EP3452514A1 - T-cell immunotherapy specific for mart-1 - Google Patents

Abstract

The present disclosure provides T cell receptors specific for human melanoma antigen recognized by T cells 1 (MART-1) epitopes for use in treating diseases or disorders, such as cancer cells that overexpress MART-1.

Description

T-CELL IMMUNOTHERAPY SPECIFIC FOR MART-1

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_443WO_SEQUENCE_LISTING.txt. The text file is 41 KB, was created on May 5, 2017, and is being submitted electronically via EFS-Web.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under K12 CA076930 awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

Locally advanced and metastatic melanomas are well-known to be resistant to current therapies. Agents for treating stage IV melanoma include BRAF inhibitors, such as vemurafenib (Zelboraf), dabrafenib (Tafmlar), trametinib (Mekinist), and cobimetimb (CoteUic), for those cancers expressing a BRAF mutation; c-KIT inhibitors, such as imatinib (Gleevec) and nilotinib (Tasigna)); alkylating agents such as DTIC (or Dacarbazine); interleukin-2 (IL-2) administered in high doses; and more recently, immunotherapies, such as ipilimumab (Yervoy). The survival rate for patients with stage IV melanoma is low. The median survival of patients with stage IV disease is less than 1 year (Tsao et al., 2004, N. Engl. J. Med. 351 :998-1012).

Adoptive immunotherapy involving the ex vivo expansion and reinfusion of tumor-reactive T-cells is an emerging treatment modality, especially in patients for whom conventional therapy fails (Stromnes et al., Immunol. Rev. 2014 257: 145-64). Consequential responses have been achieved in metastatic melanoma using tumor- reactive T-cells expanded from a tumor site (Rosenberg et al.,2011, Clin. Cancer Res. 17:4550-7). However, successful tumor-infiltrating lymphocyte (TIL) therapies require sufficient accessible tumor for adequate sampling, and has been constrained to specialized centers by toxicities associated with high-dose pre-infusion conditioning and post-infusion IL-2 (Yang, 2013, Clin. Dermatol. 31 :209-19). Endogenous antigen- specific CTL can also be obtained and expanded from peripheral blood (PB) and infused with lower-dose conditioning and a very tolerable safety profile, but have effectively reduced tumor burdens in only a limited number of patients, due in part to the short persistence of the transferred cells (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Hunder et al., 2008, N. Engl. J. Med. 358:2698-703; Wallen et al., 2009, PLoS One 4:e4749; and Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168- 73). In previous prior studies, infused CTL persisted beyond 42 days in 11-15% of patients. Median CTL persistence in vivo was <14 days, and overall response rate (inclusive of patients with CRs and PRs) was only 7%. "(Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Hunder et al., 2008, N. Engl. J. Med. 358:2698-703; Wallen et al., 2009, PLoS One 4:e4749; and Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168-73)

There is a clear need for alternative highly antigen-specific TCR

immunotherapies directed against various cancers, such as melanomas. Presently disclosed embodiments address these needs and provide other related advantages.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1D: Tumor regressions following melanoma-reactive polyclonal CTL combined with anti-CTLA4. (A) Timeline of successive therapies in "Patient Z." (B) Kinetics of response for 3 target lesions (y-axis) spanning 5 years (x-axis). (C) Serial PET (leftmost image) and CT images at indicated time-points. Arrows indicate the location of the right hilar (upper row) and subcarinal (lower row) masses. (D)

Photograph of the Patient Z's depigmented eyelashes and eyebrows ~5 months after the start of the combined treatment.

Figures 2A-2F: Kinetics, clonality, phenotype and function of monoclonal and polyclonal CTL in vivo. (A) Percent multimer⁺CD8⁺ T cells (left y-axis) in peripheral blood mononuclear cells (PBMCs) (solid circles) collected before and at defined timepoints after monoclonal (dashed line) and polyclonal (solid line) CTL infusions (indicated). Grey shaded areas indicate anti-CTLA4 treatment. (B, C) Inset pie plots represent individual clonotypes composing the monoclonal (B) and polyclonal (C) infused CTL. Graphs track the corresponding unique (B) and sum of clonotypes (C) as a percent of total CD8⁺ T cells (y-axis). Time-points in which the corresponding clones were assessed but not detected (nd) are indicated. *Only clone TCR-13 was detected immediately prior to the polyclonal infusion with a frequency of 0.054%. (D) Percent expression of CD28, CD62L, CCR7 (long-lived memory markers - left), PD1 (activation/exhaustion marker - middle), IFNy, T Fa, and IL2 (functional markers - right) on polyclonal (upper graph) and monoclonal (lower graph) infused CTL. (E, F) The same analysis performed on multimer⁺ cells 1 day (E) and 86 days (F) in vivo after infusion.

Figure 3: Reactivity to non-targeted epitopes. Heat-map summarizing responses of CD8⁺ and CD4⁺ T cells independent of HLA restriction to pools of 20-30 peptides spanning MARTI, NY-ESOl, gplOO, tyrosinase, and MAGE- A3. The shading scale (light to dark) reflects the response magnitude at indicated timepoints before and after administration of monoclonal and polyclonal CTL during the patients' treatment course (upper schema). Inset numbers indicate IFNy spots per 10⁵ PBMC for each peptide pool.

Figures 4A and 4B: Purity and phenotype of infused monoclonal and polyclonal products. (A) Plots to the left: percent expression of CD8⁺ cells (x-axis) binding the specific HLA A*0201 -restricted MART- I 26-35 multimer (y-axis) within infused lymphocytes. Plots to the left: percent expression of CD4⁺ T cells (y-axis) and cells expressing either CD 16 (identified natural killer cells) or CD 19 (identifies B cells) (x-axis) within infused lymphocytes. (B) Percent expression of CD45RO, CD27, CD28, CD62L, CCR7 (markers associated with memory /long-lived memory), PD1 (activation/exhaustion marker), CD57 (exhaustion marker) on monoclonal (graph to the left) and polyclonal (graph to the right) infused CTL.

Figure 5: Patient Characteristics. Tumor size by mWHO in response to ipilimumab alone (Stromnes et al., 2014, Immunol. Rev. 257: 145-64): +8% at 12 weeks and +63% at 28 weeks (Rosenberg et al.,2011, Clin. Cancer Res. 17:4550-7). +43% at 12 weeks (Yang, 2013, Clin. Dermatol. 31 :209-19). Received a 1^st course of ipilimumab with no evaluable disease in the neo-adjuvant setting immediately after 1^st surgery, second course: +56% at 12 weeks. (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7) Received 1 dose of ipilimumab 4 weeks before the start of the trial while the CTL products were being generated in the lab. No documented response to 1 dose of ipilimumab (Hunder et al., 2008, N. Engl. J. Med. 358:2698-703).

Leukapheresis performed 3 or 4 weeks after last surgery and CTL product generated and frozen in anticipation of progressive disease (Wallen et al., 2009, PLoS One 4:e4749). No treatment for 6 weeks between Leukapheresis and the start of

experimental therapy due to local wound infection. LN: lymph node, s.c: subcutaneous, HD: High-dose

Figures 6A and 6B: Tumor regressions following melanoma-reactive polyclonal CTL combined with anti-CTLA-4. (A) Spider plot of all treated patients showing changes from baseline in the tumor burden (y-axis), measured as the products of the perpendicular diameters of all target lesions, assessed weeks after the CTL infusion (x-axis). The dashed lines above the solid baseline indicate 25% progression (mWHO PD) and the dashed line below the solid baseline indicate 50% reduction

(mWHO PR). Solid lines indicate patients with PD, small dashed lines patients with SD, large dashed lines patients with PRs and _ _ _ -medium dashed lines patients with CRs. Squares indicate the occurrence of new lesions, asterisks indicate the start of an alternate treatment, pound (#) signs indicate disease progression sufficient to transition to comfort care, and horizontal arrows indicate continued monitoring. (B) Serial images of CTs performed before infusion (left), 64 and 55 weeks after treatment (right) for Patients 1 (top) and 7 (bottom) respectively. Arrowheads indicate the location of the largest index lesions for each patient.

Figures 7A and 7B: Kinetics of in vivo persistence of melanoma-reactive polyclonal CTL. (A) Percent multimer⁺CD8⁺ T-cells (left y-axis) in PBMCs (solid circles) collected 7 days {+1-2 days) before and at defined time-points after infusions are shown for patients who achieved CR, PR or SD after treatment. Vertical downward arrows indicate CTL infusions, vertical arrows pointing upwards indicate anti-CTLA-4 infusions, asterisks indicate the start of an alternate treatment, pound signs indicate comfort care, horizontal arrows indicate concurrent corticosteroid therapy, horizontal arrows indicate ongoing monitoring. (B) The same analysis performed for patients who progressed after treatment.

Figures 8A-8D: Phenotypic and functional characteristics of transferred melanoma-reactive CTL. (A) Expression of CD27, CD28, CD127, CD62L, CCR7, CD57 and PD1 (y-axis) on gated multimer⁺ cells for CD8⁺ CTL products immediately before infusion and at after 3, 6, 9 and 23 weeks in vivo for patients who achieved CR, PR or SD. (B) The same analysis performed for patients who progressed. (C) Open symbols indicate patients who achieved CR, OR or SD (left column), solid symbols indicate patients who had PD (right column). Left plots: percent of cells within the infusion products producing IFNy in response to MARTI peptide, and in PBMC before, 6 and 12 weeks after the CTL infusion; plots to the right: respective percentages of TNFa and IL-2 cells amongst IFNy⁺ cells. (D) Mean intranuclear Ki-67 expression of endogenous CD8⁺multimer^" cells (black columns), pre-infusion CTL products and multimer⁺ CD8⁺ T-cells at indicated timepoints (grey columns), for all patients combined. Two-tailed paired t-tests were used for statistical analysis. *p<0.05,

**p<0.005, ***p<0.0005 n.s.: not significant.

Figures 9A and 9B: Reactivity to non-targeted epitopes. (A) IFNy spots/10⁵ PBMC (y-axis) at indicated time-points (x-axis) before and after the infusion of polyclonal CTL generated in the presence of IL-21 for patients who achieved CR, PR or SD, and (B) who progressed after receiving the treatment. Pound (#) signs indicate patients who had received prior ipilimumab, vertical downward arrows indicate CTL infusions, horizontal lines indicate ipilimumab administration with the total number of doses received indicated immediately above. The scale of the y-axis for graphs for Patients 1 (max 700 spots/10⁵ PBMC) and 4 (max 3000 spots/10⁵ PBMC) are different from all others (max 400 spots/10⁵ PBMC). Two-tailed paired t-tests were used for statistical analysis. *p<0.05, **p<0.005, n.s.: not significant.

Figure 10: Treatment plan. All patients received polyclonal MARTI -specific CTL lines primed with IL-21 on day 0 (10¹⁰ cells/m²) preceded by 300mg/m² of Cyclophosphamide on day -2. Infusions were followed by low-dose subcutaneous IL-2 for 14 days (2.5 x 10⁵ R7 twice daily) starting within 6 hours of the CTL infusion.

Ipilimumab 3mg/m² intavenous was administered once every 3 weeks for a total of 4 doses starting on day 1 after the CTL infusion. Patients were evaluated for responses 6 and 12 weeks after infusion (on study) and then as clinically indicated.

Figures 11A and B: Phenotypic and functional characteristics of infused MARTl-specific CTL products. (A) From the left: Production of IFNy in response to T2 B-LCL targets pulsed with the HLA-A*0201 -restricted MARTl₂₆-35 peptide, and lysis of HLA A*0201⁺ MARTI -expressing MEL-526 cell line (solid circles) and the HLA A*0201⁺ MARTI -negative MEL-375 cell line (open circles) at decreasing effector to target ratios in ⁵¹Cr-release assays. (B) Expression of CD45RO, CD27, CD28 and CD127 (upper row), and CD62L, CCR7, CD57 and PD1 (lower row) by MARTl-specific infused CTL for all patients.

Figures 12A and 12B: Cutaneous regression of metastatic melanoma. (A) The right inguinal region of Patient 9 before (left), 24 (middle) and 31 (right) weeks after the CTL infusion. (B) Close-up of Patient 9's lesions before (left) and 31 weeks (right) after the CTL infusion.

Figure 13: Kaplan-Meier curve for progression-free survival and overall survival. As of November 1, 2015, the median follow-up for overall survival was 187 weeks (range 220-141 weeks); 5 patients are alive, 2 of whom continue in CR (*) and did not receive additional anti-tumor treatment.

Figure 14: Functional characteristics of transferred melanoma-reactive CTL. Left column: percent CD8⁺ T cells (x axis) producing IFNy (y axis) before infusions, by the CTL product, and at indicated time points in PBMC after the CTL infusion for Pt 10 (representative) in response to MARTI peptide. Middle and right columns: respective percentages of TNFa and IL2 (x-axis) producing cells amongst IFNy⁺ cells (y axis).

Figure 15: Phenotype and function of products infused in patients who presented with disease control versus progression. Comparative surface expression of CD27, CD28, CD127, CD62L, CCR7, CD57 and PD1; intracellular production of IFNy, TNFa and IL-2 in response to cognate antigen; and intranuclear expression of Ki- 67 for products infused in patients who presented with a CR, PR or SD to the combined treatment or with PD. Figures 16A and 16B: Reactivity to melanoma-associated antigens in patients with melanoma and patients who received monoclonal CTL products. (A)

IFNy spots/10⁵ PBMC (y-axis) for 3 patients with metastatic melanoma who did not undergo therapy with anti-CTLA4 or T cell infusions and (B) for 5 patients who underwent infusions of melanoma-specific T-cells only. Vertical downward arrows indicate CTL infusions. Two-tailed paired t-tests were used for statistical analysis, n.s.: not significant.

Figures 17A-17C depict characteristics of monoclonal CTL products and concurrence of clonotype frequencies determined by TCR νβ PCR, multimer stains and HTTCS in vivo. (A) Scatter plot showing binding of the monoclonal cells to CD8 (x- axis) and HLA A*0201 -restricted MARTI27-35 (y-axis). (B) Pie graphs showing the percent of individual clonotypes composing the monoclonal CTL products. The frequency of the specific clone is overlayed on the plots. The total number of sequences detected in the products is stated above each plot. (C) Pre- and post- infusion TCR-VP copies / 100 CD8 T cells (solid squares), % multimer⁺ T cells (solid circles) and % clonotypes in PBMC (solid hexagons) are shown for 2 patients who received monoclonal CTL products. Vertical arrows indicate CTL infusions. Dashed lines represents limit of detection of frequencies by HTTCS. Significance of the correlation (R² and p values) between HTTCS/multimer and HTTCS/TCR copied per 100 cells are depicted below each graph.

Figures 18A and 18B depict characteristics of polyclonal products and concurrence of clonotype frequencies determined by HTTCS and multimer staining. (A) Pie graphs show the frequency of the first 25 TCR clonotypes in polyclonal products (shaded in color) as well as the remainder of the clones (shaded in grey). The total number of clonotypes in the products is indicated above each plot. The frequency of the most prevalent clonotype (TCR seq 1) is indicated. (B) Percent antigen-specific clonotypes obtained by HTTCS (open circles), and % multimer⁺ T cells (solid black circles) before (left-most timepoint) and after infusions (infused on day 0) are shown for 10 patients who received polyclonal antigen-specific products. Vertical arrows indicate CTL infusions. Dashed lines represent the limit of detection for HTTCS and dotted lines represents limit of detection for multimer staining, which is dependent on the ratio of CD8⁺ to CD4⁺ T cells and varies for each patient. Significance of the correlation (R² and p values) between HTTCS and multimer are depicted on the right side of each graph.

Figure 19 depicts a bar graph showing naive vs antigen-experienced phenotype based on clonotype frequency in PBMC. The propensity of clonotypes present in 3 normal donor PBMC above or below the threshold of 0.001% with a CD3⁺CD45RO⁺ (antigen experienced) versus a CD3⁺CD45RA⁺ (naive) phenotype was determined by tracking clonotypes across whole PBMCs, sorted CD3⁺CD45RA⁺ and sorted

CD3⁺CD45RO⁺ populations using HTTCS. The proportion of clonotypes with PBMC frequencies <0.001% (bar to the left) and >0.001% (bar to the right) that were also in the CD3⁺CD45RA⁺ sorted population are shown.

Figures 20A-20C depict the composition of infused products and

persistence/frequency of TCR subsets in vivo. (A) Percent clonotypes detected at any time-point after infusions (open circles). The individual percent contribution of the most prevalent 25 antigen-specific clonotypes in the infused products (left y axis) are shown (colored lines) as well as the sum of the remaining sequences in the products (grey lines), before (left-most timepoint) and at selected time-points after infusions (day 0). Arrows indicate the immunodominant clonotypes for Pts P2225-1 and 7 who achieved a CR (indicated) as best response. (B) Half-lives of clonotypes for each Pt grouped according to their best response. Boxes from left to right: CRs, PR, SD, PD. Arrows pointing to circles indicate the immunodominant clonotypes for patients P2225- 1 and 7. (C) Box and whisker plots of the t½ grouped according to patient best responses. *p<0.05, ***p<0.0005.

Figures 21A and 21B depict persistence/frequency of different clonotype subsets in vivo. (A) Pie graphs showing the number (top row) and percentage (bottom row) of clonotypes composing the infused polyclonal products. Clonotypes in the infusion products which were below the limit of detection either before or at any time- point after infusions (grey area). Clonotypes detected at any time-point after infusions which were not detected in PBMC before infusions (white area). The number of clonotypes composing the blue area for each patient are shown on the top row and their respective percentage are shown on the bottom row. Sum of clonotypes detected in PBMC before infusions expanded in the infusion product (orange area). Sum of clonotypes detected in PBMC before not expanded in the infusion product (d area). (B) Percent total antigen-specific TCR sequences (solid circles); percent total TCR sequences detected at any time-point after infusions which were not detected in PB before infusions (open circles); percent total TCR sequences detected in PB before infusions expanded in the infusion product (solid squares), before and at selected timepoints after infusions.

DETAILED DESCRIPTION

In certain aspects, the present disclosure provides T cell receptors (TCRs) that are specific for MART-1 peptide antigen associated with a major histocompatibility complex (MHC) (e.g., human leukocyte antigen, HLA), for use in, for example, adoptive immunotherapy to treat cancer.

By way of background, patients with metastatic melanoma rarely display tumor-specific immune cells sufficient for thwarting disease progression (Restifo et al., 2012, Nat Rev Immunol 12:269-281). Adoptive transfer of autologous peripheral blood (PB)-derived antigen-specific T cells can increase the frequency of melanoma-specific T cells, with a very tolerable safety profile (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Wallen et al., 2009, PLoS One 4:e4749; Yee et al., 2002, Proc Natl Acad Sci USA 99: 16168-16173). When used as monotherapy, this approach has been effective in delaying disease progression. But sustained, complete tumor regression is rare, in part due to the short in vivo survival of transferred cells as well as inhibitory signals limiting full T-cell activation (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Wallen et al., 2009, PLoS One 4:e4749; Yee et al., 2002, Proc Natl Acad Sci USA 99: 16168-16173).

The compositions and methods described herein will in certain embodiments have therapeutic utility for the treatment of diseases and conditions associated with MART-1 overexpression (e.g., detectable MART-1 expression at a level that is greater in magnitude, in a statistically significant manner, than the level of MART-1 expression that is detectable in a normal or disease-free cell). Such diseases include various forms of hyperproliferative disorders, such as melanoma. Non-limiting examples of these and related uses are described herein and include in vitro, ex vivo and in vivo stimulation of MART-1 antigen-specific T cell responses, such as by the use of recombinant T cells expressing a TCR specific for a MART-1 peptide (e.g., EAAGIGILTV (SEQ ID NO: 1))·

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 limits the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic characteristics of a claimed invention. 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%, 10%, 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, 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 γδ T cell, a regulatory T cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may 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.

"Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to a cell surface. MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non-covalently associated β2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a 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).

A "T cell" (or "T lymphocyte") is an immune system cell that matures in the thymus and produces T cell receptors (TCRs), which can be obtained (enriched or isolated) from, for example, peripheral blood mononuclear cells (PBMCs) and are referred to herein as "bulk" T cells. After isolation of T cells, both cytotoxic (CD8+) and helper (CD4+) T cells can be sorted into naive, memory, and effector T cell subpopulations, either before or after expansion. T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD 127, and CD45RA, and decreased expression of CD45RO as compared to TC_M), memory T cells (T_M) (antigen-experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). T_M can be further divided into subsets of central memory T cells (TC_M, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (T_EM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or T_CM)- Effector T cells (T_E) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that has decreased expression of CD62L ,CCR7, CD28, and are positive for granzyme and perforin as compared to T_CM- Helper T cells (T_H) 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 action is induced will depend on presence of other cells and signals. T cells can be collected in accordance with 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.

