wherein the polynucleotide comprises DNA, RNA (optionally mRNA), or both.

58. The polynucleotide of claim 57, comprising DNA.

59. A vector comprising the polynucleotide of any one of claims 50-58.

60. The vector of claim 59, wherein the vector comprises a viral vector.

61. The vector of claim 60, wherein the viral vector comprises a lentiviral vector or a y-retroviral vector.

62. The vector of any one of claims 59-61, wherein the vector is capable of delivering the polynucleotide to a host cell.

63. The vector of claim 62, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

64. The vector of claim 63, wherein the human immune system cell is a CD4⁺ T cell, a CD8⁺ T cell, a CD4'CD8‘ double negative T cell, a yδ T cell, a natural killer cell, a natural killer T cell, a macrophage, a monocyte, a dendritic cell, or any combination thereof.

65. The vector of claim 64, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

66. A host cell comprising the polynucleotide of any one of claims 50-58 and/or the vector of any one of claims 59-65, and/or express the binding protein of any one of claims 1-49.

67. The host cell of claim 66, wherein the modified cell comprises a hematopoietic progenitor cell and/or an immune cell, optionally a human immune cell.

68. The host cell of claim 67, wherein the immune cell comprises a T cell, a NK cell, a NK-T cell, a dendritic cell, a macrophage, a monocyte, or any combination thereof.

69. The host cell of claim 68, wherein the immune cell comprises a CD4⁺ T cell, a CD8⁺ T cell, a CD4' CD8‘ double negative T cell, a yδ T cell, or any combination thereof, wherein, optionally, the immune cell comprises a CD4⁺ T cell and a CD8⁺ T cell, wherein, further optionally, the CD4⁺ T cell, the CD8⁺ T cell, or both comprise (i) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (ii) a polynucleotide encoding a polypeptide that comprises an extracellular portion of a CD8 co-receptor p chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii).

70. The host cell of claim 68 or 69, wherein the immune cell comprises a CD8⁺ T cell and/or a CD4⁺ T cell.

71. The host cell of any one of claims 66-70, wherein the modified cell comprises a chromosomal gene knockout of a PD-1 gene; a LAG3 gene; a TIM3 gene; a CTLA4 gene; an HLA component gene; a TIGIT gene; a TCR component gene, a FasL gene, or any combination thereof.

72. The host cell of claim 71, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from an al macroglobulin gene, an α2 macroglobulin gene, an α3 macroglobulin gene, a pi microglobulin gene, or a P2 microglobulin gene.

73. The host cell of claim 71 or 72, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR a variable region gene, a TCR P variable region gene, a TCR constant region gene, or a combination thereof.

74. The host cell of any one of claims 66-73, wherein the polynucleotide encoding the binding protein is heterologous to the host cell and is comprised in an endogenous TCR gene locus.

75. The host cell of any one of claims 66-74, further comprising a heterologous polynucleotide encoding:

(i) a safety switch protein;

(ii) a selection marker;

(iii) a CD8 co-receptor β-chain;

(iv) a CD8 co-receptor a-chain; or

(v) any combination thereof.

76. A composition, comprising:

(i) the binding protein of any one of claims 1-49;

(ii) the polynucleotide of any one of claims 50-58;

(iii) the vector of any one of claims 59-65; and/or

(iv) the host cell of any one of claims 66-75, optionally comprising CD4+ T cells, CD8+ T cells, or both, and a pharmaceutically acceptable carrier, excipient, or diluent.

77. The composition of claim 76, comprising the host cell, wherein the host cell comprises an immune cell, optionally CD8+ T cells and/or CD4+ T cells, wherein, further optionally, the CD8+ T cells and CD4+ T cells are present in about a 1 : 1 ratio, and/or the composition comprises substantially no naive T cells.

