NZ786619A

NZ786619A - Chimeric antigen and T cell receptors and methods of use

Info

Publication number: NZ786619A
Application number: NZ786619A
Authority: NZ
Inventors: Jed Wiltzius
Original assignee: Kite Pharma Inc
Filing date: 2017-03-31
Publication date: 2022-04-29

Abstract

The invention provides a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising extracellular domain disclosed herein. Some aspects of the invention relate to a polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) comprising the extracellular domain disclosed herein. Other aspects of the invention relate to cells comprising the CAR or the TCR and their use in a T cell therapy.

Description

CHIMERIC ANTIGEN AND T CELL RECEPTORS AND METHODS OF USE CROSS REFERENCE TO RELATED APPLICATIONS This application is a divisional of NZ 746739 and claims the benefit of U.S.

Provisional Patent Application No. ,258, filed April 1, 2016, which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on February 28, 2019, is named _02US_SL.txt and is 450,469 bytes in size.

BACKGROUND OF THE INVENTION Human cancers are by their nature comprised of normal cells that have undergone a genetic or etic conversion to become abnormal cancer cells. In doing so, cancer cells begin to express proteins and other antigens that are ct from those expressed by normal cells. These aberrant tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. r, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells.

Current therapies T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in a patient. To increase the ability of T cells to target and kill a ular cancer cell, methods have been developed to engineer T cells to express constructs which direct T cells to a ular target cancer cell. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), which se binding domains capable of interacting with a particular tumor antigen, allow T cells to target and kill cancer cells that express the ular tumor antigen.

A need exists for improved CARs and TCRs for targeting and killing cancer cells.

Y OF THE INVENTION The present invention addresses this need by providing compositions and methods comprising genetically engineered immune cells that express antigen receptors (CARs) or T cell receptors (TCRs) which speciﬁcally target and kill cancer cells.

A CAR may comprise, for example, (i) an antigen-speciﬁc component ("antigen binding molecule"), (ii) one or more costimulatory domains (which includes a hinge domain), and (iii) one or more activating domains. Each domain may be heterogeneous, that is, comprised of sequences d from different protein . CAR-expressing immune cells (such as T cells) may be used in various therapies, ing cancer therapies. id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8" id="p-8"

[0008] As described in more detail below, including the Examples section, CARs sing a costimulatory domain which includes a truncated hinge domain ("THD") provides ctedly superior properties when compared to a CAR sing a costimulatory domain which includes a complete hinge domain ). Polynucleotides encoding such CARs can be transduced into T cells and the CARs are expressed in T cells, e.g., a patient’s own T cells. When the uced T cells are transplanted back to a patient, the CARS direct the T cells to recognize and bind an epitope present on the surface of cancer cells, thus, ng binding of cancer cells rather than ncerous cells. This binding leads to activation of cytolytic mechanisms in the T cell that specifically kill the bound cancer cells.

Prior to the present invention, it was unknown that a CARs comprising a THD is superior to a CAR comprising a CHD. Thus, the present invention satisﬁes an unmet need that exists for novel and improved therapies for treating cancer.

An aspect of the present invention is an isolated polynucleotide encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which comprises (i) an antigen binding molecule, (ii) a costimulatory domain, and (iii) an activating domain. The costimulatory domain may comprise an extracellular domain, a transmembrane , and an intracellular domain, wherein the extracellular domain comprises a truncated hinge domain consisting essentially of or consisting of (i) an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 123 to 152 of SEQ ID NO: 1 and, optionally, (ii) one to six amino acids.

In some embodiments, the one to six amino acids are heterologous amino acids.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley In some embodiments, the truncated hinge domain consists essentially of or consists of an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 123 to 152 of SEQ ID NO: 1.

In some embodiments, the amino acid sequence is encoded by a nucleotide ce at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 2.

In some embodiments, the transmembrane domain is a transmembrane domain of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T cell receptor, or any combination thereof.

In some embodiments, the transmembrane domain comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% cal to SEQ ID NO: 5.

In some embodiments, the transmembrane domain is encoded by a nucleotide sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% cal to SEQ ID NO: 4.