"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, 3^rd Ed., Current Biology Publications, p. 4:33, 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 a and β chains (also known as TCRa and TCR , respectively), or γ and δ chains (also known as TCRy and TCR6, respectively). Like immunoglobulins, 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_a, β- 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^th ed.) at the N-terminus, and one constant domain {e.g., a-chain constant domain or C_a, typically amino acids 117 to 259 based on Kabat, β-chain constant domain or C , typically amino acids 117 to 295 based on Kabat) adjacent to the cell membrane. Also like

immunoglobulins, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) {see, e.g., Jores et al., Proc. Nat'lAcad. Sci. U.S.A. 57:9138, 1990; Chothia et al, EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In certain embodiments, a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. 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 a TCR a-chain or β-chain (or γ chain and δ chain for γδ TCRs) that is involved in binding of the TCR to antigen. The variable domains of the a-chain and β-chain (V_a and V_a, respectively) of a native TCR generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. The V_a domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (V-J); the ν_β 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 V_a or V domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs that bind a particular antigen may be isolated using a V_a or V domain from a TCR that binds the antigen to screen a library of complementary V_a or V domains, respectively.

The terms "complementarity determining region," and "CDR," which are synonymous with "hypervariable region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within TCR variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each a- chain variable region (a CDR1, a CDR2, aCDR3) and three CDRs in each β-chain variable region ^CDRl, βCDR2, βCDR3). CDR3 is thought to be the main CDR responsible for recognizing processed antigen. CDR1 and CDR2 mainly interact with the MHC. However, the CDR1 loops of the alpha chain and beta chain have also been shown to interact with the N-terminal and C-terminal ends of the antigenic peptide, respectively, and may contribute to both peptide specificity and MHC binding. "CD3" is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al., pl72 and 178, 1999). In mammals, the complex comprises a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of CD3ζ chains. The CD3y, CD35, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD35, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine- based activation motif or ITAM, whereas each 0)3ζ chain has three. Without wishing to be bound by theory, it is believed 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 CD35 chain, two CD3s chains, a homodimer of CD3ζ chains, a TCRa chain, and a

TCRP chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of 0)3ζ chains, a TCRy chain, and a TCR5 chain.

A "component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCRa, TCRp, TCRy or TCR5), a CD3 chain (i.e., CO3j, CD35, CD3s or CO3Q, 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 TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCRp, CD3y, CD35, and two CD3s chains).

"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. Exemplary antigens include MART- 1.

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. For example, a MART-1 protein or fragment thereof may be an antigen that contains one or more antigenic epitopes.

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., MART-1, MART-1 peptide:MHC complex). A binding domain includes any naturally occurring, synthetic, semisynthetic, 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. Exemplary binding domains include single chain

immunoglobulin variable regions (e.g., scTCR, scFv), receptor ectodomains, ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex or other target of interest.

As used herein, "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 (e.g., MART-1 peptide: :HLA or a tetramer such an HLA complex) 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_on] to the off-rate [k₀ff] 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 10¹⁰ 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 may be defined as an equilibrium dissociation constant (K_d) of a particular binding interaction with units of M (e.g., 10^"5 M to 10^"13 M).

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 K_d

(dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (k₀ff) for the target antigen that is less than that of the wild type binding domain, or a combination thereof. In certain embodiments, enhanced affinity TCRs may be codon optimized to enhance expression in a particular host cell, such as T cells (Scholten et al., Clin. Immunol. 779: 135, 2006).

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 fusion protein affinities, such as Western blot, ELISA, analytical

ultracentrifugation, spectroscopy, surface plasmon resonance (Biacore®) analysis, MHC tetramer assay (see, e.g., Scatchard et al, Ann. N. Y. Acad. Sci. 57:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al, Cancer Res. 53:2560, 1993; Altman et al., Science 274:94-96, 1996; and U.S. Patent Nos. 5,283, 173, 5,468,614, or the equivalent).

The term "MART-1 -specific binding protein" refers to a protein or polypeptide that specifically binds to MART-1 or a peptide or fragment thereof. In some embodiments, a protein or polypeptide binds to MART-1 or a peptide thereof, such as a MART-1 peptide in complexed with an MHC or HLA molecule, e.g., on a cell surface, with at or at least about a particular affinity. In certain embodiments, a MART-1- specific binding protein binds a MART-1 -derived peptide:HLA complex (or MART-1- derived peptide:MHC complex) with a ¾ 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 MART-1 specific binding protein provided herein, such as any of the MART-1 -specific TCRs provided herein, for example, as measured by the same assay. In certain embodiments, a MART-1 -specific binding protein comprises a MART-1 -specific immunoglobulin superfamily binding protein or binding portion thereof.

Assays for assessing affinity or apparent affinity or relative affinity are known. In certain examples, apparent affinity for a TCR is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers. In some examples, apparent K_D of a TCR is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent K_D being determined as the concentration of ligand that yielded half-maximal binding.

The term "MART-1 binding domain" or "MART-1 binding fragment" refer to a domain or portion of a MART-1 -specific binding protein responsible for the specific MART-1 binding. A MART-1 -specific binding domain alone (i.e., without any other portion of a MART-1 -specific binding protein) can be soluble and can bind to MART-1 with a K_d 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. Exemplary MART-1 -specific binding domains include MART-1 -specific scTCR (e.g., single chain o^TCR proteins such as Va-L-Υβ, Υβ-L-Va, Va-Ca-L-Va, or Va-L-V -C , wherein Va and νβ are TCRa and β variable domains respectively, Ca and C are TCRa and β constant domains, respectively, and L is a linker) and scFv fragments as described herein, which can be derived from an anti-MART-1 TCR or antibody.

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.) 201 1 Garland Science, NY; chapters 6, 9 and 16). For example, processed antigen peptides originating in the cytosol (e.g., tumor antigen, intrcellular pathogen) are generally from about 7 amino acids to about 1 1 amino acids in length and will associate with class I MHC 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 molecules.

"MART-1 antigen" or " MART-1 peptide antigen" refer to a naturally or synthetically produced portion of a MART-1 protein ranging in length from about 7 amino acids to about 15 amino acids, which can form a complex with a MHC (e.g., HLA) molecule and such a complex can bind with a TCR specific for a MART-1 peptide:MHC (e.g., HLA) complex. MART-1 antigen peptides are presented in the context of class I MHC. In particular embodiments, a MART-1 peptide is

EAAGIGILTV (SEQ ID NO: 1), which is known to associate with human class I HLA (and, more specifically, associates with allele HLA-A*0201).

A "linker" refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex). In certain embodiments, a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids. "Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-10) 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 may result from the construct design of 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).

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 TCRa chain, TCR chain, TCRa constant domain, TCR 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%).

As used herein, "nucleic acid" or "nucleic acid molecule" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and 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 may 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 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 term "recombinant" or "genetically engineered" refers to a cell, microorganism, nucleic acid molecule, or vector that has been genetically modified by human intervention - that is, modified by introduction of an exogenous or 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. Human generated genetic alterations may include, for example, modifications that introduce nucleic acid molecules (which may include an expression control element, such as a promoter) that encode 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^m 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 construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may 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).

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 picornavirus 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, 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).

"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.

The term "operably-linked" 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 is capable of affecting 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.

As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule that is operably-linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid," "expression plasmid," "virus" and "vector" are often used interchangeably.

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 may 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 may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or 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 may 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 may 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, electroporation, or the like, wherein the added molecule may 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 may 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.

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. For example, as disclosed herein, a host cell can be modified to express two or more heterologous or exogenous nucleic acid molecules encoding desired TCR specific for a MART-1 antigen peptide (e.g., TCRa and TCR ). 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 cell.

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., promoter, translational attenuation sequences) may 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 may be homologous to a native host cell gene, and may 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 BLAST2.0 software as defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, with the parameters set to default values.

As used herein, a "hematopoietic progenitor stem cell" refers to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to other cell types including cells of the T cell lineage. Hematopoietic stem cells may be isolated, for example, but not limited to, from fetal liver, bone marrow, cord blood.

As used herein, a "hematopoietic progenitor cell" is a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types {e.g., immune system cells). Exemplary hematopoietic progenitor cells include those with a CD24^Lo Lin^" CD117⁺ phenotype or those found in the thymus (referred to as progenitor thymocytes).

As used herein, the term "host" refers to a cell {e.g., T cell) or microorganism targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., high or enhanced affinity anti- MART-1 TCR). In certain embodiments, a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to biosynthesis of the heterologous or exogenous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous TCR; increased co- stimulatory factor expression). In certain embodiments, a host cell is a human hematopoietic progenitor cell transduced with a heterologous or exogenous nucleic acid molecule encoding a TCRP, TCRa chain or both, specific for a MART-1 antigen peptide. In certain embodiments, a host cells are autologous, allogeneic or syngeneic to subject to receive the host cells containing a polynucleotide encoding a binding protein of this disclosure.

As used herein, "hyperproliferative disorder" refers to excessive growth or proliferation as compared to a normal or undiseased cell. Exemplary hyperproliferative disorders include tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis, as well as autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, or the like). In certain embodiments, a hyperproliferative disorder is a melanoma. Binding Proteins Specific for MART-1 Antigen

"MART-1 " or "Protein melan-A" or "melanoma antigen recognized by T cells 1 " refers to a 118 amino acid transmembrane protein encoded by the MLANA gene. The transcript sequence for human MLANA is set forth in NCBI Reference identifier

M_005511.1 (SEQ ID NO:2), and protein sequence is set forth in NCBI Reference identifier NP_005502 (SEQ ID NO:3). MART-1 is an antigen specific for cells of the melanocyte lineage, found in skin, the retina, and melanocytes, but not in other normal tissues. MART-1 is an attractive target for tumor therapy because it is the antigen most commonly expressed by melanoma tumors, and MART-1 epitopes presented in the HLA-A*0201 context are most frequently recognized by tumor infiltrating lymphocytes (TILs) from melanoma patients (Labarriere et al., 1998, Int. J. Cancer 78:209-215; Benlalam et al. 2001, Eur. J. Immunol. 31 :2007-2015). In certain aspects, the present disclosure provides a binding protein (e.g., an immunoglobulin superfamily binding protein or portion thereof), comprising (a) a T cell receptor (TCR) a-chain variable (V_a) domain, and a TCR β-chain variable domain comprising a CDR3 amino acid sequence set forth in any one of SEQ ID NOS:4-16 and 24-151, wherein the binding protein is capable of specifically binding to a MART-1 peptide (SEQ ID NO: l):HLA complex. In some embodiments, the νβ CDR3 amino acid sequence consists of the amino acid sequence set forth in any one of SEQ ID NOS:4-16 and 24-151. In particular embodiments, a νβ CDR3 amino acid sequence comprises or consists of the amino acid sequence set forth in any one of SEQ ID

NOS:4-16 and 24. In a particular embodiment, the ν_β CDR3 amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO:4. An exemplary νβ amino acid sequence that comprises the CDR3 amino acid sequence of SEQ ID NO:4 is set forth in SEQ ID NO: 173. An exemplary TCR β-chain amino acid sequence that comprises the ν_β amino acid sequence of SEQ ID NO: 173 is set forth in SEQ ID NO: 172. In certain embodiments, a binding protein (e.g., an immunoglobulin

superfamily binding protein or portion thereof) as described herein includes variant polypeptide species that have up to five amino acid substitutions, insertions, or deletions in the amino acid sequence of any one of SEQ ID NOS:4-16 and 24-151 as presented herein, provided that the binding protein retains or substantially retains its specific binding function to MART-1 peptide EAAGIGILTV (SEQ ID NO: 1). In some embodiments, a variant binding protein has one, two, three, four, or five amino acids substitutions, insertions, or deletions in the amino acid sequence of any one of SEQ ID NOS:4-16 and 24-151, provided that the binding protein retains or substantially retains its ability to specificaly bind to MART-1 peptide EAAGIGILTV (SEQ ID NO: 1):HLA complex.

In other embodiments, a binding protein provided herein may comprise a variant TCR νβ domain having an amino acid sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID

NO: 173, provided that the binding protein retains or substantially retains its binding function to MART-1 peptide (SEQ ID NO: 1):HLA complex. In yet another

embodiment, a binding protein provided herein may comprise a variant TCR β-chain having an amino acid sequence with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 172, provided that the binding protein retains or substantially retains its ability to specifically bind to MART-1 peptide (SEQ ID NO: l):HLA complex.

Binding proteins provided herein that comprise a νβ CDR3 amino acid sequence comprising of the amino acid sequence of SEQ ID NO:4, or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, may be paired with a V_a domain comprising a CDR3 amino acid sequence as set forth in Table 1 A (e.g., any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169). Accordingly, an exemplary binding protein comprises: (a) a T cell receptor (TCR) a-chain variable (V_a) domain comprising a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169; and a TCR β-chain variable (ν_β) domain comprising a CDR3 amino acid sequence set forth in SEQ ID NO:4, or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, wherein the binding protein is capable of specifically binding to a MART-1 peptide (SEQ ID NO: 1):HLA complex. In certain embodiments, a binding protein as described herein comprises a νβ CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 155; a ν_β CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 157; a νβ CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 159; a ν_β CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 161; a ν_β CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 163; a νβ CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 165; a ν_β CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 167; or a ν_β CDR3 amino acid sequence comprising SEQ ID NO:4 or a ν_β domain comprising an amino acid sequence set forth in SEQ ID NO: 173, and a V_a CDR3 amino acid sequence of SEQ ID NO: 169.

Table 1A TCR chain gene segment and CDR sequence information

TCR CDR3 nucleotide CDR3 amino acid V gene J gene chain sequence sequence segment segment

GGGAAG

(SEQ ID NO: 166)

TGTGCCCTCCCAGCCTGGA GACTCTGCAGTGTACTTCT GTGCAGCATTCGGAGGAGG CAAFGGGADGLTF TCRAJ45- TGCTGACGGACTCACCTTT TCRAV29-01

(SEQ ID NO: 169) 01*01 GGCAAA

(SEQ ID NO: 168)

GAGTCCGCCAGCACCAACC AGACATCTATGTACCTCTG TGCCAGCAAAATACAGGAC CASKIQDGYASEQYF TCRBJ02-

V, GGCTATGCGTCCGAGCAGT TCRBV28-01

(SEQ ID NO:4) 07 ACTTCGGGCCG

(SEQ ID NO: 170)

CASSYGGGQPQHF TCRBJ01-

V, TCRBV06-05

(SEQ ID NO:5) 05

CASSPFELSLSEQFF TCRBJ02-

V, TCRBV28-01

(SEQ ID NO: 6) 01

CASSQPLYNSPLHF TCRBJ01-

V, TCRBV04-01

(SEQ ID NO:7) 06

CASSFWDKAKNIQYF TCRBJ02-

V, TCRBV05-05

(SEQ ID NO: 8) 04

CASSSPINRAGGLDTQY F TCRBJ02-

V, TCRBV28-01

03

(SEQ ID NO: 9)

CASRTGLGQPQHF TCRBJ01-

V_R TCRBV28-01

(SEQ ID NO: 10) 05

CASSYSINQPQHF TCRBJ01-

V_R TCRBV06-05

(SEQ ID NO: 11) 05

CASSLDGGASGNEQFF TCRBJ02-

V_R TCRBV05-04

(SEQ ID NO: 12) 01

CASSSLLDRGIDEQYF TCRBJ02-

V_R

(SEQ ID NO: 13) 07

CASGLGPLFADTQYF TCRBJ02-

V_R TCRBV27-01

(SEQ ID NO: 14) 03

CASKQGALTGELFF TCRBJ02-

V_R TCRBV06-05

(SEQ ID NO: 15) 02

CASSYSGVGQPQHF TCRBJ01-

V_R TCRBV06-06

(SEQ ID NO: 16) 05

Moreover, binding proteins provided herein that may comprise a V_a domain comprising a variant of the CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169 that has up to five (one, two, three, four, or five) amino acid substitutions, insertions, or deletions in the amino acid sequence of any one of SEQ ID NO S: 155, 157, 159, 161, 163, 165, 167, and 169, respectively, provided that the binding protein retains or substantially retains its ability to specifically bind to MART-1 peptide (SEQ ID NO: 1):HLA complex.

In certain aspects, the present disclosure further provides a binding protein (e.g., an immunoglobulin superfamily binding protein or portion thereof), comprising (a) a T cell receptor (TCR) a-chain variable (V_a) domain, and a TCR β-chain variable (\¾) domain comprising a CDR3 amino acid sequence encoded by a nucleotide sequence contained in SEQ ID NO: 153, wherein the binding protein is capable of specifically binding to a Tyrosinase peptide (SEQ ID NO:23):HLA complex.

Tyrosinase refers to refers to a 529 amino acid protein encoded by the TYR gene. The transcript sequence for human TYR is set forth in NCBI Reference identifier NM_000372.4, and protein sequence is set forth in NCBI Reference identifier

NP 000363.1. Tyrosinase is an oxidase that is involved in melanin synthesis.

Tyrosinase is an attractive target for melanoma therapy because it an antigen commonly expressed by melanoma tumors.

In certain embodiments, a binding protein {e.g., an immunoglobulin superfamily binding protein or portion thereof) as described herein includes variant polypeptide species that have up to five (one, two, three, four, or five) amino acid substitutions, insertions, or deletions in the amino acid sequence of νβ CDR3 encoded by the nucleotide acid sequence contained in SEQ ID NO: 153 as presented herein, provided that the binding protein retains or substantially retains its ability to specifically bind to Tyrosinase peptide SEIWRDIDF (SEQ ID NO:23).

Peptide-MHC complexes, such as MART-1 peptide (SEQ ID NO: 1):MHC complexes are recognized by and bound through the TCR Va and TCR νβ domains. During lymphocyte development, Va exons are assembled from different variable and joining gene segments (V-J), and νβ exons are assembled from different variable, diversity, and joining gene segments (V-D-J). The TCRa chromosomal locus has 70-80 variable gene segments and 61 joining gene segments. The TCRP chromosomal locus has 52 variable gene segments, and two separate clusters of each containing a single diversity gene segment, together with six or seven joining gene segments. Functional Va and νβ gene exons are generated by the recombination of a variable gene segment with a joining gene segment for Va, and a variable gene segment with a diversity gene segment and a joining gene segment for νβ.

The Va and νβ domains each comprise three hypervariable loops, also referred to as complementary determining regions (CDRs) that contact the peptide-MHC complex. CDR1 and CDR2 are encoded within the variable gene segment, whereas CDR3 is encoded by the region spanning the variable and joining segments for Va, or the region spanning variable, diversity, and joining segments for νβ. Thus, if the identity of the variable gene segment of a Va or νβ is known, the sequences of their corresponding CDR1 and CDR2 can be deduced. Compared with CDR1 and CDR2, CDR3 is significantly more diverse because of the addition and loss of nucleotides during the recombination process.

TCR variable domain sequences can be aligned to a numbering scheme (Kabat, Chothia, Enhanced Chothia, and Aho), allowing equivalent residue positions to be annotated and for different molecules to be compared using ANARCI software tool (2016, Bioinformatics 15:298-300). A numbering scheme provides a standardized delineation of framework regions and CDRs in the TCR variable domains.

Table 1A provides the identities of the variable gene segment and joining gene segment for a TCR Va comprising a CDR3 comprising an amino acid sequence of any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169 and the identities of the variable gene segment and joining gene segment for a TCR νβ comprising a CDR3 comprising an amino acid sequence of any one of SEQ ID NOS:4-16. Accordingly, the CDR1 and CDR2 sequences may be deduced from the corresponding variable gene segments.