78. A method for treating a disease or disorder associated with expression of

(1) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3),

(2) a PBK antigen comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO : 10), and/or (3) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5) in a subject, the method comprising administering to the subject an effective amount of:

(i) the binding protein of any one of claims 1-49;

(ii) the polynucleotide of any one of claims 50-58;

(iii) the vector of any one of claims 59-65;

(iv) the host cell of any one of claims 66-75; and/or

(v) the composition of claim 76 or 77.

79. The binding protein of any one of claims 1-49, the polynucleotide of any one of claims 50-58, the vector of any one of claims 59-65, the host cell of any one of claims 66-75, and/or the composition of claim 76 or 77, for use in a method of treating a disease or disorder associated with expression of

(1) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3),

(2) a PBK antigen comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO : 10), and/or

(3) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5) in a subject.

80. The binding protein of any one of claims 1-49, the polynucleotide of any one of claims 50-58, the vector of any one of claims 59-65, the host cell of any one of claims 66-75, and/or the composition of claim 76 or 77, for use in the manufacture of a medicament for treating a disease or disorder associated with expression of

(1) a PBK antigen comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO.:3),

(2) a PBK antigen comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO : 10), and/or (3) an OIP5 antigen comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO.:4) or KSLMKILSEV (SEQ ID NO.:5) in a subject.

81. The method of claim 78 or the binding protein, polynucleotide, vector, host cell, and/or composition for use of claim 79 or 80, wherein the subject is HLA- A*02:01⁺ or HLA-B*07:02⁺.

82. The method of claim 78 or 81 or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-81, wherein the disease or condition is a cancer.

83. The method of claim 78, 81, or 82, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-82, wherein the cancer comprises a solid tumor or a hematological malignancy.

84. The method of claim 78, 81, 82, or 83, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-83, wherein the cancer comprises glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, neuroblastoma, hepatoblastoma, PNET, glioma, cranipharyngioma, choroid plexus tumors, schwannomas, meningiomas, Wilm’s tumor, germ cell tumors, hepatocellular carcinoma, or any combination thereof.

85. The method of claim 78, 81, 82, 83, or 84, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-84, wherein the subject is human.

86. The method of claim 78, 81, 82, 83, 84, or 85, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-85, wherein the subject is a pediatric subject.

87. The method of claim 78, 81, 82, 83, 84, 85, or 86, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-86, wherein the subject has previously received one or more of:

(i) surgery;

(ii) radiation therapy;

(iii) chemotherapy; or

(iv) a hematopoietic stem cell transplant (HSC).

88. The method of claim 78, 81, 82, 83, 84, 85, 86, or 87, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-87, wherein one or more of the modified immune cells comprised in the composition is autologous to the subject.

89. The method of claim 78, 81, 82, 83, 84, 85, 86, 87, or 88, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-88, wherein the method further comprises administering an inhibitor of an immune checkpoint molecule to the subject.

90. The method of claim 78, 81, 82, 83, 84, 85, 86, 87, 88, or 89, or the binding protein, polynucleotide, vector, host cell, and/or composition for use of any one of claims 79-89, wherein the method comprises administering the binding protein, polynucleotide, vector, modified host cell, and/or composition to the subject intrathecally, intracerebroventricularly, into the cerebrospinal fluid (CSF), intracranially, intraventricularly, intravenously, locally, or systemically.

91. An immunogenic composition, comprising:

(i) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3);

(ii) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO : 10);

(iii) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4);

(iv) an isolated peptide or polypeptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5); and/or

(v) a variant of the isolated peptide or polypeptide of SEQ ID NO.:3, SEQ ID NO.: 10, SEQ ID NO.:4, or SEQ ID NO.: 5, wherein the variant comprises one, two, or three differences in amino acid sequence as compared to SEQ ID NO.:3, 10, 4, or 5, respectively, wherein the isolated peptide or polypeptide of any one of (i)-(vii) does not comprise an isolated full-length human PBK or OIP5.