In some embodiments, the ellular domain comprises a signaling region of 4-1BB/CD137, activating NK cell receptors, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, ha, CD8beta, CD96 (Tactile), CD1 la, CD1 1b, CD1 lc, CDl ld, CDS, CEACAMl, CRT AM, cytokine ors, DAP-10, DNAMl (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-l, ICAM-l, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, Immunoglobulin-like proteins, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-l, LFA-l, a ligand that speciﬁcally binds with CD83, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNF SF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-l (CDl la/CD18), MHC class Dolecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRFl), OX-40, [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley ed set by Anne.Headley p, programmed death-l (PD-l), PSGLl, SELPLG (CD162), signaling lymphocytic activation molecules (SLAM proteins), SLAM (SLAMFl, CD150, lPO-3 ), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF receptor ns, TNFR2, a Toll ligand receptor, TRANCE/RANKL, VLAl, or VLA-6, or a combination thereof.

In some embodiments, the intracellular domain ses a 4-lBB/CDl37 signaling region.

In some ments, the intracellular domain comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 7.

In some embodiments, the intracellular domain comprises an amino acid sequence encoded by a nucleotide sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 6. id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20" id="p-20"

[0020] In some embodiments, the antigen g molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH ses 3 complementarity determining regions (CDRs) and the VL comprises 3 CDRs.

In some embodiments, the antigen binding molecule speciﬁcally binds an antigen selected from the group consisting of 5T4, alphafetoprotein, B cell maturation antigen (BCMA), CA-l25, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30 , CD33, CD56, CDl23, CD138, c-Met, CSPG4, C-type lectin-like molecule 1 (CLL-l), EGFRvIII, epithelial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HERl-HER2 in ation, HER2-HER3 in combination, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp4l, HIV-1 pe glycoprotein gpl20, IL-llRalpha, kappa chain, lambda chain, melanoma-associated antigen, mesothelin, MUC-l, mutated p53, d ras, prostate- speciﬁc antigen, RORl, or VEGFR2, or a combination thereof.

In some embodiments, the antigen binding molecule speciﬁcally binds BCMA, CLL-l, or FLT3.

In some embodiments, the activation domain comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 9 or SEQ ID NO: 251.

[Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley In some embodiments, the activation domain is encoded by a nucleotide ce at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 8.

In some embodiments, the CAR or TCR further comprises a leader peptide.

In some embodiments, the leader peptide comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 11. id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27" id="p-27"

[0027] In some ments, the leader peptide is encoded by a nucleotide sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 10.

Another aspect of the present invention is a vector comprising the polynucleotide of an above aspect or embodiment.

In some embodiments, the vector is an adenoviral vector, an adenovirus- associated vector, a DNA vector, a lentiviral vector, a plasmid, a retroviral vector, or an RNA vector, or any combination thereof.

Yet another aspect of the t invention is a polypeptide encoded by the polynucleotide of an above aspect or ment or the vector of an above aspect or embodiment.

In another aspect, the present ion is a cell sing the polynucleotide of an above aspect or embodiment, the vector of an above aspect or embodiment, or the polypeptide of an above aspect or ment, or any combination thereof. id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32" id="p-32"

[0032] In some embodiments, the cell is a T cell.

In some embodiments, the T cell is an allogeneic T cell, an autologous T cell, an engineered autologous T cell (eACTTM), or a tumor-inﬁltrating cyte (TIL).

In some embodiments, the T cell is a CD4-- T cell.

In some embodiments, the T cell is a CD8-- T cell. id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36" id="p-36"

[0036] In some embodiments, the T cell is an in vitro cell.

In some embodiments, the T cell is an autologous T cell.

An aspect of the present invention is a composition comprising the polynDotide of an above aspect or embodiment, comprising the vector of an above aspect or ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley embodiment, comprising the polypeptide of an above aspect or embodiment, or comprising the cell of an above aspect or ment.

In some embodiments, the composition is formulated to be delivered to a subject, optionally, comprising at least one pharmaceutically-acceptable excipient.

Another aspect of the present invention is a method of making a cell expressing a CAR or TCR comprising transducing a cell with the polynucleotide of an above aspect or embodiment under le conditions.

In some embodiments, the method further comprises isolating the cell.

Yet another aspect of the present invention is a method of inducing an immunity against a tumor comprising administering to a subject an effective amount of a cell comprising the polynucleotide of an above aspect or embodiment, comprising the vector of an above aspect or ment, or the polypeptide of an above aspect or embodiment, or any combination thereof.

In another aspect, the present invention is a method of treating a cancer in a subject in need thereof comprising administering to the subject the polynucleotide of an above aspect or embodiment, the vector of an above aspect or embodiment, the polypeptide of an above aspect or embodiment, the cell of an above aspect or embodiment, or the composition of an above aspect or ment.