In some embodiments, a TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 155 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-02 and a joining gene segment of TCRAJ34- 01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 155 and a CDR1 and CDR2 sequence encoded by a

TCRAV12-02 gene. In another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 157 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV29-01 and a joining gene segment of TCRAJ26-01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 157 and a CDRl and CDR2 sequence encoded by a TCRAV29-01 gene. In another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 159 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-02 and a joining gene segment of TCRAJ30-01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 159 and a CDRl and CDR2 sequence encoded by a TCRAV12-02 gene. In another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 161 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-01 and a joining gene segment of TCRAJ24-01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 161 and a CDRl and CDR2 sequence encoded by a TCRAV12-01 gene. In another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 163 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV13-02 and a joining gene segment of TCRAJ38-01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 163 and a CDRl and CDR2 sequence encoded by a TCRAV13-02 gene. In another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 165 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV12-02 and a joining gene segment of TCRAJ17-01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 165 and a CDRl and CDR2 sequence encoded by TCRAV 12-02. In another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 167 is contained in an amino acid sequence encoded by a variable gene segment of TCRAV25-01 *01 and a joining gene segment of a TCRAJ28-01 *01 gene. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 167 and a CDRl and CDR2 sequence encoded by a TCRAV25-01 *01 gene. In yet another embodiment, the TCR Va comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 169 is contained in an amino acid sequence encoded by a a variable gene segment of

TCRAV29-01 and a joining gene segment of TCRAJ45-01 *01. Accordingly, the TCR Va comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 169 and a CDRl and CDR2 sequence encoded by a TCRAV29-01 gene.

In still more embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:4 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV28-01 and a joining gene segment of TCRBJ02- 07. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:4 and a CDRl and CDR2 sequence encoded by a TCRBV28-01 gene. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:5 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV06-05 and a joining gene segment of TCRBJOl-05. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 5 and a CDRl and CDR2 sequence encoded by TCRBV06-05. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:6 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV28-01 and a joining gene segment of TCRBJ02-01. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 6 and a CDRl and CDR2 sequence encoded by TCRBV28-01. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:7 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV04-01 and a joining gene segment of TCRBJOl-06. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:7 and a CDRl and CDR2 sequence encoded by TCRBV04-01. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:8 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV05-05 and a joining gene segment of TCRBJ02-04. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:8 and a CDRl and CDR2 sequence encoded by TCRBV05-05. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO:9 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV28-01 and a joining gene segment of TCRBJ02-03. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO:9 and a CDRl and CDR2 sequence encoded by TCRBV28-01. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 10 is contained in an amino acid sequence encoded by a variable gene segment of TCRB V28-01 and a j oining gene segment of TCRB JO 1 - 05. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 10 and a CDRl and CDR2 sequence encoded by a TCRBV28-01 gene. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 11 is contained in an amino acid sequence encoded by a variable gene segment of TCRB V06-05 and a j oining gene segment of TCRB JO 1 -05. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 11 and a CDRl and CDR2 sequence encoded by a TCRBV06-05 gene. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 12 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV05-04 and a joining gene segment of TCRBJ02-01. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 12 and a CDRl and CDR2 sequence encoded by a TCRBV05-04 gene. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 14 is contained in an amino acid sequence encoded by a variable gene segment of TCRB V27-01 and a j oining gene segment of TCRB J02-03. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 14 and a CDRl and CDR2 sequence encoded by a TCRBV27-01 gene. In some embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 15 is contained in an amino acid sequence encoded by a a variable gene segment of TCRBV06-05 and a joining gene segment of TCRBJ02-02. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 15 and a CDRl and CDR2 sequence encoded by a TCRBV06-05 gene. In yet other embodiments, the TCR νβ comprising a CDR3 comprising an amino acid sequence of SEQ ID NO: 16 is contained in an amino acid sequence encoded by a variable gene segment of TCRBV06-06 and a joining gene segment of TCRB JO 1-05. Accordingly, the TCR νβ comprises a CDR3 comprising an amino acid sequence of SEQ ID NO: 16 and a CDR1 and CDR2 sequence encoded by a TCRBV06-06 gene.

Methods of identifying binding pairs of TCR Va and νβ domains are known in the art and include, for example, PCT Patent Publication No. WO 2016/161273;

Redmond et al., 2016, Genome Med. 8: 80; Munson et al., 2016, Proc. Natl. Acad. Sci. 113 :8272-7; Kim et al., 2012, PLoS ONE 7:e37338 (each of the methods from which are incorporated by reference in its entirety). Accordingly, a V_a domain for the MART- 1 specific νβ domains described herein (e.g., a νβ domain comprising CDR3 as set forth in any one of SEQ ID NOS:4-16) may be identified using methods known in the art.

Conservative substitutions of amino acids are well known and may occur naturally or may be introduced when the binding protein or TCR is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY, 2001). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide- directed mutagenesis may be used to prepare immunogen polypeptide variants (see, e.g., Sambrook et al., supra).

A variety of criteria known to persons skilled in the art indicate whether an amino acid that is substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids may be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In certain circumstances, substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide (e.g., using GE EWORKS, Align, the BLAST algorithm, or other algorithms described herein and practiced in the art).

In certain embodiments, any of the MART-1 specific binding proteins disclosed herein is capable of specifically binding to MART-1 "EAAGIGILTV" peptide (SEQ ID NO: l): HLA-A*0201 complex.

In certain embodiments, any of the Tyrosinase specific binding proteins disclosed herein is capable of specifically binding to Tyrosinase "SEIWRDIDF" peptide (SEQ ID NO:23): HLA-B*4402 complex.

In certain embodiments, any of the MART-1 or Tyrosinase specific binding proteins disclosed herein are each a T cell receptor (TCR), a chimeric antigen receptor or an antigen-binding fragment of a TCR, any of which can be chimeric, humanized or human. In further embodiments, an antigen-binding fragment of the TCR comprises a single chain TCR (scTCR) or a chimeric antigen receptor (CAR). In certain embodiments, a MART-1 or Tyrosinase specific binding protein is a TCR. In further embodiments, a MART-1 or Tyrosinase specific binding protein is an enhanced affinity or high affinity TCR.

Binding proteins according to the present disclosure, e.g., TCRs, may further comprise a TCR constant domain, e.g., joined to the C-terminus of a V_a domain, a νβ domain, or both. A TCR β-chain constant domain may be encoded by a TRBCl gene or TRBC2 gene. In a particular embodiment, the TCR β-chain constant domain is a TRBC2 constant domain having an amino acid sequence as set forth in SEQ ID

NO: 175. A TCR a-chain constant domain may be encoded by a TRAC gene.

A cell expressing a high affinity or enhanced affinity TCR specific for a MART- 1 peptide (or Tyrosinase peptide) is capable of binding to a MART-1 :HL A complex (or Tyrosinase:HLA complex) independent of or in the absence of CD8, is capable of more efficiently associated with a CD3 protein as compared to endogenous TCR, or both. Methods of generating enhanced affinity TCRs for use in gene therapy are known in the art, and include techniques involving generation of libraries of TCR mutants that have undergone rounds of mutagenesis and subsequent screening for mutations that confer higher affinity for the target peptide/MHC ligand (Richman & Kranz, 2007, Biomol. Eng. 24:361-373; Udyavar et al., 2009, J. Immunol. 182:4439-4447; Zhao et al., 2007, J. Immunol. 179:5845-5854). Methods of generating enhanced affinity TCRs wherein the TCRa chain from an antigen-specific TCR is used to select de novo generated TCRP chains that pair with an antigen-specific TCRa chain during T cell development in vitro have also been disclosed (PCT Published Application WO2013/166321, incorporated by reference in its entirety).

In certain embodiments, any of the MART-1 binding proteins disclosed herein is capable of specifically binding to MART-1 "EAAGIGILTV" peptide (SEQ ID NO: 1): HLA-A complex with a Kd of less than or equal to 10^"8M.

In certain embodiments, there is provided a composition comprising a MART-

1 -specific or Tyrosinase-specific binding protein or high affinity recombinant TCR according to any one of the aforementioned embodiments and a pharmaceutically acceptable carrier, diluent, or excipient.

Compositions disclosed herein can further comprise at least one additional binding protein comprising a V_a domain and a ν_β domain, wherein the additional binding protein is capable of specifically binding to a MART-1 peptide EAAGIGILTV (SEQ ID NO: l):HLA complex. The composition can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50 or more additional binding proteins comprising a V_a domain and a ν_β domain, wherein the additional binding protein is capable of specifically binding to a MART-1 peptide EAAGIGILTV (SEQ ID NO: 1):HLA complex. In certain embodiments, the additional binding proteins within the composition are also binding proteins provided herein, e.g., comprising a V_a domain, and a ν_β domain comprising CDR3 of any one of the sequences set forth in SEQ ID NOS:4-16 and 24-151. Each of the binding domains present in the composition can be at a frequency of at least 0.1% in the composition. Moreover, the frequency of each of the binding domains may be unevenly distributed in the composition. In some embodiments, compositions comprising more than one MART-1 specific binding protein represent polyclonal compositions. A polyclonal composition is composed of at least two or more binding domains that are produced by different host cell (e.g., T cell) lineages. In certain embodiments, a polyclonal composition is composed of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75,

100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 binding domains that are produced by different T cell lineages (representing clonotypes >0.001% as detected by HTTCS) A monoclonal composition is composed of a binding domain that is produced from a single host cell lineage.

Methods useful for isolating and purifying recombinantly produced soluble

TCR, by way of example, may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant soluble TCR into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant soluble TCR described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.

In certain embodiments, nucleic acid molecules encoding an immunoglobulin superfamily binding protein or high affinity TCR specific for MART-1 are used to transfect/transduce a host cell (e.g., T cells) for use in adoptive transfer therapy, wherein the host cells are autologous, allogeneic or syngeneic to the subject to receive the adoptive transfer therapy. Advances in TCR sequencing have been described (e.g., Robins et al, Blood 114:4099, 2009; Robins et al, Sci. Translat. Med. 2:47ra64, 2010; Robins et al, J Immunol. Methods 375: 14-9, 2012; Warren et al, Genome Res. 21 :790, 2011) and may be employed in the course of practicing the embodiments according to the present disclosure. Similarly, 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 antigen- specificity {e.g., Schmitt et al, Hum. Gen. 20: 1240, 2009; Dossett et al, Mol. Ther. 77:742, 2009; Till et al, Blood 172:2261, 2008; Wang et al, Hum. Gene Ther. 75:712, 2007; Kuball et al, Blood 709:2331, 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, including those directed to high affinity TCRs specific for MART-1 peptide antigens complexed with an HLA receptor.

The MART-1 -specific binding proteins or domains as described herein {e.g., comprising a ν_β domain comprising CDR3 as shown in any one of the sequences set forth in SEQ ID NOS:4-16 and 24-151) may be functionally characterized according to any of a large number of art accepted methodologies for assaying T cell activity, including determination of T cell binding, activation or induction and also including determination of T cell responses that are antigen-specific. Examples include determination of T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC restricted T cell stimulation, CTL activity {e.g., by detecting ⁵¹Cr release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T-cell functions. Procedures for performing these and similar assays are may be found, for example, in Lefkovits {Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See, also, Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular

Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281 : 1309 (1998) and references cited therein. "MHC-peptide tetramer staining" refers to an assay used to detect antigen- specific T cells, which features a tetramer of MHC molecules, each comprising an identical peptide having an amino acid sequence that is cognate (e.g., identical or related to) at least one antigen (e.g., MART-1), wherein the complex is capable of binding T cell receptors specific for the cognate antigen. Each of the MHC molecules may be tagged with a biotin molecule. Biotinylated MHC/peptides are tetramerized by the addition of streptavidin, which can be fluorescently labeled. The tetramer may be detected by flow cytometry via the fluorescent label. In certain embodiments, an MHC- peptide tetramer assay is used to detect or select enhanced affinity TCRs of the instant disclosure.

Levels of cytokines may be determined according to methods described herein and practiced in the art, including for example, ELISA, ELISPOT, intracellular cytokine staining, and flow cytometry and combinations thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from an antigen-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and/or cell viability, such as by incorporation of tritiated thymidine or non-radioactive assays, such as MTT assays and the like. The effect of an immunogen described herein on the balance between a Thl immune response and a Th2 immune response may be examined, for example, by determining levels of Thl cytokines, such as IFN-γ, IL-12, IL-2, and TNF-β, and Type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

Polynucleotides Encoding Binding Proteins Specific for MART-1 Antigen Peptides Isolated, recombinant or engineered nucleic acid molecules encoding binding protein (e.g., immunoglobulin superfamily binding protein) or high affinity recombinant T cell receptor (TCR) specific for MART-1 or Tyrosinase as described herein may be produced and prepared according to various methods and techniques of the molecular biology or polypeptide purification arts. Construction of an expression vector that is used for recombinantly producing a binding protein or high affinity engineered TCR specific for a MART-1 or Tyrosinase peptide of interest can be accomplished by using any suitable molecular biology engineering techniques known in the art, including the use of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing as described in, for example, Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003). To obtain efficient transcription and translation, a polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operably {i.e., operatively) linked to the nucleotide sequence encoding the immunogen.

Certain embodiments relate to nucleic acids that encode the polypeptides contemplated herein, for instance, binding proteins or high affinity engineered TCRs specific for MART-1 or Tyrosinase. As one of skill in the art will recognize, a nucleic acid may refer to a single- or a double-stranded DNA, cDNA or RNA in any form, and may include a positive and a negative strand of the nucleic acid which complement each other, including anti-sense DNA, cDNA and RNA. Also included are siRNA, microRNA, RNA— DNA hybrids, ribozymes, and other various naturally occurring or synthetic forms of DNA or RNA.

In certain embodiments, provided herein are isolated polynucleotides that encode a binding protein or high affinity engineered TCR of this disclosure specific for a MART-1 peptide or a MART-1 peptide :HLA complex. In particular embodiments related to encoded MART-1 specific binding proteins, a polynucleotide encodes a V_a domain, and a polynucleotide encodes a νβ domain that comprises a nucleotide sequence that encodes a CDR3 as set forth in any one of SEQ ID NOS:4-16 and 24-151.

In further embodiments, a polynucleotide encodes two or more binding proteins according to any of the embodiments disclosed herein.

In another embodiment, provided herein are isolated polynucleotides that encode a binding protein or high affinity engineered TCR of this disclosure specific for a Tyrosinase peptide. In particular embodiments related to encoded Tyrosinase specific binding proteins, a polynucleotide encodes a V_a domain, and a polynucleotide encodes a νβ domain and comprises a νβ CDR3 nucleotide sequence contained or set forth in SEQ ID NO: 153. In any of the embodiments disclosed herein, a polynucleotide encoding a binding protein of the instant disclosure is codon optimized for efficient expression in a target host cell. An exemplary codon optimized polynucleotide sequence of a TCR νβ domain is set forth in SEQ ID NO: 174. An exemplary codon optimized polynucleotide sequence of a TCR β-2 chain constant domain is set forth in SEQ ID NO: 176. An exemplary codon optimized polynucleotide sequence of a TCR β-chain is set forth in SEQ ID NO: 171.

In any of the embodiments disclosed herein, a polynucleotide encoding a binding protein of the instant disclosure encodes an additional sequence disposed between the TCR a-chain encoding polynucleotide and a TCR β-chain encoding polynucleotide, or TCR γ- chain polynucleotide and TCR δ-chain encoding polynucleotide, allowing multicistronic expression. Sequences that may be used for multicistronic expression include protease sites, viral self-cleaving 2A peptides, furin cleavage sites, and internal ribosome entry sites (IRES). Examples of self-cleaving peptides include those encoded by any one of SEQ ID NOS: 17-21.

Standard techniques may be used for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays and tissue culture and

transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well- known in the art and as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology techniques that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience;

Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and Applications, Edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes,

(Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, Eds., 1985); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Animal Cell Culture (R. Freshney, Ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley- VCH); PCR

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Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental

Immunology, Volumes I-IV (D. M. Weir andCC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and

Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001);

Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008). Certain embodiments include nucleic acid molecules contained in a vector. One of skill in the art can readily ascertain suitable vectors for use with certain embodiments disclosed herein. An exemplary vector may comprise a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, or which is capable of replication in a host organism. 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)). 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 or high affinity recombinant TCRs specific for MART-1, or variants thereof, as described herein) is 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, the nucleic acid encoding binding proteins or high affinity recombinant binding proteins (e.g., TCRs) specific for MART-1 or Tyrosinase 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 (i.e., 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, polynucleotides encoding binding proteins of the instant disclosure are contained in an expression vector that is a viral vector, such as a lentiviral vector or a γ-retroviral vector.

In particular embodiments, a polynucleotide encoding a binding protein or recombinant expression vector comprising a polynucleotide encoding a binding protein is delivered to an appropriate cell, for example, a T cell or an antigen-presenting cell, i.e., a cell that displays a peptide/MHC complex on its cell surface {e.g., a dendritic cell) and lacks CD8. In certain embodiments, the host cell is a hematopoietic progenitor cell, hematopoietic stem cell, or a human immune system cell. For example, the immune system cell can be a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof . In certain embodiments, wherein a T cell is the host, the T cell can be naive, a central memory T cell, an effector memory T cell, or any combination thereof. The recombinant expression vectors may therefore also include, for example, lymphoid tissue-specific transcriptional regulatory elements (TREs), such as a B lymphocyte, T lymphocyte, or dendritic cell specific TREs. Lymphoid tissue specific TREs are known in the art {see, e.g., Thompson et al., Mol. Cell. Biol. 72: 1043, 1992); Todd et al., J. Exp. Med. 777: 1663, 1993); Penix et al, J. Exp. Med. 775: 1483, 1993).

In addition to vectors, certain embodiments relate to host cells modified {i.e., genetically engineered) to contain a heterologous polynucleotide encoding a binding protein {e.g., TCR) or a vector comprising a heterologous polynucleotide encoding a binding protein {e.g., TCR) that are presently disclosed. A modified or genetically engineered host cell comprising a heterologous polynucleotide encoding at least one binding protein expresses on its cell surface at least one binding protein of the instant disclosure. Host cells can be modified ex vivo or in vivo. A host cell may include any individual cell or cell culture that may receive a vector or the incorporation of a nucleic acid or protein, as well as any progeny cells. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector,

transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989). In any of the aforementioned embodiments, a host cell containing a polynucleotide encoding a binding protein of this disclosure are comprised of cells that are autologous, allogeneic or syngeneic to the subject receiving the modified host cells, such as in an adoptive immunotherapy procedure.

In certain embodiments, a binding protein of the instant disclosure is expressed at a higher level on the surface of a host T cell containing a polynucleotide encoding the binding protein {e.g., TCR) as compared to an endogenous TCR. A host cell comprising one or more polynucleotides encoding one or more binding proteins of this disclosure may express a single binding protein or two or more different binding proteins of the instant disclosure.

Methods of Treatment

In certain aspects, the instant disclosure is directed to methods for treating a hyperproliferative disorder or a condition characterized by MART-1 overexpression by administering to human subject in need thereof an effective amount of a composition comprising a binding protein or high affinity recombinant TCR specific for human MART-1 according to any of the binding proteins or TCRs of this disclosure.

In other aspects, the instant disclosure is directed to methods for treating a hyperproliferative disorder or a condition characterized by Tyrosinase overexpression by administering to human subject in need thereof an effective amount of a composition comprising a binding protein or high affinity recombinant TCR specific for human Tyrosinase according to any of the binding proteins or TCRs of this disclosure.

The presence of a hyperproliferative disorder or malignant condition in a subject refers to the presence of dysplastic, cancerous and/or transformed cells in the subject, including, for example neoplastic, tumor, non-contact inhibited or oncogenically transformed cells, or the like {e.g., solid cancers; hematologic cancers including lymphomas and leukemias, such as acute myeloid leukemia, chronic myeloid leukemia, etc.), which are known in the art and for which criteria for diagnosis and classification are established {e.g., Hanahan and Weinberg, Cell 144:646, 2011; Hanahan and Weinberg, Cell 100:57, 2000; Cavallo et al., Cane. Immunol. Immunother. 60:319, 2011; Kyrigideis et al., J. Carcinog. 9:3, 2010). In certain embodiments, such cancer cells may be melanoma cells or metastatic melanoma.

As understood by a person skilled in the medical art, the terms, "treat" and "treatment," refer to medical management of a disease, disorder, or condition of a subject {i.e., patient, host, who may be a human or non-human animal) {see, e.g.,

Stedman's Medical Dictionary). In general, an appropriate dose and treatment regimen provide one or more of a binding protein or high affinity recombinant TCR specific for human MART-1 {e.g., comprising νβ CDR3 of any one of the sequences in Table IB, such as SEQ ID NO S: 4- 16, and variants thereof) or a host cell expressing the same, and optionally an adjunctive therapy {e.g., a cytokine such as IL-2, IL-15, IL-21 or any combination thereof), in an amount sufficient to provide therapeutic or prophylactic benefit. Therapeutic or prophylactic benefit resulting from therapeutic treatment or prophylactic or preventative methods include, for example an improved clinical outcome, wherein the object is to prevent or retard or otherwise reduce {e.g., decrease in a statistically significant manner relative to an untreated control) an undesired physiological change or disorder, or to prevent, retard or otherwise reduce the expansion or severity of such a disease or disorder. Beneficial or desired clinical results from treating a subject include abatement, lessening, or alleviation of symptoms that result from or are associated the disease or disorder to be treated; decreased occurrence of symptoms; improved quality of life; longer disease-free status {i.e., decreasing the likelihood or the propensity that a subject will present symptoms on the basis of which a diagnosis of a disease is made); diminishment of extent of disease; stabilized {i.e., not worsening) state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; and remission (whether partial or total), whether detectable or undetectable; or overall survival.