92. The immunogenic composition of claim 91, wherein

(a) one or more copies of any one of (i)-(v) and/or

(b) one or more of any of (i)-(v) is/are present in a fusion polypeptide, wherein the fusion polypeptide optionally further comprises the amino acid sequence of a self-cleaving peptide.

93. The immunogenic composition of claim 91 or 92, wherein the immunogenic composition is capable of eliciting an immune response in a subject against cancer cells, wherein, optionally, the cancer cells comprise glioblastoma cells, medulloblastoma cells, breast cancer cells, colorectal cancer cells, lung cancer cells, esophageal cancer cells, lymphoma cells, leukemia (e.g., acute myeloid leukemia) cells, melanoma cells, cholangiocarcinoma cells, hepatocellular carcinoma cells, or any combination thereof.

94. The immunogenic composition of any one of claims 91-93, further comprising an adjuvant.

95. An isolated polynucleotide encoding the immunogenic composition of any one of claims 91-93, wherein the polynucleotide is optionally contained in a vector.

96. The polynucleotide of claim 95, wherein the polynucleotide is codon- optimized for expression in a host cell, wherein the host cell is optionally a dendritic cell or a T cell.

97. A host cell comprising the isolated polynucleotide of claim 95 or 96, wherein the polynucleotide is heterologous to the host cell, and wherein the host cell is optionally an immune cell and is further optionally a professional antigen-presenting cell.

98. The host cell of claim 97, wherein the host cell is a dendritic cell or a T cell.

99. A method of eliciting an immune response in a subject against glioblastoma, medulloblastoma, breast cancer, colorectal cancer, lung cancer (e.g., nonsmall cell lung cancer), esophageal cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), melanoma, cholangiocarcinoma, bladder cancer, cervical cancer, pancreatic cancer, and/or hepatocellular carcinoma, the method comprising administering to the subject the binding protein of any one of claims 1-49, the polynucleotide of any one of claims 50-58, the vector of any one of claims 59-65, the host cell of any one of claims 66-75, the composition of claim 76 or 77, the immunogenic composition of any one of claims 91-94, and/or the host cell of claim 97 or 98.

100. A method for expanding a population of T cells that bind to a peptide (e.g., a peptide comprised in a peptide:HLA complex) selected from: (i) a peptide comprising or consisting of the amino acid sequence

MMTLSIPHI (SEQ ID NO:3);

(ii) a peptide comprising or consisting of the amino acid sequence

RPSAAHIVEA (SEQ ID NO : 10);

(iii) a peptide comprising or consisting of the amino acid sequence

SLMKILSEV (SEQ ID NO:4); and

(iv) a peptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO: 5), the method comprising contacting a sample comprising one or more T cells that bind to the peptide with the immunogenic composition of any one of claims 91-94, the polynucleotide of claim 95 or 96, the host cell of claim 97 or 98, and/or antigen- presenting cells that express or have been contacted with a peptide or polypeptide comprising or consisting of the amino acid sequence set forth in any one of SEQ ID NOs.:3-5 and 10.

101. A method for generating and/or isolating T cells, the method comprising contacting a sample comprising T cells, wherein the sample optionally comprises peripheral blood cells, with:

(a) an immunogenic composition of any one of claims 91-94; and/or

(b) antigen-presenting cells (APCs) that express or have been contacted with

(i) a peptide or polypeptide comprising or consisting of the amino acid sequence MMTLSIPHI (SEQ ID NO:3);

(ii) a peptide or polypeptide comprising or consisting of the amino acid sequence RPSAAHIVEA (SEQ ID NO : 10);

(iii) a peptide or polypeptide comprising or consisting of the amino acid sequence SLMKILSEV (SEQ ID NO:4); and

(iv) a peptide or polypeptide comprising or consisting of the amino acid sequence or KSLMKILSEV (SEQ ID NO:5); or

(v) any combination of (i)-(iv); and optionally sorting T cells from other cells in the sample, thereby isolating and/or generating T cells.

102. A T cell isolated and/or generated by the method of claim 101.