In some embodiments, the cancer is a hematologic cancer. id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45" id="p-45"

[0045] In some embodiments, the cancer is of the white blood cells.

In some ments, the cancer is of the plasma cells.

In some embodiments, the cancer is ia, lymphoma, or myeloma.

In some embodiments, the cancer is acute lymphoblastic leukemia (ALL) (including non T cell ALL), acute myeloid ia, B cell phocytic leukemia, B-cell acute lymphoid leukemia ("BALL"), blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic ia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse large B cell ma (DLBCL), follicular lymphoma (FL), hairy cell leukemia, Hodgkin's Disease, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, monoclonal gammapathy of undetermined signif1cance , multiple myeloma, myelodysplasia and myelodysplastic me, non-Hodgkin's lymphoma (NHL), plasma cell proliferative er (including asymptomatic myeloma ering multiple myelrn or indolent myeloma), plasmablastic lymphoma, plasmacytoid dendritic cell [Annotation] Anne.Headley None set by eadley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley neoplasm, plasmacytomas (including plasma cell dyscrasia, solitary myeloma, solitary cytoma, extramedullary plasmacytoma, and multiple plasmacytoma), POEMS syndrome (also known as Crow-Fukase me; Takatsuki disease; and PEP syndrome), primary mediastinal large B cell lymphoma (PlVﬂ3C), small cell- or a large cell-follicular lymphoma, c marginal zone lymphoma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute lymphoid leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, or Waldenstrom macroglobulinemia, or a combination thereof.

Generally, the present invention relates to Engineered Autologous Cell Therapy, abbreviated as "eACTTM," also known as adoptive cell transfer. eACTTM, is a process by which a patient's own T cells are collected and subsequently genetically engineered to recognize and target one or more antigens expressed on the cell surface of one or more specific cancer cells. T cells may be engineered to express, for example, a CAR or TCR. CAR positive (CAR+) T cells are engineered to express a CAR. CARs may comprise, e.g., an extracellular single chain variable nt (scFv) with specificity for a particular tumor n, which is directly or indirectly linked to an intracellular signaling part sing at least one costimulatory domain, which is directly or indirectly linked to at least one ting domain, the components may be arranged in any order. The costimulatory domain may be derived from a ulatory protein known in the art, e. g., SEQ ID NO: 1, and the activating domain may be derived from, e.g., any form of CD3-zeta. In some embodiments, the CAR is designed to have two, three, four, or more costimulatory domains. In some embodiments, a CAR is engineered such that the costimulatory domain is expressed as a te polypeptide chain.

Examples ofCAR T cell ies and constructs are described in US. Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and 050708, ational Patent ations Nos. W02012033885, W02012079000, W0201412726l, W02014186469, W02015080981, W02015142675, W02016044745, and W02016090369, and Sadelain el al, Cancer Discovery, 3: 388-3 98 (2013), each ofwhich is incorporated by reference in its entirety.

Any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein. While the present invention has been described in conjunction with the detailed description f, the foregoing description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modiﬁcations are within the scope of the follov'u claims.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley The patent and scientiﬁc literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, dictionaries, documents, manuscripts and scientiﬁc literature cited herein are hereby incorporated by reference.

Other features and advantages of the invention will be apparent from the Drawings and the ing Detailed ption, including the Examples, and the claims.

BRIEF DESCRIPTION OF THE FIGURES id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53" id="p-53"

[0053] The above and further features will be more y iated from the following detailed description when taken in conjunction with the accompanying drawings.

The drawings however are for ration purposes only, not for limitation. shows a costimulatory protein having the amino acid sequence of SEQ ID NO: 1. The costimulatory protein’s hinge domain (solid underline), transmembrane domain d underline), and signaling domain (dashed underline) are labeled. A novel ted hinge domain ("THD") is bolded. FIGs. 1B and 1C provide ribbon diagrams of the extracellular domain of the costimulatory protein having the amino acid sequence of SEQ ID NO: 1. shows an example of a region within the amino acid sequence of SEQ ID NO: 1 used to derive one ment of a hinge region in the context of CAR, i.e., a region containing amino acids 114 to 152 of SEQ ID NO: 1 (herein referred to as a complete hinge domain or "CHD", it is marked in black and dark grey). shows the THD which contain amino acids 123 to 152 of SEQ ID NO: 1 d in . In , the n of the hinge region that is excluded from is marked dark grey and circled. id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55" id="p-55"

[0055] FIGs. 2A-2H show CLUTSTAL W (2.83) multiple sequence alignments of eight example binding molecules disclosed herein. shows a sequence alignment of example anti-CLL-l binding molecules comprising a VH domain. CDRs and framework regions FRs are shown, as determined by Chothia numbering (). is a table providing the SEQ ID NO for each VH and CDR illustrated in . shows a sequence alignment of example anti-CLL-l binding les comprising a VL domain.