"Treatment" can also mean prolonging survival when compared to expected survival if a subject were not receiving treatment. Subjects in need of the methods and compositions described herein include those who already have the disease or disorder, as well as subjects prone to have or at risk of developing the disease or disorder.

Subjects in need of prophylactic treatment include subjects in whom the disease, condition, or disorder is to be prevented {i.e., decreasing the likelihood of occurrence or recurrence of the disease or disorder). The clinical benefit provided by the

compositions (and preparations comprising the compositions) and methods described herein can be evaluated by design and execution of in vitro assays, preclinical studies, and clinical studies in subjects to whom administration of the compositions is intended to benefit, as described in the examples.

Genetically engineered or modified host cells comprising a heterologous polynucleotide and expressing the binding protein or high affinity recombinant TCR specific for human MART-1 as described herein may be administered to a subject in a pharmaceutically or physiologically acceptable or suitable excipient or carrier.

Pharmaceutically acceptable excipients are 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.

In certain embodiments, genetically modified host cells expressing the binding proteins disclosed herein promote an antigen-specific T cell response against human MART-1 in a class I HLA-restricted manner. In some embodiments, the HLA- restricted response is transporter-associated with antigen processing (TAP)

independent. TAP independent pathways for MHCI class presentation of endogenous peptides can be advantageous where tumors inhibit TAP function to avoid immune detection.

In certain embodiments, an antigen-specific T cell response induced by the methods disclosed herein comprises at least one of a CD4+ helper lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In some embodiments, the CTL response is directed against a MART-1 overexpressing cell.

Genetically engineered host cells {e.g., T cells) expressing the binding proteins described herein can be exposed ex vivo to IL-21. Priming the MART-1 specific T cells with IL-21 (Hinrichs et al., 2008, Blood 111 :5326-33) can promote expansion of CTLs that phenotypically exhibit a less terminally differentiated phenotype, with a majority of cells expressing CD28 after ex vivo culture (Li et al., 2005, J. Immunol. 175:2261-9; Li et al., 2008, Blood 111 :229-35) and exhibiting enhanced persistence after adoptive transfer. The genetically engineered host cells may be cultured for a shorter duration of time, e.g., no more than 6 weeks, which is significantly less than the standard >12 week production time. For example a clinical grade sorter (Pollack et al., 2014, Journal for Immunotherapy of Cancer 2:36, incorporated by reference in its entirety) can be used to select CTLs for a polyclonal composition, thereby limiting the ex vivo expansion steps usually needed to achieve target CTL numbers for adoptive therapy.

Genetically engineered host cells used in adoptive transfer may represent a monoclonal composition or a polyclonal composition.

In certain embodiments, genetically engineered host cells may be further distinguished by their (a) expression of CD28 prior to infusion; (b) production of IFNy, TNFa, IL-2 or any combination thereof prior to infusion; (c) low expression of PD-1 or CD57, or both prior to infusion; or any combination of (a), (b), and (c).

A therapeutically effective dose is an amount of host cells (expressing a binding protein or high affinity recombinant TCR specific for human MART-1) used in adoptive transfer that is capable of producing a clinically desirable result (i.e., a sufficient amount to induce or enhance a specific T cell immune response against cells overexpressing MART-1 (e.g., a cytotoxic T cell response) in a statistically significant manner) in a treated human or non-human mammal. As is well known in the medical arts, the dosage for any one patient depends upon many factors, including the patient's size, weight, body surface area, age, the particular therapy to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Doses will vary, but a preferred dose for administration of a host cell comprising a recombinant expression vector as described herein is about 10⁷ cells/m², about 5 x 10 7 cells/m 2 , about 108 cells/m2 , about 5 x 108 cells/m2 , about 109 cells/m2 , about 5 x 10⁹ cells/m², about 10¹⁰ cells/m², about 5 x 10¹⁰ cells/m², or about 10¹¹ cells/m².

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 disease, 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). 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.

A condition associated with MART-1 overexpression includes any disorder or condition in which underactivity, over-activity or improper activity of a MART-1 cellular or molecular event is present, and typically results from unusually high (with statistical significance) levels of MART-1 expression in afflicted cells {e.g., melanoma cells), relative to normal cells. A subject having such a disorder or condition would benefit from treatment with a composition or method of the presently described embodiments. Some conditions associated with MART -1 overexpression thus may include acute as well as chronic disorders and diseases, such as those pathological conditions that predispose the subject to a particular disorder.

Some examples of conditions associated with MART-1 overexpression include hyperproliferative disorders, which refer to states of activated and/or proliferating cells (which may also be transcriptionally overactive) in a subject including tumors, neoplasms, cancer, malignancy, etc. In addition to activated or proliferating cells, the hyperproliferative disorder may also include an aberration or dysregulation of cell death processes, whether by necrosis or apoptosis. Such aberration of cell death processes may be associated with a variety of conditions, including cancer (including primary, secondary malignancies as well as metastasis), or other conditions.

According to certain embodiments, virtually any type of cancer that is characterized by MART-1 overexpression may be treated through the use of compositions and methods disclosed herein, including melanomas. Furthermore, "cancer" may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, blood or lymph or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease following transplantation of organs or stem cells; multi-drug resistant cancers, primary or secondary malignancies, angiogenesis related to malignancy, or other forms of cancer. Also contemplated within the presently disclosed embodiments are specific embodiments wherein only one of the above types of disease is included, or where specific conditions may be excluded regardless of whether or not they are characterized by MART-1 overexpression.

Certain methods of treatment or prevention contemplated herein include administering a host cell (which may be autologous, allogeneic or syngeneic to the subject being treated) comprising a desired heterologous nucleic acid molecule encoding a bidning protein as described herein that is stably integrated into the chromosome of the cell. For example, such a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) in order to administer a desired,

MART-1 -targeted T-cell composition to a subject as an adoptive immunotherapy.

As used herein, administration of a composition or therapy 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., MART-1 specific recombinant (i.e., engineered) host cells with one or more cytokines (e.g., JL-2, IL-15, IL-21); alkylating agents, such as cyclophosphamide, an inhibitor of an immune checkpoint molecule, or any combination thereof).

In certain embodiments, a plurality of doses of a genetically engineered host cell containing a heterologous polynucleotide encoding a binding protein as described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks. In further embodiments, a cytokine (e.g., IL-2, IL-15, IL-21) is administered sequentially, provided that the subject was administered the genetically engineered host cell containing a heterologous

polynucleotide encoding a binding protein as described herein at least three or four times before cytokine administration. In certain embodiments, the cytokine is administered subcutaneously (e.g., IL-2, IL-15, IL-21). In still further embodiments, the subject being treated is further receiving a chemotherapy (e.g., Dacarbazine or temozolomide), a BRAF inhibitor, a c-KIT inhibitor, an inhibitor of an immune checkpoint molecule or any combination thereof. In some embodiments, an inhibitor of an immune checkpoint molecule blocks the activity or expression of the immune checkpoint molecule. An immune checkpoint molecule can be CTLA-4, A2AR, B7- H3, B7-H4, BTLA, HVEM, GAL9, IDO, KIR, LAG-3, PD-1, PD-L1, Tim-3, VISTA, TIGIT, LAIR1, CD 160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, CEACAM-5, CD244, or any combination thereof. In a specific embodiment, an immune checkpoint inhibitor molecule is CTLA-4, PD-1, PD-L1, or a combinatin thereof. In some embodiments, an inhibitor of an immune checkpoint molecule is an antibody or antigen binding fragment thereof, a fusion protein, a small molecule, an RNAi molecule, (e.g., siRNA, shRNA, miRNA) a ribozyme, an aptamer, or an antisense oligonucleotide. In other embodiments, the genetically modified host cells are further modified to reduce or inhibit expression of the gene encoding the immune checkpoint inhibitor molecule, for example, by using an RNA-guided endonuclease (e.g., CRISPR/Cas system), a zinc finger nuclease, a Transcription activator-like effector nuclease (TALEN), an RNAi molecule, or an antisense oligonucleotide. In specific embodiments, the inhibitor of the immune checkpoint molecule is ipilimumab or tremelimumab. In other embodiments, the inhibitor of the immune checkpoint molecule is nivolumab or pembrolizumab. In yet other embodiments, the inhibitor of the immune checkpoint molecule is durvalumab or atezolizumab. In still further embodiments, the inhibitor of the immune checkpoint inhibitor molecule is administered concurrently or subsequent to administration of the genetically engineered host cell (e.g., within a week) and may comprise multiple doses administered at intervals of about two to four weeks.

In another aspect, methods of treatment involving a polyclonal T cell composition specific for MART-1 according to the embodiments disclosed herein in conjunction with an inhibitor of immune checkpoint molecule inhibitor may be capable of inducing a de novo antigen-specific T cell response to a non-targeted antigen (e.g., NY-ESOl, gplOO, MAGE A3, tyrosinase, or any combination thereof). This phenomenon of "epitope spreading" may be advantageous for treating melanoma, which is a highly mutated tumor. Multivalent responses induced by epitope spreading may block outgrowth of antigen-loss tumor variants and improve tumor eradication.

Polyclonal T cell compositions specific for MART-1 comprising two or more different binding proteins according to the embodiments disclosed herein may be particularly useful for treating subjects afflicted with a proliferative disorder, e.g., melanoma, more particularly metastatic melanoma, that is refractory to monotherapy with MART-1 specific monoclonal adoptive immunotherapy, monotherapy with an inhibitor of an immune checkpoint inhibitor molecule, or a combination thereof.

An effective amount of a therapeutic or 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.

The level of a CTL immune response may be determined by any one of numerous immunological methods described herein and routinely practiced in the art. The level of a CTL immune response may be determined prior to and following administration of any one of the herein described MART-1 -specific binding proteins expressed by, for example, a T cell. Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods routinely practiced in the art (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). Antigen-specific T cell responses are generally determined by comparisons of observed T cell responses according to any of the herein described T cell functional parameters (e.g., proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.) that may be made between T cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells) and T cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen. A response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.

A biological sample may be obtained from a subject for determining the presence and level of an immune response to a MART- 1 -derived antigen peptide as described herein. A "biological sample" as used herein may be a blood sample (from which serum or plasma may be prepared), biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source. Biological samples may also be obtained from the subject prior to receiving any immunogenic composition, which biological sample is useful as a control for establishing baseline (i.e., pre-immunization) data.

The pharmaceutical compositions described herein may be presented in unit- dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to preserve the stability of the formulation until. In certain embodiments, a unit dose comprises a genetically engineered host 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 recombinant cells or active compound calculated to produce the desired therapeutic 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 therapeutic or prophylactic 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 in the art and may be performed using samples obtained from a subject before and after treatment.

EXAMPLES

EXAMPLE 1

SINGLE PATIENT STUDY OF IL-21 PRIMED POLYCLONAL CTL + CTLA4 BLOCKADE IN

REFRACTORY METASTATIC MELANOMA PATIENT

Materials and Methods

Clinical protocols. All clinical investigations were conducted according to the Declaration of Helsinki principles. Patient "Z" was first enrolled in protocol FHCRC #2271 (monoclonal CTL plus denileukin diftitox, 3 patients treated) then #2225 (polyclonal CTL plus ipilimumab, 10 patients treated; see, Example 2). Of the 10 patients treated on protocol #2225, two, including Patient Z, achieved ongoing CRs (beyond 12 weeks), two achieved partial responses, three achieved stable disease and three had progressed at 12 weeks. Both protocols were approved by the FHCRC Institutional Review Board and the U.S. Food and Drug Administration, and registered at clinicaltrials.org as NCT00945269 and NCT00871481.

Treatment Plans. Enrolled in protocol #2271, Patient Z received 10¹⁰/m² monoclonal (Wallen et al., 2009, PLoS One 4:e4749; Yee et al., 2002, Proc Natl Acad Sci USA 99: 16168-16173) A*0201 -restricted MARTI -specific T cells 28 days apart. The second infusion was preceded by Ontak (18mcg/kg i.v. 6, 4, and 2 days prior to infusion), and both infusions were followed by low-dose IL-2 (250,000U/m² s.c. twice daily x 14 days). In protocol #2225, Patient Z received CY (300mg/m² iv) before the infusion of 10¹⁰ polyclonal, IL-21 primed (Pollack et al., 2014, J. Immunother, Cancer 2:36) MARTI₂₆-35 CTL/m², immediately followed by low-dose subcutaneous IL-2 and ipilimumab (3mg/kg every 3 weeks x 4 doses) (Hodi et al., 2010, N. Engl. J. Med. 363 :711-723). Radiologic responses were evaluated according to the mWHO-based irRC Criteria (Wolchok et al., 2009, Cancer Res. 15:7412-7420).

Isolation and expansion of monoclonal MARTI -specific CTL (without IL-21).

PBMCs were collected by leukapheresis and all ensuing ex vivo manipulations were performed in the clinical Good Manufacturing Practices (cGMP) Cell Processing Facility of the FHCRC. Donor PBMC were stimulated three times for 7-10 day cycles with autologous dendritic cells (DC) pulsed with the HLA*0201 -restricted MART-1₂₆- 35 (EAAGIGILTV - SEQ ID NO: 1) peptide (Anaspec) at a DC to effector ratio of 1 :2- 10 to obtain sufficient frequencies (>5%) of MARTI -reactive CD8⁺ T-cells. On Day 2 of each stimulation, the y_c-chain cytokines IL-2 (12.5R7/ml), IL-7 (5ng/ml) and IL-15 (lng/ml) were added. Cultures that contained > 5% specific CD8⁺ T cells, assessed by multimer analysis, were cloned by limiting dilution and then stimulated twice using the Rapid Expansion Protocol (Ho et al., 2006, J. Immunol. Methods 2006, 310:40-52; Riddell et al., 1992, Science 257:238-241). CTL products were frozen, thawed and washed before infusion, for a total production time of 12-13 weeks.

Isolation and expansion of polyclonal MARTI -specific CTL (with IL-21).

PBMCs were depleted of CD25⁺ T cells (Miltenyi Biotec Inc.) to eliminate regulatory T cells, and stimulated twice for seven days with autologous DC pulsed with MARTI₂₆-35 (EAAGIGILTV - SEQ ID NO: l). Dendritic Cell (DC) stimulations were supplemented with the same y_c-chain cytokines plus IL-21 (30 ng/mL) on Day 1. Cultures that contained > 5% specific CD8⁺ T cells were clinical-grade sorted (BD Influx cell sorter, BD Biosciences) and stimulated twice using the Rapid Expansion Protocol. The total production time was 6 weeks. The purity and phenotype as well as the νβ repertoire of each CTL product immediately before infusion are shown in Figure 4 and Table IB, respectively. There was no overlap in the Υβ repertoire of both products.

Table IB. Υβ Repertoire of Infused Monoclonal and Polyclonal CTL Products

νβ Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

(SEQ ID NO: 36)

C AS S SKFPF VMQDTQ YF

0.006476782 0

(SEQ ID NO: 37)

CASSGTDTYEQYF

0.006201174 0

(SEQ ID NO: 38)

CAWSKRGVGQPQHF

0.005925567 0

(SEQ ID NO: 39)

CASSLGGRGNQPQHF

0.005649959 0

(SEQ ID NO:40)

CASSQSGASFNEKLFF

0.005512155 0

(SEQ ID NO:41)

CASSYRASTEAFF

0.005098743 0

(SEQ ID NO:42)

CATSDLTGDEQYF

0.005098743 0

(SEQ ID NO:43)

C AS S STDRGAYEQYF

0.004960939 0

(SEQ ID NO:44)

CASRSAGGGHEQYF

0.004685332 0

(SEQ ID NO:45)

CAS SFEVEGSPQRTGIYF

0.004685332 0

(SEQ ID NO:46)

CASKYSVFAQSRANVLTF

0.004547528 0

(SEQ ID NO:47)

CASSNQPQHF

0.004547528 0

(SEQ ID NO:48)

CASSQGGNSPLHF

0.004409724 0

(SEQ ID NO:49)

CSARQVDSPLHF

0.00427192 0

(SEQ ID NO:50)

CAQSRWGALAGMGETQYF

0.004134116 0

(SEQ ID NO:51)

CAS SFNNSPLHF

0.004134116 0

(SEQ ID NO: 52)

CASTTGPANEKLFF 0.003996312 0 νβ Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

(SEQ ID NO:53)

CSALAGGDTQYF

0.003996312 0

(SEQ ID NO: 54)

CSATGQGGEQYF

0.003996312 0

(SEQ ID NO: 55)

CASRLGTAWDEQYF

0.003858508 0

(SEQ ID NO:56)

C AS SIMAGAKNIQ YF

0.003720705 0

(SEQ ID NO:57)

CAWSPGTSGGYEQYF

0.003720705 0

(SEQ ID NO: 58)

CASRQTGGRRMNTEAFF

0.003582901 0

(SEQ ID NO:59)

CASWCNCVQSRANVLTF

0.003582901 0

(SEQ ID NO: 60)

CRPVSATGEETQYF

0.003582901 0

(SEQ ID NO:61)

CSVEKRQGSEKLFF

0.003582901 0

(SEQ ID NO: 62)

CAS SL AGNGEQFF

0.003445097 0

(SEQ ID NO:63)

CASSLSPSGGYEQYF

0.003445097 0

(SEQ ID NO: 64)

CASRGVYDGTSGRRSSYNEQFF

0.003307293 0

(SEQ ID NO:65)

CASTATGDNSPLHF

0.003307293 0

(SEQ ID NO: 66)

CAS SRDRVDEKLFF

0.003169489 0

(SEQ ID NO: 67)

CAWKGQGTLGRNSPLHF

0.003169489 0

(SEQ ID NO: 68)

CSARDPSGGATDTQYF

0.003169489 0

(SEQ ID NO: 69)

CAS SIGTGERNQPQHF 0.003031685 0 β Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

(SEQ ID NO: 70)

CASSPQGRAFF

0.003031685 0

(SEQ ID NO:71)

CAS SISRGPKLFF

0.002893881 0

(SEQ ID NO: 72)

CAS SLTRGEQFF

0.002893881 0

(SEQ ID NO:73)

C AS SRYTISIRTEAFF

0.002893881 0

(SEQ ID NO: 74)

CASIGQLNTEAFF

0.002756077 0

(SEQ ID NO:75)

CAS SDRGNQPQHF

0.002756077 0

(SEQ ID NO: 76)

CAS SRT YTMKSRANVLTF

0.002756077 0

(SEQ ID NO: 77)

CASRAPGQVRTDTQYF

0.002618274 0

(SEQ ID NO: 78)

CAS SERGAETQ YF

0.002342666 0

(SEQ ID NO: 79)

CASSLLREGTDTQYF

0.002342666 0

(SEQ ID NO: 80)

CAS SPPGPRNEQ YF

0.002342666 0

(SEQ ID NO:81)

CASGKPRAGQSRANVLTF

0.002204862 0

(SEQ ID NO: 82)

CAS S ARI AGGAKNIQ YF

0.002204862 0

(SEQ ID NO:83)

CASSRGQGGPYLNQPQHF

0.002204862 0

(SEQ ID NO: 84)

CSVRGVTDTQYF

0.002204862 0

(SEQ ID NO:85)

CAS S VGQGNEQ YF

0.002067058 0

(SEQ ID NO: 86)

CAISYGQGFGYTF 0.001929254 0 Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

(SEQ ID NO:87)

CASASGGGSDTQYF

0.001929254 0

(SEQ ID NO:88)

CAS SMRLHF VLDEQ YF

0.001929254 0

(SEQ ID NO:89)

CAS S VVDEYNEQFF

0.001929254 0

(SEQ ID NO: 90)

CASRPGPTGELFF

0.00179145 0

(SEQ ID NO:91)

CAS SEDPGGNEKLFF

0.00179145 0

(SEQ ID NO: 92)

CASSGQGYNEQFF

0.00179145 0

(SEQ ID NO:93)

CAS SPQRGQLNYGYTF

0.00179145 0

(SEQ ID NO: 94)

CASSPTGYTEAFF

0.001653646 0

(SEQ ID NO:95)

CSGEGTGEDNEQFF

0.001653646 0

(SEQ ID NO: 96)

SASSYGQGFGYTS

0.001653646 0

(SEQ ID NO: 97)

CASGSDRAGETQYF

0.001515843 0

(SEQ ID NO: 98)

CASKGEEIRKTYGYTF

0.001515843 0

(SEQ ID NO: 99)

CASSDSASGHTQYF

0.001515843 0

(SEQ ID NO: 100)

SASSYGQGFGYTF

0.001515843 0

(SEQ ID NO: 101)

CASSFWFQETQYF

0.001378039 0

(SEQ ID NO: 102)

CASSIQLSTDTQYF

0.001378039 0

(SEQ ID NO: 103)

CAS SLRS S YNEQFF 0.001378039 0 β Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

(SEQIDNO:104)

CASSRTSGGATGYEQYF

0.001378039 0

(SEQIDNO:105)

CATGQPQHF

0.001378039 0

(SEQIDNO:106)

CATSRDRGLAN EQFF

0.001378039 0

(SEQIDNO:107)

SASSYGQGFGYTY

0.001378039 0

(SEQIDNO:108)

CAS SMLGDGTGELFF

0.001240235 0

(SEQIDNO:109)

CASSPAGTDYEQYF

0.001240235 0

(SEQIDNO:110)

CASSWRDSPYGYTF

0.001240235 0

(SEQIDNO:lll)

CASTPQGA EQYF

0.001240235 0

(SEQIDNO:112)

CSAVASARSGELFF

0.001240235 0

(SEQIDNO:113)

CASRGNIATVLDEQYF

0.001102431 0

(SEQIDNO:114)

C ASRQTGRGAS S YNEQFF

0.001102431 0

(SEQIDNO:115)

CAS SDDGL AGALPETQ YF

0.001102431 0

(SEQIDNO:116)

CASSDSWTSGRREFF

0.001102431 0

(SEQIDNO:117)

CAS SIRGPPL VAAYNEQFF

0.001102431 0

(SEQIDNO:118)

CASSIRTGRNQPQHF

0.001102431 0

(SEQIDNO:119)

CASSPS EQFF

0.001102431 0

(SEQIDNO:120)

CASSTRGGSSYNEQFF 0.001102431 0 νβ Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

(SEQIDNO:121)

CASTPPGGTGKTDTQYF

0.001102431 0

(SEQIDNO:122)

CSVDGREGLPETQYF

0.001102431 0

(SEQIDNO:123)

SASSYGQGFGYTC

0.001102431 0

(SEQIDNO:124)

YAS S YGQGFGYTY

0.001102431 0

(SEQIDNO:125)

CASKIQDGYASEQYF (TCR-1)

0 45.24154391 (SEQIDNO:4)

CAS S YGGGQPQHF (TCR-2)

0 23.60886973 (SEQIDNO:5)

CASSPFELSLSEQFF (TCR-3)

0 14.24224955 (SEQIDNO:6)

CASSQPLYNSPLHF (TCR-4)

0 9.951973856 (SEQIDNO:7)

CAS SF WDKAKNIQ YF (TCR-5)

0 4.22890913 (SEQIDNO:8)

CASSSPINRAGGLDTQYF (TCR- 6) 0 0.429902403

(SEQIDNO:9)

CASRTGLGQPQHF (TCR-7)

0 0.34363672 (SEQIDNO:10)

CAS S YSINQPQHF (TCR-8)

0 0.310021304 (SEQIDNO:ll)

CASSLDGGASGNEQFF (TCR-9)

0 0.263635874 (SEQIDNO:12)

CASSSLLDRGIDEQYF (TCR-10)

0 0.215904546 (SEQIDNO:13)

CASGLGPLFADTQYF (TCR-11)

0 0.188938509 (SEQIDNO:14)

CASKQGALTGELFF (TCR-12)

0 0.173380564 (SEQIDNO:15) νβ Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

CASSYSGVGQPQHF (TCR-13)

0 0.119224173 (SEQIDNO:16)

CAS SQDGGLDKGGELFF

0 0.096087537 (SEQIDNO:126)

CSAQGQGPTTDTQYF

0 0.094485277 (SEQIDNO:127)

CASASGMGQPQHF

0 0.08070584 (SEQIDNO:128)

CASGTSGGSWIDEQFF

0 0.043885904 (SEQIDNO:129)

CASSQTGVHTGELFF

0 0.042059327 (SEQIDNO:130)

CASSYGDGPPLHF

0 0.02827989 (SEQIDNO:131)

CAS S SPGLPETQ YF

0 0.026004681 (SEQIDNO:132)

CASTVQGLGSPLHF

0 0.013811482 (SEQIDNO:133)

CASTETGTLSYEQYF

0 0.012962284 (SEQIDNO:134)

CSARNGLSEQYF

0 0.009453334 (SEQIDNO:135)

CASSSLSSGNTIYF

0 0.008668227 (SEQIDNO:136)

CASSQ FLELFF

0 0.007899142 (SEQIDNO:137)

CASSYSSQGSQPQHF

0 0.006008475 (SEQIDNO:138)

CASSPQGD EQFF

0 0.005768136 (SEQIDNO:139)

CATSDFPGLAGD EQFF

0 0.004950984 (SEQIDNO:140)

CASSQEDLLGDGYTF

0 0.004406215 (SEQIDNO:141) νβ Percent in Percent in CDR3 amino-acid sequence

monoclonal product polyclonal product

CASSYSSIGQPQHF

0 0.003797356 (SEQIDNO:142)

CASSQDAGMSYEQYF

0 0.003540995 (SEQIDNO:143)

CASSLSSGEAFF

0 0.003396791 (SEQIDNO:144)

CASSTGLAGFTDTQYF

0 0.002868046 (SEQIDNO:145)

CAS SLGLIMNEQFF

0 0.002803955 (SEQIDNO:146)

CAS SPRP AEQFF

0 0.002803955 (SEQIDNO:147)

CASSLGTSGSPLHF

0 0.002499526 (SEQIDNO:148)

CASSYRQATEAFF

0 0.002163051 (SEQIDNO:149)

CAS SEQRAGINKNEQFF

0 0.002082938 (SEQIDNO:150)

CASSLGVTSEKLFF

0 0.001954757 (SEQIDNO:151)

T-cell tracking by peptide-Major Histocompatibility Complex (MHC) multimers. Allophycocyanin-conjugated MARTI -specific antigen peptide-MHC (pMHC) multimers (FHCRC Immune Monitoring Core Facility) were used to detect transferred CTL in PBMCs collected after infusions, with a staining sensitivity of 0.05% of total CD8⁺ T cells, as previously described (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7).

T-cell tracking by HTTCS. To guarantee that all tracked clonotypes were tumor- specific, only pMHC multimer-binding cells within the CTL infusion products were selected by flow cytometry, before DNA isolation for HTTCS. The HTTCS limit of detection was set at 0.001% of all TCR reads, below which frequencies could not be reliably determined (Robins et al., 2012, J Immunol Methods, 375:14-9). The HTTCS frequency of each clonotype is based on all TCR νβ chain reads, from both CD4⁺ and CD8⁺ T cells. (Robins et al., 2009, Blood 114:4099-4107) To compare tracking by HTTCS versus multimer staining, expressed as a percent of CD8⁺ T cells, HTTCS results are reported as a percent of CD8⁺ cells using the formula: (HTTCS frequencies) x ([%total CD8⁺ T cells]+[%total CD4⁺ T cells] / [%total CD8⁺ T cells in each sample]).

Flow Cytometry. Monoclonal and polyclonal CTL products pre-transfer and

PBMCs post-transfer were identified by binding to a specific pMHC multimer, and analyzed by flow cytometry after staining with fluorochrome-conjugated mAbs to CD4, CD 16, CD 19 (exclusion channel), CD8, CD28, CD62L, CCR7, and PDl (BD- Pharmingen). Assessments of the intracellular cytokine expression of IFNy, TNFa and IL-2 in response to cognate antigen were performed, as previously described (Papagno et al., 2007). Cells were analyzed on an LSRII instrument (Becton Dickinson) using fluorescence-activated cell sorting (FACS)-Diva software.

Enzyme -Linked Immunospot (ELISpots) Assays. Amino acid (aa) peptides, 15 aa in length offset by 5 aas, were grouped into pools of 20-30 peptides spanning

MARTI, NY-ESO-1, gplOO, tyrosinase, and MAGE A3 (2, 2, 5, 4 and 3 pools, respectively) (Sigma-Aldrich, St. Louis, Mo). Peptide pools were used to stimulate PBMC at indicated time-points and T cell reactivity was quantified using a human IFNy ELISpot assay, as previously described (Scheibenbogen et al., 2000, J Immunol

Methods 244:81-89). This method quantifies CD8⁺ and CD4⁺ T cell reactivity to peptide pools independent of HLA restriction.

Results

Clinical Evaluation. This 53 year-old male presented with stage III melanoma on his lower right thigh. He underwent wide local excision and inguinal node dissection (Clark level 4, Breslow 1.5mm, no ulceration, 3/8 lymph nodes involved), followed by 12 months of adjuvant interferon (IFN) alpha treatment. Four years later, he developed metastatic disease with supraclavicular, subcarinal and right hilar masses uniformly positive for melanoma tumor antigen 1 (MART-1). In anticipation of adoptive transfer studies, his PB mononuclear cells (PBMCs) were collected. He then received four cycles of high-dose Interleukin 2 (IL-2), but experienced disease progression.

He received two infusions of monoclonal MARTI -specific T cells (10¹⁰ cells

/m²) 30 days apart, each followed by low-dose subcutaneous (s.c.) IL-2 (250,000 U/m² every 12h). The second infusion was preceded by selective CD25 T-regulatory cell depletion (denileukin diftitox); the melanoma progressed. The patient received anti- CTLA4 monoclonal antibody (ipilimumab 3 mg/kg x 4 doses) that partially slowed tumor growth initially, but four months later he developed new metastases.

Finally, he received IL-21 primed polyclonal MART-1 specific T cells at a dose of 10¹⁰/m² immediately followed by a single course of ipilimumab (same dosing as above) (Pollack et al., 2014, J. Immunother. Cancer 2:36). The T-cell infusion was preceded by low-dose cyclophosphamide (CY) conditioning (300 mg/m² x 1) and followed by a two-week course of low-dose subcutaneous IL-2. Although the entire regimen could be administered in the ambulatory setting, he was hospitalized for monitoring of potential cell-infusion-associated adverse events (AEs).

No serious adverse events were observed apart from expected transient (<24 hours) culture-negative fevers (>38.3°C), associated with CTL-induced cytokine release syndrome, and lymphopenia lasting 10 days (Chapuis et al., 2013, Science translational medicine 5: 174ral27). Prior to CTL infusions (Figure 1 A), the patient presented with PET⁺ subcarinal and right hilar masses (Figure IB, 1C). Serial imaging demonstrated progressive disease after the first course of CTL infusions followed by ipilimumab monotherapy. The patient had bulky disease in the paratracheal, supraclavicular and subcarinal regions at the time the combined therapy with IL-21 primed, polyclonal MART-1 -specific T cells plus ipilimumab was initiated. Twelve weeks (day 384) after the start of the combined treatment, progressive tumor reduction was associated with the development of vitiligo (Figure ID), manifest as depigmentation of the eyebrows and eyelashes. The patient achieved a CR by Response Evaluation Criteria In Solid Tumors (RECIST) and immune-related response criteria (irRC) (Wolchok et al., 2009, Cancer Res. 15:7412-7420) at year 3, and remains disease-free five years later, with no additional therapy or long-term immune side effects other than persistent vitiligo.

Persistence, clonality, phenotype and function of monoclonal and polyclonal CTL in vivo. The first course of monoclonal MARTI -specific CTL yielded a peak CTL frequency of 2.1% of total CD8⁺ T cells one day after the second infusion; CTL rapidly disappeared. Combined therapy resulted in a 4% peak one week post-infusion, and frequencies of 1.2% and 4%, respectively, 2 and 4 years later (Figure 2A). Tracking of the monoclonal CTL (Clone 120) (Figure 2B, inset pie-chart), using high-throughput TCRP sequencing (HTTCS), confirmed that the peak response was due to the infused clone, which was not detected in pre-infusion PBMC samples or in PBMC samples taken at any time-point after Day 29 (Figure 2B, graph). Polyclonal CTL included 13 clonotypes with frequencies >0.1% (Figure 2C, inset pie-chart). HTTCS tracking revealed that one clonotype (TCR-1) represented the majority of detected antigen- specific CTL after infusions (Figure 2C, graph). To assess phenotypic and functional differences that could account for the preferential survival of long-lived IL-21 -primed CTL, compared to monoclonal CTL, we assessed surface expression of markers associated with long-lived memory T cells (CD28, CD62L, C-C Chemokine Receptor 7 [CCR7]), activation/exhaustion (Programmed cell death protein 1 (PD1)) and function (IFNy, Tumor Necrosis Factor (TNFa) and IL-2 production in response to cognate antigen). The polyclonal CTL product expressed CD28 (55.1%), low PDl (12%) and produced IFNy, TNFa and IL-2 (Figure 2D, upper graph), whereas monoclonal CTL expressed none of the memory markers, a higher fraction of PD1 (44%), and produced only IFNy in response to cognate antigen (Figure 2D, lower graph). Following adoptive transfer, the polyclonal CTL additionally expressed CD62L in vivo (Figure 2E). After 12 weeks (day 385), they expressed all three memory markers (CD28, CD62L and CCR7), maintained low PD1 expression (12%) and produced IFNy, TNFa and IL-2 in response to cognate antigen (Figure 2F), all features consistent with central memory T cells (Restifo et al., 2012).

Response to non-targeted antigens (antigen-spreading). The reactivity of CD8⁺ and CD4⁺ T cells to overlapping peptides spanning known melanoma-associated proteins MART-1, New York Esophageal Protein 1 (NY-ESOl), glycoprotein (gp) 100, tyrosinase, and Melanoma Associated- Antigen (MAGE) A3, independent of Human Leukocyte Antigen (HLA)-restriction, was tested at indicated timepoints throughout the patient's treatment course (Figure 3). PBMCs taken before and up to 80 days after the first infusion of monoclonal CTL showed low/no reactivity to the overlapping peptides (<10 IFNy spots O⁵ PBMC). Following the initial ipilimumab treatment, antigen- spreading was still not detected (day 295). However, 6, 12 and 27 weeks (days 349, 390 and 495) following the start of combined therapy the patient developed a marked response to multiple peptides within each melanoma-associated protein. Frequencies matched or exceeded MARTI -specific responses in some cases, suggesting antigen- spreading, coincident with a >80% reduction in tumor burden (Figure 1).

Thus, it has been demonstrated that IL-21 -primed, polyclonal MARTI -specific CTLs plus ipilimumab achieved complete, durable tumor eradication with minimal side effects in a patient whose melanoma was refractory to monoclonal MARTI -specific CTL and subsequent single-agent ipilimumab.

Without wishing to be bound by theory, several factors are hypothesized to be involved in immune-mediated tumor regression. First, polyclonal IL-21 -primed CTL achieved higher peak frequencies and longer persistence in vivo, compared to identical doses of monoclonal CTL. Reduced ex vivo manipulation (<6 weeks vs >12 weeks) plus IL-21 addition during priming (Pollack et al., 2014, J. Immunother. Cancer 2:36) generated CTL that had undergone fewer divisions and had characteristics associated with enhanced survival. Specifically, expression of CD28 and a retained capacity to secrete IL-2 after exposure to cognate antigen (Topp et al., 2003, J. Exp. Med. 198:947- 955) likely facilitated the robust persistence of transferred tumor-specific cells.

Second, ipilimumab exposure likely enhanced the antitumor activity of the transferred cells. By enabling unobstructed engagement of B7 with CD28 (instead of CTLA4), the CD28⁺ CTL subset may have preferentially survived/expanded through continued production/secretion of autocrine IL-2. Consistent with this hypothesis, CTL examined in vivo months after transfer nearly all expressed CD28⁺, retained the capacity to secrete IL-2 in response to cognate antigen and had low PD1 expression (Freeman et al., 2000, J. Exp. Med. 192: 1027-1034). In contrast, CD28^", IL-2^" and PD1^M monoclonal cells did not survive beyond one day post-transfer without ipilimumab exposure. With ipilimumab, the transferred cells further acquired the canonical markers of long-lived memory cells CD62L and CCR7, suggesting that the remaining cells were now programmed to persist (Unsoeld et al., 2002, J. Immunol. 169:638-641; Wolfl et al., 2010, Cancer Immunol. Immunother. 60: 173-186).

Finally, epitope spreading was observed when both the targeted immune response provided by tumor-specific CTL, plus the pro-inflammatory context fostered by anti-CTLA4 blockade, were present (Ribas et al., 2003, Trends Immunol. 24:58-61). While epitope spreading has been demonstrated in some patients receiving anti-CTLA4 antibody monotherapy (Kvistborg et al., 2014, Sci. Transl. Med. 6:254ral28), no evidence of antigen spreading or a clinical response was evident in this patient prior to receiving the combination therapy. Although delayed responses can occur after ipilimumab alone, this usually occurs by 3 months (Wolchok et al., 2009, Cancer Res. 15:7412-7420). Patient Z demonstrated unequivocal disease progression with the appearance of a new lesion seven months following ipilimumab monotherapy. As melanoma is a highly mutated tumor, antigen-spreading may have increased the number and strength of T cells targeting multiple antigens beyond the ones assessed here (Schreiber et al., 2011, Science 331 : 1565-1570). Multivalent responses may have blocked the outgrowth of antigen-loss tumor variants such that complete tumor eradication could occur.

EXAMPLE 2

TEN PATIENT STUDY OF T CELL THERAPY USING IL-21 PRIMED CTL COMBINED WITH

CTLA-4 BLOCKADE

Materials and Methods

Clinical protocol, patient characteristics, generation of melanoma-specific CTL products. Between August 2011 and April 2013, 10 patients with progressive metastatic melanoma (Figure 5) received treatment on protocol #2225, approved by the Fred Hutchinson Cancer Research Center (FHCRC) Institutional Review Board and the U.S. Food and Drug Administration (NCT 00871481). All treated patients provided written informed consent. Eligibility required HLA A*0201 and tumor expression of MART-1. (Kawakami et al., 1994, J. Exp. Med. 180:347-52)

A total of 14 patients underwent leukapheresis in anticipation of entering this trial. Of the 4 who underwent leukapheresis and did not receive treatment, 4 started an alternate treatment while waiting for the cells to be generated: 2 received a B-RAF inhibitor, one started a trial of IL-21 and ipilimumab and 1 received high-dose (HD) IL- 2. One patient remains in CR after HD IL2. By the time the 3 remaining patients had progressive disease, the trial was closed to accrual. Treatment Plan. Patients received a single outpatient infusion of

cyclophosphamide (CY) 300mg/m² 48 hours before the infusion of 10¹⁰ melanoma- specific CTL/m² (determined safe from previous studies) (Wallen et al., 2009, PLoS One 4:e4749), followed by low-dose s.c. IL-2 (250,000 IU/m² twice daily for 14 days) to enhance the survival of transferred T-cells. (Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168-73) On day 1 after CTL infusion, patients received ipilimumab (anti- CTLA-4, Yervoy^®, BMS) 3mg/kg every 3 weeks for a total of 4 doses (Figure 10). Patients were monitored for toxicities based on Common Toxicity Criteria v4.0.

(National Cancer Institute NIoH: Common Terminology Criteria for Adverse Events (CTCAE) v4.0, (ed 10/01/2009), Cancer Therapy Evaluation Program, 2009) Staging studies were obtained 6 and 12 weeks after the infusion and then as clinically indicated.

Assessment of clinical responses. Both the immune-related Response Criteria (irRC) and the response evaluation criteria in solid tumors (RECIST) criteria were used to assess clinical responses. (Eisenhauer et al., 2009, Eur. J. Cancer 45:228-47;

Wolchok et al., 2009, Clin. Cancer Res. 15:7412-20) Discrepancies (Table 2) were due to differences in the specific measures of tumor size and classification of new lesions, (Wolchok et al., 2009, Clin. Cancer Res. 15:7412-20) with the latter are incorporated in the global tumor burden in irRC, but classified as PD in RECIST criteria.