CDﬁand FRs are shown, as determined by Chothia numbering (). is a [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley table providing the SEQ ID NO for each VL and CDR sequence illustrated in . shows a sequence alignment of example anti-BCMA binding molecules comprising a VH domain. Complementarity determining s (CDRs) and framework s (FRs) are shown, as determined by Chothia numbering (). SEQ ID NOS 253-260, CDR1 in order from top to bottom; SEQ ID NOS 8, CDR2 from top to bottom, SEQ ID NOS 269-276, CDR3 from top to bottom. is a table providing the SEQ ID NO for each VH and CDR numbered by an alternative method. shows a sequence alignment of example anti-BCMA binding molecules comprising a VL domain. CDRs and FRs are shown, as determined by Chothia numbering (). SEQ ID NOS 37-44, CDR1 in order from top to bottom; SEQ ID NOS 45-52, CDR2 from top to bottom, SEQ ID NOS 4, CDR3 from top to bottom. is a table providing the SEQ ID NO for each VL and CDR sequence numbered by an alternative method. depicts CAR sion in primary human T cells electroporated with mRNA encoding for various CARs. Data obtained from CAR having a complete hinge domain ("CHD") is shown and data obtained from CAR having a ted hinge domain ("THD") is shown.

FIGs. 4A-4X show IFNγ, IL-2, and TNFα production by electroporated anti- FLT3 CAR T cells following 16 hours of ture with the indicated target cell lines. FIGs. 4A-4B, 4G-4H, 4M-4N, and 4S-4T show IFNγ tion following co-culture with Namalwa, EoL-1, HL60, and MV4;11 target cells, respectively. FIGs. 4C-4D, 4I-4J, 4O-4P, and 4U-4V show IL-2 production following co-culture with Namalwa, EoL-1, HL60, and MV4;11 target cells, respectively. FIGs. 4E-4F, 4K-4L, 4Q-4R, and 4W-4X show TNFα production following co-culture with Namalwa, EoL-1, HL60, and MV4;11 target cells, respectively. id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58" id="p-58"

[0058] FIGs. 5A-5H show cytolytic activity of electroporated anti-FLT3 CAR T cells against Namalwa (FIGs. 5A-5B), EoL1 (FIGs. 5C-5D), HL60 (FIGs. 5E-5F), and MV4;11 (FIGs. 5G-5H) target cell lines following 16 hours of co-culture.

FIGs. 6A-6B depict CAR expression in lentivirus transduced primary human T cells from two healthy donors. id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60" id="p-60"

[0060] FIGs. 7A-7F show IFNγ (FIGs. 7A-7B), TNFα (FIGs. 7C-7D), and IL-2 (FIGs. 7E-7F) production by lentivirus uced CAR T cells from two healthy donors ing 16 hours of co-culture with the indicated target cell lines.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley FIGs. 8A-8D show the average cytolytic activity over time from two healthy donors expressing the anti-FLT3 CAR constructs co-cultured with Namalwa (), EoL1 (), MV4;11 (), and HL60 () target cell lines for 16, 40, 64, 88, or 112 hours.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by eadley FIGS. 9A-9B depict proliferation of CF SE-labeled lentivirus transduced CAR T cells from two healthy donors following 5 days of co-culture with CD3-CD28 beads or the indicated target cell lines.

FIGs. lOA-lOD depict CAR expression in lentivirus transduced y human T cells used for in vivo studies. FIGs. lOE-lOF show graphical representations of measured bioluminescence imaging of labeled acute myeloid leukemia (AML) cells following intra- venous injection of either control (mock) or anti-FLT3 CAR T cells (lOE3-CHD, lOE3- THD, or D) in a xenogeneic model, performed in duplicate. FIG. lOG provides the p-values for the respective data points in E. FIGs. lOH-lOK show survival curves of mice injected with mock or lOE3-CHD (FIG. lOH), mock or lOE3-THD (FIG. lOI), mock or 8B5-THD (]), or lOE3-THD or 8B5-THD (K) CAR T cells.