Table 2. Assessment of Clinical Response.

irRC ; RECIST ; Received 2 or 3 of 4 planned ipilimumab doses due to progressive disease 4Patient initiated alternate treatment modality; ND: not done; ⁵Received 3/4 planned ipilimumab doses due to ipilimumab-induced hypophysitis ⁶Patient had brain MRI but no restaging scans performed. X: patient died due to progressive disease; (mets): development of brain metastasis. Isolation and expansion of polyclonal MARTI -specific CTL. PBMCs were collected by leukapheresis and all ensuing ex vivo manipulations involving processing of products destined for infusion were performed in the cGMP Cell Processing Facility of the FHCRC based on a protocol established for in vitro enrichment of low frequency antigen-specific CTL. (Stromnes et al., 2014, Immunol. Rev. 257: 145-64; Rosenberg et al., 2011, Clin. Cancer Res. 17:4550-7; Yang, 2013, Clin. Dermatol. 31 :209-19) Briefly, PBMCs were depleted of CD25⁺ T cells (Miltenyi Biotec Inc.) to eliminate regulatory T cells, and stimulated twice for 7 days with autologous dendritic cells pulsed with the HLA*0201 -restricted MART-l_26-35 (EAAGIGILTV) peptide (SEQ ID NO: l) (Anaspec, San Jose, CA). DC stimulations were supplemented with the y_c-chain cytokines IL-2 (10 IU/mL), IL-7 (5 ng/mL), and IL-21 (30 ng/mL). Cultures that contained more than 5% specific CD8⁺ T cells assessed by multimer analysis were clinical-grade sorted (BD Influx cell sorter, BD Biosciences) and stimulated twice using the Rapid Expansion Protocol. (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Hunder et al., 2008, N. Engl. J. Med. 358:2698-703) CTL products were freshly infused or frozen, thawed and washed before infusion, for a total production time of 6 weeks.

Cytotoxicity assays. Cytotoxic activity of infusion products was examined by assessing the capacity of the CTL to lyse HLA A*0201⁺ MART1⁺ MEL-526 compared to MARTl^~MEL-375 melanoma cells at an E:T ratio ranging from 100: 1 to 1 : 1 (Suppl. Figure 11). (Wallen et al., 2009, PLoS One 4:e4749)

T-cell monitoring by MHC-peptide multimer s. MARTI -specific pMHC multimers (produced by the FHCRC immune monitoring core facility) were used to detect transferred CTL in PBMCs collected after infusions, with a staining sensitivity of 0.05% of total CD8⁺ T-cells, as previously described. (Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168-73)

Flow Cytometry. Autologous CTL products pre-transfer and in PBMCs post- transfer were identified by binding to specific multimer and analyzed by flow cytometry after staining with fluorochrome-conjugated mAbs to CD4, CD 16, CD 19 (exclusion channel), CD8, CD27, CD28, CD62L, CCR7, CD127 (IL7Ra), CD57 and PD-1 (BD- pharmingen) (Figure 11). Assessment of the intracellular cytokine expression of IFNy, TNFa and IL-2 in response to cognate antigen, and intranuclear Ki-67 expression were performed as described. (Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168-73; Chapuis et al., 2013, Sci. Transl. Med. 5: 174ra27) Cells were analyzed on an LSRII flow cytometer (Becton Dickinson) using FACS-Diva software.

ELISpots. For each patient, reactivity of whole PBMC to peptides 15 amino- acids (aa) in length offset by 5 aa and spanning MARTI, NY-ESO-1, Tyrosinase, gplOO and MAGE A3 (Sigma- Aldrich, St. Louis, Mo) was quantified using a human IFNy ELISpot assay as described. (Klebanoff et al., 2012, J. Immunother. 35:651-60) Results are presented as the number of spot forming cells/10⁵ PBMCs.

Statistical Analysis. Statistical tests were performed with the Graph-Pad prism software version 3.0 or with the R-package for statistical analysis (http://www.r- project.org).

Results

Polyclonal IL-21 -primed MARTI -specific CTL demonstrate ex vivo anti-tumor activity and express phenotypic characteristics associated with in vivo survival. All polyclonal CTL products demonstrated intracellular production of IFNy in response to antigen-presenting targets (HLA-A*0201⁺, transporter for antigen presentation (TAP)- deficient, B-lymphoblastoid cells, or T2 B-LCL), pulsed with the HLA-A*0201- restricted MARTl₂₆-35 peptide (SEQ ID NO: 1) (Figure 11 A, left columns), as well as specific lysis (range 27-66%, median 54%) of the HLA A*0201⁺ MART1⁺ MEL-526 cell line³¹ (Figure. 11 A, right columns). Immediately before infusion, multimer-binding CTL expressed CD45RO (median 98.9%), consistent with an antigen-experienced phenotype (Figure 1 IB). In accordance with our previous studies, (Li et al., 2005, J. Immunol. 175:2261-9; Li et al., 2008, Blood 111 :229-35) a subset of the expanded CTLs retained expression of CD27, CD28 and CD127 (with medians of 63.5%, 72.4%, and 36.2%, respectively) consistent with IL-21 exposure during priming. (Chapuis et al., 2013, Sci. Transl. Med. 5: 174ra27) Little or no expression of the lymph node homing markers, CD62L and CCR7, or the activation/exhaustion markers, CD57 and PD1, was detected.

Concurrent transfer of MARTI -specific CTL does not alter the safety /toxicity profile of anti-CTLA-4. Ten patients with metastatic melanoma whose disease progressed after previous systemic or surgical therapy, including ipilimumab monotherapy for 3 patients (Figure 5), received combined CTL plus anti-CTLA-4 treatment (Figure 10). Seven patients experienced transient (<24 hours) sterile fevers (>38.3°C) +/- chills associated with a CTL infusion-induced cytokine-release syndrome (Table 3). Lymphopenia lasting <10 days, (Chapuis et al., 2013, Sci. Transl. Med.

5: 174ra27) and self-limiting moderately erythematous maculo-papular skin rashes were observed in 9/10 patients. Ipilimumab therapy was associated with self-limited nausea/diarrhea (grade <1) in 8/10 patients and transient (< 14 days) moderately- elevated (grade < 2) liver enzymes were detected in 5/10 patients. Patient 9 developed pituitary insufficiency secondary to ipilimumab-induced hypophysitis, (Weber et al., 2012, J. Clin. Oncol. 30:2691-7) and Patient 5 experienced a drug fever attributed to the combined effect of ipilimumab plus vemurafenib introduced for progressive disease. (Ribas et al., 2013, N. Engl. J. Med. 368: 1365-6; Harding et al., 2012, N. Engl. J. Med. 366:866-8) Both patients received systemic steroids as treatment for immune-related adverse events. (Weber et al., 2012, J. Clin. Oncol. 30:2691-7) Overall, the toxicities could be attributed to either CTL infusions or ipilimumab alone, but no unexpected toxicities were associated with the combination.

Table 3. List of adverse events related to treatment

'National Cancer Institute Common Terminology Criteria for Adverse Events

:*Lymphopenia is a predictable, transient side effect of T cell infusions¹.

Adoptive transfer of MARTI -specific CTL with concurrent anti-CTLA-4 can produce sustained clinical responses. Two of 10 patients reached a sustained CR as defined by irRC and RECIST criteria,(Kawakami et al., 1994, J. Exp. Med. 180:347-52; Ho et al., 2006, J. Immunol. Methods 310:40-52) at 12 and 104 weeks following the CTL infusion respectively (Table 2, Fig. 6A green lines, Fig. 6B). Patient 1 had progressed after salvage ipilimumab monotherapy initiated 7 months before (Example 1). The time to response after ipilimumab alone is outside the expected range and the appearance of new lesions during the interim precluded any beneficial effect of ipilimumab monotherapy. (Wolchok et al., 2009, Clin. Cancer Res. 15:7412-20) Neither patient received additional anti-tumor therapy and both are alive and disease-free 220 and 169 weeks (11/01/15), respectively, after the start of treatment.

Two patients experienced PR as best responses by irRC (Fig. 11 A, blue lines). After demonstrating a 41% reduction in tumor burden at 6 weeks, Patient 9 was diagnosed with new sub-centimetric brain metastasis at 7 weeks along with ipilimumab- induced hypophysitis treated with systemic steroids. Despite the new brain lesions, the patient exhibited a reduction in overall tumor burden of 71% at 16 weeks, associated first with flattening then central blanching of numerous cutaneous metastases (Fig. 12A and 12B). However, as a possible consequence of systemic steroids, or the

characteristics of the brain parenchyma in which CTL may be excluded in the absence of inflammation, (Ransohoff et al., 2012, Nat. Rev. Immunol. 12:623-35) the brain lesions progressed. The patient elected to receive comfort care and died 2 months later. Patient 10 experienced a PR at 6 weeks (by irRC) which was maintained at 28 weeks (- 79%) but developed new brain metastases at 46 weeks. Patients 2, 4 and 6 exhibited SD at 12 weeks (Fig. 11 A, purple lines): Patients 2 and 6 elected to undergo alternate treatments due to persistent disease. Patients 3, 5 and 8, progressed at 6, 6, and 12 weeks, respectively (Fig. 11 A, red lines).

Overall, by irRC, 7 of 10 patients achieved best responses of CR, PR or SD. Of 3 patients who progressed after 4 doses of ipilimumab monotherapy (Patients 1, 2 and 3), one reached a CR, one had SD and one progressed. With a median follow-up of 187 weeks (range 140-220 weeks), 5/10 patients are alive and 2/10 remain in sustained CRs (Figure 13).

Polyclonal MARTI -specific IL-21-primed CTL persist in vivo when transferred with concurrent anti-CTLA-4. The majority of patients had nearly undetectable pre- existing PBMC frequencies of endogenous MARTI -specific multimer-binding CTL (range <0.05 -0.21%, median <0.05%). Persistence of the infused CTL was documented for 10/10 patients at 6 weeks and for 7/7 evaluable patients at 12 weeks, with median frequencies of 1.6% (range 0.3-2.9%) and 1.1% (range 0.3-2.2%), respectively (Fig. 7). The transferred CTL could be detected for as long as the patients donated PBMC for analysis, regardless of their tumor response at 12 weeks, with the exceptions of patients 9 and 5 who each received the equivalent of lmg/kg prednisone. Both experienced a gradual decline in the frequency of transferred cells to undetectable levels at 6 and 17 weeks, respectively.

Transferred CTL detected in PB exhibit/acquire phenotypic and functional characteristics of long-lived memory T-cells in patients who achieved CR PR or SD. Regardless of the levels detected on multimer⁺ CTL at the time of infusion (Fig. 1 IB), CTL tracking in patients who achieved CR, PR or SD documented a significant increase in the frequency of multimer⁺ cells expressing CD28 (p<0.05), CD27 (p<0.05), CD127 (p<0.005), CD62L (p<0.05) and CCR7 (p<0.005) at 12 weeks (Fig. 8A, left column). By contrast, although the statistical power to detect differences was limited due to the small sample size (n=3), the expression of CD28 was lost (p<0.05), CD27 and CD 127 decreased, and CD62L and CCR7 were never acquired on the transferred cells in patients with PD (Fig. 8 A, right column). The expression of CD57 and PD1, associated with an exhausted and activated/exhausted phenotype respectively, was increased on transferred cells found in PBMC of patients with PD, but not in patients who achieved a CR, PR or SD (Fig. 8B). We were unable to obtain tumor tissue after treatment to assess the expression of these markers on cells that had reached the tumor

microenvironment.

The functional profile of transferred CTL was determined by gating for∑FNy⁺ cells (Fig. 8C). Before CY infusions, <0.2% of IFNy-producing cells could be detected in PBMC, including in patients with low but detectable levels of MARTI -specific cells, attesting to the lack of pre-existing, functional PB CTL. Consistent with the

maintenance of CD28 expression on the generated cells, pre-infusion CTL secreted IL-2 in addition to IFNy and TNFa upon exposure to cognate antigen. (Ragheb et al., 1999, J. Immunol. 163 : 120-9; Topp et al., 2003, 198:947-55) In patients with CR, PR or SD, transferred CTL continued to produce all three cytokines 6 and 12 weeks post-transfer (Fig. 8C, left column) and for as long as transferred CTL could be detected by multimer staining (Fig. 14). However, no ΙΡΝγ-secreting cells could be detected post-transfer in patients who had PD (Fig. 8C, right column).

The ability of infused CTL to maintain the potential to divide was investigated, as reflected by the expression of Ki-67 in vivo. (Scholzen et al., 2000, J. Cell Physiol. 182:311-22) Before infusions, multimer^" CD8⁺ T-cells contained a mean of 1.1% Ki- 67^4' cells and polyclonal multimer⁴ CTL products contained a mean of 81.2% Ki-67⁴ cells 14 days after in vitro stimulation (Fig 8D, left columns). Between 0 and 11 weeks after transfer, Ki-67 expression in transferred multimer⁴ CTL progressively decreased and expression in host multimer^" CD8⁴ T-cells transiently increased (Fig 8D, middle columns). By 12 weeks following CTL infusion, after the completion of exogenous s.c. IL-2 and anti-CTLA-4, a small fraction of the transferred CTL were Ki-67⁴ (mean 3.9%) (Fig. 8D, right columns). Expression of this proliferation marker in the multimer⁴ cells was significantly higher compared to host multimer^" CD8 T-cells, which had returned to pre-treatment levels (mean 1.14%; p<0.0005). Direct comparison of cell products administered to patients with PD did not appear less functional than products infused into patients with CR, PR or SD (Fig. 15). Although no correlation with overall survival could be detected, the quantity of TNFa secreted by CTL products in response to cognate antigen was associated with a decreased likelihood of progression-free survival (PFS), and CD57 expression on infused cells after 3-weeks in vivo was associated with an increased likelihood of PFS (Table 4).

Table 4: Associations between OS, PFS and CTL parameters

Overall Survival HR(95% CI) P-value

IL-2 0.95(0.85-1.07) 0.43

CD27 1.03(0.86-1.22) 0.76

CD28 0.93(0.86-1) 0.06

CD127 1.01(0.96-1.05) 0.74

Phenotype of

Infused CTL CD62L 0.82(0.54-1.26) 0.37 after 3 weeks CCR7 0.62(0.33-1.17) 0.14 in vivo

CD57 1.05(0.98-1.11) 0.17

PD1 1.12(0.97-1.29) 0.12

Persistence 1.21(0.87-1.67) 0.25

*Model didn't converge, gray-shaded area: the association

reached statistical significance for this series.

Evidence of epitope spreading inpatients who achieved CR, PR or SD.

Reactivity of pre and post therapy to melanoma antigen epitopes that could bind to all expressed MHC antigens and could be recognized by host CD4⁺ and CD8⁺ T cells was identified and assessed. ELISpot analyses were used to assess reactivity of patient- derived T-cells towards overlapping peptides spanning the melanoma-associated proteins MARTI, NY-ESOl, gplOO, Tyrosinase and MAGE A3 (Fig. 9). All patients who achieved CR, PR or SD demonstrated a significantly increased reactivity to the melanoma-associated proteins at one or more timepoint after CTL infusion (Fig. 9A). In contrast, patients who progressed (Fig. 9B) either developed no new reactivity (Patient 3) or did not significantly increase reactivity (Patients 5 and 8). Detectable reactivity to melanoma-associated antigens was also not observed in 3 other patients with metastatic melanoma who declined therapy (Fig. 16 A), or in 5 patients who received CTL clones alone (Figure 16B).

Induction of durable antitumor responses in patients with metastatic melanoma was demonstrated in an ambulatory setting of antigen-specific PB-derived CTL in combination with CTLA-4 blockade. (Butler et al., 2011, Sci. Transl. Med. 3 :80ra34) Although the number of patients who participated in this initial study is limited, the results are encouraging compared to each individual modality.^{4 17}'²¹ Seven of 10 patients achieved a CR, PR or SD as best response by irRC. Two patients remain in sustained CRs without additional therapy >3 and 4 years later. These results were achieved with a toxicity profile comparable to ipilimumab monotherapy. (Weber et al., 2012, J. Clin. Oncol. 30:2691-7)

Compared to previous trials, infused CTLs in this study consisted of polyclonal, pMHC multimer-sorted T-cell lines that had undergone shorter ex vivo manipulation (<6 weeks) and fewer cell divisions. (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7; Wallen et al., 2009, PLoS One 4:e4749; Yee et al., 2002, Proc. Natl. Acad. Sci. USA 99: 16168-73; Pollack et al., 2014, J. Immunother. Cancer 2:36) The transferred CTL persisted in vivo {111 evaluable patients at 12 weeks), which is in sharp contrast with previous results (2/11 CTL clones beyond 2 weeks). (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7) CTL were exposed to IL-21 during primary stimulation, which has been shown to promote retention of CD27, CD28 and CD 127 on naive T-cells. (Chapuis et al., 2013, Sci. Transl. Med. 5: 174ra27; Hinrichs et al., 2008, Blood 111 :5326-33; Li et al., 2005, J. Immunol. 175:2261-9; Li et al., 2008, Blood 111 :229-35; Cui et al., 2011, Immunity 35:792-805) It is likely that the infused cells in the current trial originated mostly from the autologous IL-21 -responsive naive T-cell pool as few if any MARTI -multi mer⁺ cells were detected pre-infusion.

Patients who achieved a CR, PR or SD shared a number of biological correlates. Compared to the infused product, persisting CTL expressed/acquired phenotypic and functional characteristics associated with long-lived memory T-cells (including markers associated with survival, CD28, CD27 and CD 127; (McAdam et al., 1998, Immunol. Rev. 165:231-47; Kimura et al., 2013, Nat. Immunol. 14: 143-51) lymph-node homing, CD62L and CCR7; and production of IFNy, TNFa and IL-2), (Kimura et al., 2013, Nat. Immunol. 14: 143-51; Kaech et al., 2002, Nat. Rev. Immunol. 2:251-62) suggesting the preferential survival/expansion of this subset. These favorable characteristics may also have been facilitated by CTLA-4 blockade such that infused CD28⁺ CTL experienced unopposed stimulation/enhanced signaling through binding of the co-stimulatory ligands, CD80 and/or CD86. Although these ligands are present at low levels on >80% of melanoma cells, (Bernsen et al., 2003, Br. J. Cancer 88:424-31) higher expression can be induced by inflammatory cytokines, such as IFNy, which would have been provided by the responding transferred CTL. (Hersey et al., 1994, Int. J. Cancer 48:527- 32) Consequently, transferred CD28⁺ cells may have gained proliferative/survival advantages related to Bcl-X_L expression and autocrine production of IL-2. (McAdam et al., 1998, Immunol. Rev. 165:231-47) In contrast, no IFNy-secreting cells could be detected post-transfer in patients with PD.

Epitope spreading was observed in all patients with CR, PR and SD, likely a consequence of transferred CTL-induced tumor-lysis and heightened T-cell activation fostered by anti-CTLA-4. (Kvistborg et al., 2014, Sci. Transl. Med. 6:254ral28) Released tumor antigens presented by local antigen-presenting cells may have promoted activation of new responses to non-targeted melanoma-associated proteins, (Ribas et al., 2003, Trends Immunol. 24:58-61; Kvistborg et al., 2014, Sci. Transl. Med. 6:254ral28; Robert et al., 2014, Clin. Cancer Res. 20:2424-32). Whether such T-cell responses, towards wild-type- or non-evaluated tumor-specific mutations, (Schreiber et al., 2011, Science 331 : 1565-70) induced the decrease in tumor size observed in some patients cannot be ascertained. Yet the combination may represent a strategy to specifically increase the number and strength of T-cells targeting multiple antigens of the patient's own tumor, which may be particularly relevant when targeting non-oncogenic antigens such as MART-1. (Chandran et al., 2015, Clin. Cancer Res. 21 :534-43) EXAMPLE 3

VALIDATION OF HTTCS FOR TRACKING THE FREQUENCY OF TRANSFERRED

MONOCLONAL CTL IN VIVO

Materials and Methods

Clinical Protocols: Protocol #2140: Patients M-2140-1 and M-2140-2 with metastatic melanoma received cyclophosphamide (CY) 4000mg/m² administered over 2 days before the infusion of 10¹⁰ monoclonal melanoma-specific CTL/m² followed by low-dose subcutaneous (s.c.) IL-2 (250,000 U/m²) twice daily for 14 days (NCT 00438984) (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7). Pts Ml and M2 received CTL specific for HLA A*0201 -restricted MARTl_26-35

(AAGIGILTV)(SEQ ID NO:22) and HLA B*4403 -restricted Tyrosinasei_92-2oo

(SEIWRDIDF)(SEQ ID NO:23), respectively. 2 of 11 patients who received monoclonal products and demonstrated post-infusion in vivo persistence were analyzed for a direct assessment of the equivalence of multimer staining, CDR3 PCR and HTTCS on a unique CTL clone.

Generation and expansion of monoclonal tumor-specific CTL products (without

IL-21): For protocol #2140, which involved monoclonal tumor-specific cells, cell processing was as previously described (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7). Briefly, PBMCs were collected by leukapheresis and all ensuing ex vivo manipulations were performed in the clinical Good Manufacturing Practices (cGMP) Cell Processing Facility of the FHCRC. (Pollack et al., J. Immunother. Cancer 2:36) Donor PBMC were stimulated three times for 7-10 day cycles with autologous dendritic cells (DC) pulsed with the HLA*0201 -restricted MART-l₂6-₃₅

(EAAGIGILTV) peptide (SEQ ID NO: 1) (Anaspec) at a DC to effector ratio of 1 :2-10 to obtain sufficient frequencies (>5%) of MARTI -reactive CD8⁺ T cells. On Day 2 of each stimulation, the y_c-chain cytokines, IL-2 (12.5IU/ml), IL-7 (5ng/ml) and IL-15 (lng/ml), were added. Cultures that contained > 5% specific CD8⁺ T cells, assessed by multimer analysis, were cloned by limiting dilution and then stimulated twice using the Rapid Expansion Protocol. (Ho et al. 2006, J. Immunol. Methods 310:40-52) CTL products were frozen, thawed and washed before infusion, for a total production time of 12-13 weeks.

CTL tracking by peptide-MHC multimer s: The sensitivity of multimer staining was fixed at 0.1% of total CD8⁺ T cells for monoclonal products (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7), and at 0.05% for polyclonal products (Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795), as previously described. To compare the multimer stain tracking results expressed as a percentage of CD8⁺ T cells, to HTTCS expressed as percentage of CD4⁺ and CD8⁺ T cells, multimer results are reported as a percent of CD4⁺ and CD8⁺ cells using the formula: (%Multimer⁺ CD8⁺ T cells) x ([% total CD8⁺ T cells in each sample]/([% total CD8⁺ T cells]+[% total CD4⁺ T cells])).

CTL tracking by quantitative PCR: Primers flanking the CDR3 region of infused melanoma-specific CTL clones were designed, as previously described (Hunder et al., 2008, New Engl. M. Med. 358:2698-2703; Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7). Total CD4⁺ and CD8⁺ T cells were determined by flow cytometry and the following formula was used to determine TCR copies per 100 T cells: 100/([% total CD8⁺ T cells in each sample]+[% total CD4⁺ T cells in each sample]* [TCR copies/(P-actin copies/2}* 100]).

DNA extraction and immunosequencing. DNA was extracted from CTL products and whole PBMC using Qiagen Maxi DNA isolation kits (QIAGEN Inc.). TCRP CDR3 regions were amplified and 750 ng of extracted DNA sequenced by Adaptive Biotechnologies Corp (Seattle, WA) using the "deep" resolution ImmunoSEQ assay, as previously described (Sherwood et al., 2011, Sci Transl. Med. 3 :90ra61). Raw sequence data was filtered using the Adaptive bioinformatic website based on the TCRP V, D and J gene definitions provided by the International ImMunoGeneTics

collaboration (IMGT) (Monod et al., 2004, Bioinformatics 20 Suppl l :i379-85), using the EVIGT database (www.imgt.org). Productive nucleotide sequences were used for all tracking experiments. The data was further filtered to exclude sequences with no identifiable V and J removing PCR errors such as primer dimer and mispriming, as well as sequences with a raw read count <2, removing nucleotide sequencing errors (Robins et al., 2009, Blood 114:4099-4107).

CTL tracking by HTTCS. Only cells that bound pMHC multimers were selected by flow cytometry before DNA isolation for HTTCS. The limit of detection of HTTCS was set at 0.001% of all TCR reads below which frequency could not be reliably determined (Robins et al., 2012, J Immunol Methods, 375: 14-9). Only clonotypes present in the CTL products were tracked in PBMC obtained after infusions. The frequency of each clonotype detected by HTTCS was based on all TCR νβ reads from CD4⁺ and CD8⁺ T cells.

Normal Donor Sorts: PBMC were collected from three healthy adult donors, and processed into three populations for TCR sequencing: i.e., whole PBMC, flow- sorted CD3⁺CD45RA⁺ and CD3 D45RO , representing the naive and antigen- experienced T cell populations, respectively. TCRs from ~1 million cells from each population were sequenced by HTTCS. Clonotypes were tracked in the original PBMC samples and considered part of a CD3 CD45RA versus an CD3⁺CD45RO⁺ phenotype if it was observed in one population but not the other, or if its abundance was ten-fold greater in one population compared to the other.

Statistical Analysis: Correlation between values obtained by HTTCS, multimer and TCR PCR: The Pearson's R was calculated on the log TCR frequency using frequencies obtained by HTTCS and either % multimer⁺ T cells or TCR copied per 100 cells and treating each biological sample (i.e., timepoint) as an independent

observation. P values were generated by normal approximation. Half-lives of persistent TCR clonotypes were determined using the formula t_1/2= t/(log₂pSf₀/N_t]). Only clonotypes that had at least 3 consecutive values above the limit of detection (0.001%) and for which R² was >0.3 were used in the analysis. Comparison of clonotype ty₂ obtained in patients grouped according to their best clinical response: the Wilcoxon rank sum test was used to obtain p values.

Results

Among patients who received monoclonal (M) T cells in a previous study (on Protocol #2140 (Chapuis et al., 2012, Proc. Natl. Acad. Sci. USA 109:4592-7), see Materials and Methods), two patients (of eleven infused) in whom the transferred cells were detectable in vivo for >40 days after infusions (Table 5) were analyzed. Patient M2140-1 was infused with cells expanded from a melanoma-specific CD8⁺ T cell clone: 99.4% of the cell infusion product bound the HLA A*0201 -restricted TCRl₂₆-35 (A2/MART1) pMHC multimer (Figure 17A). HTTCS revealed one clonotype comprised 99.7% of all TCR reads; 29 other clonotypes detected with frequencies >0.001% (Table 6) comprised the remaining 0.3% and were likely

bystander/contaminant clonotypes derived from the irradiated allogeneic PBMCs used to expand the cells (Figure 17B, upper pie plot). Patient M2140-2 was infused with CD8⁺ T cells recognizing the HLA B*4402-restricted Tyrosinase i9_2-2oo epitope

(B44/Tyr), for which a pMHC multimer could not be synthesized. The cell product included a total of 868 clonotypes of which the most prevalent single clonotype represented 94% of all TCR reads (Figure 17B, lower pie plot). For both patients, the most prevalent HTTCS-detected clonotype was confirmed as the infused CTL clone by TCR νβ quantitative PCR (Table 7). The post-transfer in vivo frequencies of the most prevalent clonotype in each infusion product were compared, as assessed by HTTCS, p- HLA multimer binding and/or TCR νβ quantitative PCR. For Patient M2140-1, frequencies obtained by HTTCS were 42.1%, 0.84% and 0.61% of total TCR reads in PBMC collected on days 4, 56 and 508 after transfer. By comparison, the frequencies by multimer staining were 40.0%, 0.21% and 0.74%, and 48.5%, 0.57% and 0.28% by TCR Vp-specific PCR (Figure 17C, upper graph). For Pt. M2140-2, HTTCS frequencies were 21.5%, 6.75% and 1.25% at days 4, 48 and 117, compared to 15.8, 3.0% and 0.71% using TCR Vp-specific PCR (Figure 17C, lower graph). Thus, for frequencies >0.001% of total T cells, HTTCS frequencies yielded results concordant to TCR νβ-specific PCR and pMHC multimer binding, suggesting HTTCS can be used to quantitatively track the frequency of infused monoclonal CD8⁺ T cells in vivo.

Table 5: Overview of Protocol Characteristics:

CTL Specificity MART1 /Tyrosinase/gp100* MART1

Use of peptide

Yes Yes

pulsed DCs

Use of IL-21 in

No Yes

cultures

Product clonality Monoclonal Polyclonal

Product median

52.70% 63.50%

CD27 expression

Product median

7.10% 72.40%

CD28 expression

Product median

CD127 0% 36.20% expression

Product median

CD62L 0% 0%

expression

Product median

CCR7 0% 0% expression

Pre-infusion CY 4000mg/m² over 2

CY 300mg/m² conditionning days

Number of

1 1 10

patients infused

Persistence**>40 2 of 1 1

10 of 10 (100%) days? (1 8%)

*gp100: glycoprotein 100; **%multimer⁺ CD8⁺ cells >0.1 %; ND: not done.

Table 6: Total, roductive and uni ue se uences in each sam le.

Pt M2140-2 Day +20 1720706 30270 1449629 24940

Pt M2140-2 Day +48 2168464 25686 1778915 20935

Pt M2140-2 Day +167 4324142 23205 3550291 18648

Pt P2225-1 Infusion Product 6241184 135 6237501 80

Pt P2225-1 Pre-infusion: Day -6 3710481 64057 3164580 54593

Pt P2225-1 Day +7 3853831 43527 3302492 36987

Pt P2225-1 Day +28 13737097 48019 1 1801283 40079

Pt P2225-1 Day +63 2061 176 48122 1750887 41076

Pt P2225-1 Day +83 5125021 35253 4336866 29568

Pt P2225-1 Day +140 5855242 120621 4964219 102720

Pt P2225-1 Day +280 1 1007334 78734 9362765 66288

Pt P2225-2 Infusion Product 147366 864 123320 699

Pt P2225-2 Pre-infusion: Day -4 45154 6620 37726 5475

Pt P2225-2 Day +6 6861283 47154 5763957 38057

Pt P2225-2 Day +27 2223107 44787 1861301 36781

Pt P2225-2 Day +63 6719917 57015 5652484 46306

Pt P2225-2 Day +83 5973060 54983 4956610 44623

Pt P2225-2 Day +140 6725965 1 13245 5677266 92809

Pt P2225-3 Infusion Product 1084207 158 1069728 121

Pt P2225-3 Pre-infusion: Day -2 1600325 46363 1318178 38575

Pt P2225-3 Day +5 1576723 29486 1305000 24507

Pt P2225-3 Day +19 3093978 46184 2562942 38253

Pt P2225-3 Day +34 726794 12135 588505 10095

Pt P2225-3 Day +55 345272 26206 2851 17 21973

Pt P2225-4 Infusion Product 8984081 406 8191908 259

Pt P2225-4 Pre-infusion: Day -1 1 13665366 130151 12038950 1 13781

Pt P2225-4 Day +7 7761444 1 19513 6859902 104837

Pt P2225-4 Day +29 10767658 139859 9525901 122963

Pt P2225-4 Day +72 3505967 98069 3100010 86317

Pt P2225-4 Day +44 10035618 166384 8897244 146518

Pt P2225-4 Day +106 7329490 140080 6505644 123489

Pt P2225-4 Day +184 8697668 138668 7718457 121943

Pt P2225-5 Infusion Product 6623326 1807 5660637 1277

Pt P2225-5 Pre-infusion: Day -7 4044341 78339 3319207 65132

Pt P2225-5 Day +7 5045236 92496 4150796 76851

Pt P2225-5 Day +27 6832576 124442 5684185 103455

Pt P2225-5 Day +43 7104181 24127 5856637 19513

Pt P2225-5 Day +75 6929298 51286 5575588 42167

Pt P2225-6 Infusion Product 7758478 3368 5953662 2381 Pt P2225-6 Pre-infusion: Day -21 4824146 107028 3937997 87642

Pt P2225-6 Day +7 6525587 1 13062 5373090 92693

Pt P2225-6 Day +28 5217051 106580 4320730 87448

Pt P2225-6 Day +63 5967729 102878 4963245 84101

Pt P2225-6 Day +77 3253189 105492 2713834 86909

Pt P2225-6 Day +1 12 4372405 89299 3621951 73076

Pt P2225-7 Infusion Product 3839245 171 3303721 99

Pt P2225-7 Pre-infusion: Day -6 2263424 120853 1872290 100152

Pt P2225-7 Day +8 2281451 92483 1912357 77082

Pt P2225-7 Day +30 1848791 98027 1544583 81680

Pt P2225-7 Day +58 2386558 87989 1962622 72976

Pt P2225-7 Day +84 2293006 109646 1897937 91 1 19

Pt P2225-7 Day +175 2205191 76936 1826123 63551

Pt P2225-8 Infusion Product 2503785 768 2034448 460

Pt P2225-8 Pre-infusion: Day -5 508090 6184 404913 4879

Pt P2225-8 Day +6 906471 13398 730248 10591

Pt P2225-8 Day +29 2878938 132648 2338565 107945

Pt P2225-8 Day +50 698176 1 1747 549677 9206

Pt P2225-9 Infusion Product 1623718 810 1523160 501

Pt P2225-9 Pre-infusion: Day -3 1 141855 12223 912763 9566

Pt P2225-9 Day +10 2302300 73726 1882936 59062

Pt P2225-9 Day +31 1351307 20871 1074884 16469

Pt P2225-9 Day +51 3493163 75850 2805757 60522

Pt P2225-9 Day +123 2750467 73968 2193028 59062

Pt P2225-10 Infusion Product 549783 8321 427405 6533

Pt P2225-10 Pre-infusion: Day -4 549783 8321 427405 6533

Pt P2225-10 Day +7 1 198353 39715 925759 32361

Pt P2225-10 Day +26 1487733 22364 1206056 17969

Pt P2225-10 Day +58 2480351 67443 1954765 54932

Pt P2225-10 Day +1 12 1090589 9954 877540 7825

*The total numbers of sequences, including sequences <0.001%, are captured.

Table 7: Identical results obtained by standard PCR and HTTCS for infused

Not available

EXAMPLE 4

HTTCS TRACKING OF IN VIVO FREQUENCIES OF ADOPTIVELY TRANSFERRED POLYCLONAL CTL

Materials and Methods

Clinical Protocols: Protocol #2225: Patients P2225-1-P10 with metastatic melanoma received CY 300mg/m² before the infusion of 10¹⁰ polyclonal A*0201- restricted MART127-35 CTL/m², immediately followed by low-dose s.c. IL-2 and ipilimumab (anti- Cytotoxic T-Lymphocyte Associated Protein 4, Yervoy^®, Bristol

Myers Squibb) (Hodi et al., 2010, N. Engl. J. Med. 363 :711-723) 3mg/kg every 3 weeks for a total of 4 doses (NCT 00871481) (Chapuis et al., 2016, J. Clin. Oncol. 34:3787- 3795). Radiologic responses were evaluated post-infusion according to the IrRC (Figure 19) (Wolchok et al., 2009, Clin. Cancer Res. 15:7412-7420). Compared to monoclonal products, a higher fraction of polyclonal products expressed the markers CD27, CD28 and CD 127 associated with memory, whereas 100% (10/10) of polyclonal products persisted, only 18% (2/11) of monoclonal products persisted beyond 40 days in vivo (Table 5). Generation and expansion of polyclonal tumor-speci fic CTL products (with IL-

21): For protocol #2225, polyclonal tumor-specific cell products were generated as previously described. (Pollack et al., J. Immunother. Cancer 2:36; Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795) Briefly, PBMCs were depleted of CD25⁺ T cells

(Miltenyi Biotec Inc.) to eliminate regulatory T cells, and stimulated for seven days twice with autologous DC pulsed with MARTI₂₆-35. DC stimulations were supplemented with the same y_c-chain cytokines plus IL-21 (30 ng/mL) on Day 1.

Cultures that contained > 5% specific CD8⁺ T cells were clinical-grade sorted (BD Influx cell sorter, BD Biosciences) and stimulated twice using the Rapid Expansion Protocol. The total production time was 6 weeks.

CTL tracking by peptide-MHC multimers: The sensitivity of multimer staining was fixed at 0.1% of total CD8⁺ T cells for monoclonal products (Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7), and at 0.05% for polyclonal products (Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795), as previously described. To compare the multimer stain tracking results expressed as a percentage of CD8⁺ T cells, to HTTCS expressed as percentage of CD4⁺ and CD8⁺ T cells, multimer results are reported as a percent of CD4⁺ and CD8⁺ cells using the formula: (%Multimer⁺ CD8⁺ T cells) x ([% total CD8⁺ T cells in each sample]/([% total CD8⁺ T cells]+[% total CD4⁺ T cells])).

CTL tracking by quantitative PCR: Primers flanking the CDR3 region of infused melanoma-specific CTL clones were designed, as previously described (Hunder et al., 2008, New Engl. M. Med. 358:2698-2703; Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA 109:4592-7). Total CD4⁺ and CD8⁺ T cells were determined by flow cytometry and the following formula was used to determine TCR copies per 100 T cells: 100/([% total CD8⁺ T cells in each sample]+[% total CD4⁺ T cells in each sample]* [TCR copies/(P-actin copies/2}* 100]).

DNA extraction and immunosequencing. DNA was extracted from CTL products and whole PBMC using Qiagen Maxi DNA isolation kits (QIAGEN Inc.). TCRP CDR3 regions were amplified and 750 ng of extracted DNA sequenced by Adaptive Biotechnologies Corp (Seattle, WA) using the "deep" resolution ImmunoSEQ assay, as previously described (Sherwood et al., 2011, Sci Transl. Med. 3 :90ra61). Raw sequence data was filtered using the Adaptive bioinformatic website based on the TCRP V, D and J gene definitions provided by the International ImMunoGeneTics

collaboration (IMGT) (Monod et al., 2004, Bioinformatics 20 Suppl l :i379-85), using the IMGT database (www.imgt.org). Productive nucleotide sequences were used for all tracking experiments. The data was further filtered to exclude sequences with no identifiable V and J removing PCR errors such as primer dimer and mispriming, as well as sequences with a raw read count <2, removing nucleotide sequencing errors (Robins et al., 2009, Blood 114:4099-4107).

CTL tracking by HTTCS. Only cells that bound pMHC multimers were selected by flow cytometry before DNA isolation for HTTCS. The limit of detection of HTTCS was set at 0.001% of all TCR reads below which frequency could not be reliably determined (Robins et al., 2012, J Immunol Methods, 375: 14-9). Only clonotypes present in the CTL products were tracked in PBMC obtained after infusions. The frequency of each clonotype detected by HTTCS is based on all TCR νβ reads from CD4⁺ and CD8⁺ T cells.

Normal Donor Sorts: PBMC were collected from three healthy adult donors, and processed into three populations for TCR sequencing: i.e. whole PBMC, flow- sorted CD3⁺CD45RA⁺ and CD3 D45RO , representing the naive and antigen- experienced T cell populations, respectively. TCRs from ~1 million cells from each population were sequenced by HTTCS. Clonotypes were tracked in the original PBMC samples and considered part of a CD3 CD45RA versus an CD3 D45RCT phenotype if it was observed in one population but not the other, or if its abundance was ten-fold greater in one population compared to the other.

Statistical Analysis: Correlation between values obtained by HTTCS, multimer and TCR PCR: The Pearson's R was calculated on the log TCR frequency using frequencies obtained by HTTCS and either % multimer⁺ T cells or TCR copied per 100 cells and treating each biological sample (i.e., timepoint) as an independent

observation. P values were generated by normal approximation. Half-lives of persistent TCR clonotypes were determined using the formula t_1/2= t/(log₂pSf₀/N_t]). Only clonotypes that had at least 3 consecutive values above the limit of detection (0.001%) and for which R² was >0.3 were used in the analysis. Comparison of clonotype ty₂ obtained in patients grouped according to their best clinical response: the Wilcoxon rank sum test was used to obtain p values.

Results

HTTCS Accurately Tracks the In Vivo Frequencies of Adoptively Transferred

Poly clonal CTL. PBMC from 10 patients who had each received one infusion of 10¹⁰/m² polyclonal (P) CTL specific for A2/MART1 on a previous study (on Protocol #2225 (Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795), see Materials and Methods) were analyzed (Table 5). HTTCS detected between 56 and 2036 (mean 555.4) clonotypes in the polyclonal infusion products from Patients P2225-1-10 (Figure 18A). The most prevalent clonotype comprised between 4% and 77% of the total cells in each infusion (mean 33.3%), and the 25 most prevalent clonotypes together comprised between 35.0% and 99.9% (mean 78.4%) of the total cell products. This HTTCS analysis was performed on infusion products selected for binding pMHC multimers with >99% purity. The limit of pMHC detection is typically >0.01% of CD8⁺ T cells, which is at least 10-fold less sensitive than HTTCS. Thus, HTTCS of infusion products that have been selected by pMHC multimer-binding is likely to also include

contaminant clonotypes. To avoid tracking these bystander clonotypes after adoptive transfer, those clonotypes that had higher frequencies in pre-infusion PBMC compared to their frequencies within the CTL products, as these clonotypes likely reflected non- expanded bystander cells that decreased in frequency through the cell culture process were excluded from analysis (Table 8). A total of 86 clonotypes (average 0.014% of total CTL products) with frequencies of 0.001%) to 8.85%> (mean 0.35%>) were thus identified and removed from our analysis. Tracking the sum of the frequency of all infused, expanded clonotypes by HTTCS yielded near concurrent results with those obtained by tracking pMHC multimer binding for all patients who received polyclonal products (Figure 18B). Thus, HTTCS appears to accurately track the in vivo frequencies of infused polyclonal CTL.

able 8: Clonotype composition of monoclonal and polyclonal CTL products

*Fraction in pre-infusion sample was < than in the cell product; **Fraction in pre-infusion sample was > than in the cell product

Single, immunodominant persistent clonotypes are associated with CRs after adoptive transfer. Of the ten patients with metastatic melanoma who received A2/MART1 -specific polyclonal CTL, two achieved CRs as evaluated by the immune- related response criteria (IrRC) (Wolchok et al., 2009, Clin. Cancer Res. 15:7412- 7420), two a partial remission (PR), three stable disease (SD) and three had progressive disease (PD) as best response (Table 9 and (Chapuis et al., J. Exp. Med. 213 : 1133-9; Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795). The frequencies of the first 25 most prevalent clonotypes detected in each cell product were individually tracked in vivo after adoptive transfer (Figure 20A, colored lines), as were the sum of remaining clonotypes (Figure 20A, grey lines). In the two patients who achieved a CR after infusion (Pt P2225-1, Pt P2225-7), only one individual clonotype remained detectable at sustained frequencies higher than those of other infused clonotypes (>0.056% at 280 days and 0.093% at 175 days post-infusion for Pts. P2225-1 and 7 respectively) and represented the majority (>99%) of detected antigen-specific cells post-infusion (Figure 20A, red arrows). Clonotype half-lives (t½, see Statistical Analysis) were determined for each patient and grouped according to best clinical response (Figure 20B). For patients who achieved a CR (Pts P2225-1 and 7), the immunodominant clonotypes

(Figure 20B, red arrows) had t½ of 173 and 132 days, which was respectively ~7 and 5 times longer than the median t½ of all infused clonotypes for Patients P2225-1-10 (24.8 days). Along with the higher expression of Ki67 by the transferred pMHC multimer- binding cells, - composed by a majority of the dominant clonotype - compared to multimer^" cells (Table 10 and ((Chapuis et al. 2012, Proc. Natl. Acad. Sci. USA

109:4592-7, Chapuis et al., 2016, J. Clin. Oncol. 34:3787-3795)), these findings indicate the immunodominant CTL had the capacity to expand and persist. For the remaining patients, the average t½ of clonotypes after transfer was 53 and 31 days for patients who achieved a PR as best response (Pts P2225-9 and 10), 44, 46 and 21 days for patients who achieved SD (Pts P2225-2, 4 and 6), and 13, 15 and 14 days for patients who progressed (Pts P2225-3, 5 and 8). When all clonotype t½ were grouped according to patients' best response (Figure 20C), the t½ differences were statistically significant between patients who obtained CRs, PRs, SDs and PDs. Thus, prolonged in vivo persistence of transferred clonotypes is associated with, and presumably important for, tumor control by transferred CTL.

Table 9: Clinical responses of patients with metastatic melanoma who received

Patient initiated alternate treatment modality. ²Patient had peripheral disease regression but new sub- centimeter brain metastasis. Overall, at 16 weeks, the volume of disease was lower than at baseline, but the location of the brain lesions eventually caused death. ³Patient developed brain metastasis at 46 weeks. X: Patient died due to progressive disease. Table 10: Ki67 expression of transferred multimer+ CTL in patients who achieved a complete remission.

Pt P2225-1

Pre-existing clonotype frequencies provide insights into the nature of persisting CTL. The number of clonotypes comprising the polyclonal cell products for Pts. P2225-1 to 10 was between 56 and 2036, with a median of 262.5 clonal sequences (Table 7). However, most clonotypes present in cell products (range 43 to 1275, median 160) were detected by HTTCS with frequencies below 0.001% (the limit of detection) in PBMC obtained before and after infusion, and constituted only a minor fraction of the total infusion product (range 0.82% - 24.7%, median 2.26%) (Figure 21 A, grey areas). As stated before, a clonotype with a frequency in the CTL product that was less than its corresponding frequency in the pre-infusion PBMC was defined as having not expanded during the culture process (Table 7, bottom row). These clones comprised a minor fraction of the final infusion product, with a median of 0.02% (range 0%-0.61%) (Figure 21 A, purple areas), and were not further monitored. Clonotypes detected in any post-infusion PBMC sample, but not detected in pre-infusion samples (range 10-205, median 56 sequences), were the dominant components of the final cell product (range 74.68%-98.97%, median 97.67%) (Figure 21A, blue areas), implying preferential expansion from individual parental clonotypes with frequencies < 0.001%; i.e., a very low frequency (VLF) population. The immunodominant clonotypes in both patients who achieved CRs (Patients P2225-1 and 7, Figure 20A and 20B, red arrows) were derived from respective VLF populations. Clonotypes expanded in the cell infusion product that were detectable in PBMC before infusion were less prevalent (range 0-6, median 2 sequences), comprising <10% (range 0-9.22%, median 0.03%) of the final cell products that were infused (Figure 21 A, orange areas). The frequencies of all expanded clonotypes were summed at multiple timepoints after infusion (Figure 2 IB, solid red circles) and further subdivided into previously undetected (Figure 2 IB, open blue circles) versus previously detected clonotypes (Figure 2 IB orange open circles). Previously undetected clonotypes represented the majority of cells in both infusion products and the detected clonotypes in post-transfer PBMC, whereas previously detected clonotypes comprised a small fraction of infused products and a minority of post-transfer clonotypes.

To assess the likelihood that the preferentially expanded and infused clonotypes originated from a distinct parental T cell subpopulation, the propensity for clonotypes present in three sets of normal donor PBMC to exhibit a mostly antigen-experienced or naive phenotype was assessed. Overall, clonotypes with frequencies <0.001% in PBMC were 15 times more likely (72% ± 2% vs 5% ± 3%) to have originated from a

CD3⁺CD45RA⁺ (primarily naive T cells, T_N) VLF population than from a

CD3⁺CD45RO⁺ (mostly antigen-experienced cells) population (Figure 19).

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 but not limited to, U.S. Provisional Patent Application No. 62/333,117, filed May 6, 2016, 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 above-detailed 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. A binding protein, compri

(a) a T cell receptor (TCR) a-chain variable (V_a) domain; and

(b) a TCR β-chain variable (ν_β) domain comprising a complementary determining region (CDR) 3 amino acid sequence set forth in any one of SEQ ID NOS:4-16 and 24-151, or a CDR3 amino acid sequence set forth in any one of SEQ ID NOS:4-16 and 24-151 with up to five amino acid substitutions, insertions, or deletions, wherein the binding protein is capable of specifically binding to a MART-1 peptide EAAGIGILTV (SEQ ID NO: l):human leukocyte antigen (HLA) complex.

2. The binding protein of claim 1, wherein the ν_β CDR3 amino acid sequence comprises or consists of any amino acid sequence set forth in any one of SEQ

ID NOS:4-16 and 24-151.

3. The binding protein of claim 1 or 2, wherein:

(a) the ν_β CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO:4 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV28-01 gene;

(b) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO:5 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV06-05 gene;

(c) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO:6 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV28-01 gene;

(d) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO:7 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV04-01 gene; (e) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO:8 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV05-05 gene;

(f) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO:9 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV28-01 gene;

(g) the ν_β CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO: 10 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV28-01 gene;

(h) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO: 1 1 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV06-05 gene;

(i) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO: 12 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV05-04 gene;

(j) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO: 14 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV27-01 gene;

(k) the ν_β CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO: 15 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV06-05 gene; or

(1) the νβ CDR3 amino acid sequence comprises an amino acid sequence of SEQ ID NO: 16 and comprises a CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV28-06 gene.

4. The binding protein of any one of claims 1-3, wherein the ν_β domain comprises an amino acid sequence having at least 80%, 85%>, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 173.

5. The binding protein according any one of claims 1-4, wherein:

(a) the ν_β domain comprises: (i) the CDR3 amino acid sequence comprising the amino acid sequence of SEQ ID NO:4;

(ii) the νβ domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO:4 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV28-01 gene; or

(iii) the νβ domain comprises the amino acid sequence having at least 80%, 85%, 90%, 95%, 99%, or 100% identity to SEQ ID NO: 173; and

(b) the V_a domain comprises:

(i) a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 155, 157, 159, 161, 163, 165, 167, and 169; or

(ii) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 155 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV12-02 gene; or

(iii) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 157 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV29-01 gene; or

(iv) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 159 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV12-02 gene; or

(v) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 161 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV12-01 gene; or

(vi) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 163 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV13-02 gene; or

(vii) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 165 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV12-02 gene; or

(viii) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 167 and comprises the CDRl amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV25-01 *01 gene; or (ix) the V_a domain comprises the CDR3 amino acid sequence comprising an amino acid sequence of SEQ ID NO: 169 and comprises the CDR1 amino acid sequence and CDR2 amino acid sequence encoded by a TCRBV29-01 gene.

6. The binding protein according to any one of claims 1-5, wherein the binding protein is capable of specifically binding to a EAAGIGILTV (SEQ ID

NO: 1):HLA complex with a K_d of less than or equal to 10^"8M.

7. The binding protein according to any one of claims 1-6, wherein the binding protein specifically binds to a EAAGIGILTV (SEQ ID NO: 1):HLA-A*0201 complex.

8. The binding protein according to any one of claims 1-7, wherein the binding protein is a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor.

9. The binding protein of any one of claims 8, wherein the binding protein is a TCR comprising:

(a) a TCR a-chain comprising the V_a domain and a TCR a-chain constant (C_a) domain, and

(b) a TCR β-chain comprising the ν_β domain and a TCR β-chain constant (Ο_β) domain.

10. The binding protein of claim 9, wherein: the C_a domain is encoded by a TRAC gene, the C domain is encoded by TRBC1 or TRBC2, or both.

1 1. The binding protein of claim 9 or 10, wherein the Ο_β domain comprises an amino acid sequence as set forth in SEQ ID NO: 175.

12. The binding protein of any one of claims 9-11, wherein the TCR β-chain comprises an amino acid sequence as set forth in SEQ ID NO: 172.

13. The binding protein of any one of claims 8-12, wherein the TCR, the antigen-binding fragment of the TCR, or chimeric antigen receptor is chimeric, humanized, or human.

14. The binding protein according to claim 8 or claim 13, wherein the antigen-binding fragment of the TCR comprises a single chain TCR (scTCR).

15. A composition comprising a binding protein according to any one of the preceding claims and a pharmaceutically acceptable carrier, diluent, or excipient.

16. The composition of claim 15, further comprising at least one additional binding protein comprising a V_a domain and ν_β domain, wherein the additional binding protein is capable of specifically binding to a MART-1 peptide EAAGIGILTV (SEQ ID NO: l):human leukocyte antigen (HLA) complex.

17. The composition of claim 16, wherein the composition comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or more additional binding proteins comprising a V_a domain and ν_β domain, wherein the additional binding proteins are capable of specifically binding to a MART-1 peptide

EAAGIGILTV (SEQ ID NO: l):human leukocyte antigen (HLA) complex.

18 The composition of any one of claims 15-17, comprising two or more binding proteins according to any one of claims 1-14.

19. The composition of claim 18, comprising three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or more binding proteins according to any one of claims 1-14.

20. The composition of any one of claims 15-19, wherein each of the binding proteins is at a frequency of at least 0.1% in the composition.

21. An isolated polynucleotide encoding a binding protein according to any one of claims 1-14.

22. The polynucleotide according to claim 21, wherein the isolated polynucleotide encodes two or more binding proteins according to any one of claims 1- 14.

23. The polynucleotide according to claim 21 or 22, wherein the

polynucleotide encoding a binding protein is codon optimized for expression in a host cell of interest.

24. The polynucleotide of claim 23, wherein the polynucleotide comprises a codon optimized νβ domain comprising the polynucleotide sequence as set forth in SEQ ID NO: 174.

25. The polynucleotide of claim 23 or 24, wherein the polynucleotide comprises a codon optimized C_β domain comprising the polynucleotide sequence as set forth in SEQ ID NO: 176.

26. The polynucleotide of any one of claims 23-25, wherein the

polynucleotide comprises a codon optimized TCR β-chain comprising an amino acid sequence as set forth in SEQ ID NO: 172.

27. An expression vector, comprising a polynucleotide according to any one of claims 21-26 operably linked to an expression control sequence.

28. The expression vector according to claim 27, wherein the vector is capable of delivering the polynucleotide to a host cell.

29. The expression vector according to claim 28, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

30. The expression vector according to claim 29, wherein the immune system cell is a T cell.

31. The expression vector according to claim 30, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

32. The expression vector according to any one of claims 27-31, wherein the T cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof.

33. The expression vector according to any one of claims 27-32, wherein the expression vector is a viral vector.

34. The expression vector according to claim 33, wherein the viral vector is a lentiviral vector or a γ-retroviral vector.

35. A host cell, comprising a heterologous polynucleotide according to any one of claims 21-26 or an expression vector according to any one of claims 27-34, wherein the host cell expresses on its cell surface a binding protein encoded by the heterologous polynucleotide.

36. The host cell according to claim 35, wherein the heterologous polynucleotide encodes a self-cleaving peptide disposed between a TCR a-chain encoding polynucleotide and a TCR β-chain encoding polynucleotide.

37. The host cell according to claim 36, wherein the self-cleaving peptide comprises or consists of a nucleotide sequence as set forth in any one of SEQ ID NOS: 17-21.

38. The host cell according to any one of claims 35-37, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

39. The host cell according to claim 38, wherein the immune system cell is a

T cell.

40. The host cell according to claim 38 or 39, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

41. The host cell according to claim 39 or 40, wherein the T cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof.

42. The host cell according to any one of claims 35-41, wherein the binding protein is capable or more efficiently associating with a CD3 protein as compared to endogenous TCR.

43. The host cell according to any one of claims 35-42, wherein the binding protein has higher surface expression on a T cell as compared to an endogenous TCR.

44. A method for treating melanoma, comprising administering to a human subject in need thereof a composition comprising a binding protein specific for human melanoma antigen recognized by T cells 1 (MART-1) according to any one of claims 1- 14.

45. The method according to claim 44, wherein the melanoma is metastatic melanoma.

46. The method of claim 44 or 45, wherein the binding protein is capable of promoting an antigen-specific T cell response against a human MART-1 in a class I HLA-restricted manner.

47. The method according to claim 46, wherein the class I HLA-restricted response is transporter-associated with antigen processing (TAP)-independent.

48. The method according to claim 46 or 47, wherein the antigen-specific T cell response comprises at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response.

49. The method according to claim 48, wherein the CTL response is directed against a MART-1 overexpressing cell.

50. The method according to any one of claims 44-49, wherein the composition comprises a host cell according to any one of claims 35-43.

51. An adoptive immunotherapy method for treating a human subject having melanoma, comprising administering to the subject an effective amount of a host cell according to any one of claims 35-43.

52. The method according to claim 51, wherein the host cell is modified ex vivo.

53. The method according to claim 51 or 52, wherein the host cell is an allogeneic cell, a syngeneic cell, or an autologous cell.

54. The method according to any one of claims 51-53, wherein the host cell is a hematopoietic progenitor cell, a hematopoietic stem cell, or a human immune system cell.

55. The method according to any one of claims 51-53, wherein the immune system cell is a T cell.

56. The method according to claim 55, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

57. The method according to claim 54, wherein the T cell is a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof.

58. The method of any one of claims 55-57, wherein the T cell has been exposed ex vivo to IL-21.

59. The method according to claim 55-58, wherein the host cell is a T cell that:

(a) expresses CD28 prior to administration;

(b) produces IFNy, T Fa, IL-2, or any combination thereof prior to administration;

(c) has low or no expression of PD-1, CD57, or both prior to administration; or

(d) any combination of (a), (b), and (c).

60. The method according to any one of claims 51-59, wherein the host cell is cultured for a period of no more than 6 weeks.

61. The method according to any one of claims 55-60, further comprising administering a plurality of host T cells according to any one of claims 35-43, wherein at least two host T cells express different binding proteins specific for MART-1.

62. The method according to any one of claims 51-61, wherein the melanoma is metastatic melanoma.

63. The method according to any one of claims 51-62, wherein the host cell is administered parenterally to the subject.

64. The method according to any one of claims 51-63, wherein the method comprises administering a plurality of doses of the host cell to the subject.

65. The method of claim 64, wherein the plurality of doses are administered at intervals between administrations of about two to about four weeks.

66. The method according to any one of claims 51-65, wherein the host cell is administered to the subject at a dose of about 10⁷ cells/m² to about 10¹¹ cells/m².

67. The method according to any one of claims 51-66, wherein the method further comprises administering a cytokine to the subject.

68. The method according to claim 67, wherein the cytokine is IL-2, IL-15, IL-21, or any combination thereof.

69. The method according to claim 68, wherein the cytokine is IL-2 and is administered concurrently or sequentially.

70. The method according to any one of claims 67-69, wherein the cytokine is administered sequentially, provided that the subject was administered the host cell at least three or four times before cytokine administration.

71. The method according to any one of claims 67-70, wherein the cytokine is IL-2 and is administered subcutaneously.

72. The method according to any one of claims 51-71, wherein

administration of the host cell is preceded by administration of an alkylating agent.

73. The method of claim 72, wherein the alkylating agent is

cyclophosphamide.

74. The method according to any one of claims 51-73, wherein the method further comprises administering an inhibitor of an immune checkpoint molecule to the subject.

75. The method according to claim 74, wherein the inhibitor of the immune checkpoint molecule is antibody or antigen binding fragment thereof, a fusion protein, a small molecule, an RNAi molecule, a ribozyme, an aptamer, or an antisense

oligonucleotide.

76. The method according to any one of claims 51-75, wherein the host cell is further modified to reduce or inhibit expression of a gene encoding the immune checkpoint inhibitor molecule.

77. The method according to claim 76, wherein the expression of the gene encoding the immune checkpoint inhibitor molecule is reduced or inhibited via an RNA-guided endonuclease, a zinc finger nuclease, a Transcription activator-like effector nuclease, an RNAi molecule, or an antisense oligonucleotide

78. The method according to any one of claims 74-77, wherein the immune checkpoint inhibitor molecule is CTLA-4, A2AR, B7-H3, B7-H4, BTLA, HVEM, GAL9, IDO, KIR, LAG-3, PD-1, PD-Ll, Tim-3, VISTA, TIGIT, LAIRl, CD 160, 2B4, TGFR beta, CEACAM-1, CEACAM-3, CEACAM-5, or CD244.

79. The method according to claim 78, wherein the immune checkpoint inhibitor molecule is CTLA-4.

80. The method according to claim 79, wherein the inhibitor of the immune checkpoint molecule is ipilimumab or tremelimumab.

81. The method according to claim 78, wherein the immune checkpoint inhibitor molecule is PD-1.

82. The method according to claim 81, wherein the inhibitor of the immune checkpoint inhibitor molecule is nivolumab or pembrolizumab.

83. The method according to claim 78, wherein the immune checkpoint inhibitor molecule is PD-L1.

84. The method according to claim 83, wherein the inhibitor of the immune checkpoint inhibitor molecule is durvalumab or atezolizumab.

85. The method according to any one of claims 74-84, wherein the inhibitor of the immune checkpoint inhibitor molecule is administered concurrently or subsequent to administration of the host cell.

86. The method of claim 85, wherein the inhibitor of the immune checkpoint inhibitor molecule is administered within a week of administration of the host cell.

87. The method according to claim 85 or 86, wherein a plurality of doses of the inhibitor of the immune checkpoint inhibitor molecule is administered at intervals between administrations of about two to about four weeks.

88. The method according to any one of claims 55-87, further comprising administering a plurality of host T cells according to any one of claims 31-39, wherein at least two host T cells express different binding proteins specific for MART-1.

89. The method according to claim 88, wherein the subject has a melanoma that is refractory to monotherapy using monoclonal adoptive immunotherapy specific for MART-1, monotherapy using an inhibitor of immune checkpoint inhibitor molecule, or a combination thereof.

90. The method according to claim 88 or 89, further comprising

administering an inhibitor of an immune checkpoint molecule, wherein administration of the plurality of host T cells and the inhibitor of immune checkpoint inhibitor induces a de novo antigen-specific T cell response to a non-targeted antigen.

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