FIGs. llA-l 1B shows CLL-l CAR expression determined by protein L 6 hours post mRNA electroporation.

FIGs. 12A-12C show the results from a cytokine release assay from different CLL-l CAR-T cell constructs 24 hours after mRNA electroporation. IL-2 (A), IFNy (B), and TNFu (C) production levels are shown for controls (target alone, mock, GFP, and CD19 CAR T cells) and anti-CLL-l CAR T cells (24Cl_HL-THD, 24Cl_HL_CHD, 24C8_HL-CHD, and 24C8_HL_THD) tured with Namalwa, MV4,ll, U937, HL60, and EoL-l cells, as indicated. id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66" id="p-66"

[0066] FIGs. l3A-l3E show tic activity of different CLL-l CAR-T cell constructs 24 hours after mRNA electroporation. T cells electroporated with control constructs (mock, GFP, and CD19 CAR) or LL-l CAR constructs HL-CHD and 24C8_HL_THD) were co-cultured with Namalwa (A), MV,4ll (B), EoL-l (C), HL-60 (D), and U937 target cells, and the percent of specific lysis of each target cell line was determined.

FIGs. C show the results from a cytokine release assay from different transduced anti-CLL-l CAR T cells 16 hours after co-culture with different cell lines. IFNy (A), IL-2 (B), and TNFu (C) tion levels are shown for controls (target alone and mock) and uced anti-CLL-l CAR T cells (lOE3_THD and 24Cl_LH_THD) co-cultured with Namalwa, HL-60, or MV4,ll target cells, as indicated.

FIGs. ISA-15C show cytolytic activity from anti-CLL-l CAR T cells (Cl_24Cl_LH_THD) 16 hours and 40 hours after ture with Namalwa (A), M‘Dl (B), or HL-60 (C) target cells.

[Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley FIGs F shows IFNγ, TNFα, and IL-2 production by lentivirus transduced CAR T cells from two healthy donors ing 16 hours of co-cultured with EoL-1 (Black), NCI-H929 (light grey), or MM1S (grey) target cell lines. FIGs. 16A and 16B show the IFNγ (pg/ml; y-axis) production in irus uced CAR T cells from a first donor () and a second donor (B). FIGs. 16C and 16D show the TNFα (pg/ml; y-axis) production in lentivirus transduced CAR T cells from a first donor (C) and a second donor (D). FIGs. 16E and 16F show the IL-2 production (pg/ml; y-axis) in lentivirus transduced CAR T cells from a first donor (E) and a second donor (F).

FIGs. 17A-17F show the average cytolytic activity (as a percentage of viable target cells remaining; y-axis) over time from two healthy donors expressing the indicated CARs co-cultured with EoL1 (FIGs. 17A and 17B), NCI-H929 (FIGs. 17C and 17D), or MM1S (FIGs. 17E and 17F) target cells for 16 hours, 40 hours, 64 hours, 88 hours, or 112 hours. FIGs. 17A and 17B show the average cytolytic activity of transduced CAR T cells from a first donor (A) and a second donor (B) co-cultured with EoL1 target cells for 16 hours, 40 hours, 64 hours, 88 hours, or 112 hours. FIGs. 17C and 17D show the average cytolytic activity of transduced CAR T cells from a first donor (C) and a second donor (D) co-cultured with NCI-H929 target cells for 16 hours, 40 hours, 64 hours, 88 hours, or 112 hours. FIGs. 17E and 17F show the average cytolytic activity of transduced CAR T cells from a first donor (E) and a second donor (F) co- ed with MM1S target cells for 16 hours, 40 hours, 64 hours, 88 hours, or 112 hours.

FIGs. 18A and 18B show proliferation of CFSE-labeled lentivirus transduced CAR T cells from a first healthy donor (A) and a second healthy donor (B) following 6 days of co-culture with CD3-CD28 beads (top row), EoL-1 (second row), NCIH929 (third row), or MM1S (bottom row) target cell lines. id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72" id="p-72"

[0072] FIG 19A and B are graphs showing thermostability of chimeric antigen receptors (CARs) of the t invention. A: In a phosphate-buffered saline (PBS) solution, a CAR comprising an extracellular domain with a truncated hinge domain ("THD") has a higher melting ature relative to a CAR comprising an extracellular domain with a complete hinge domain ("CHD"). B: In the ce of 50 mM NaCl, a CAR comprising an extracellular domain with a THD has a higher melting temperature relative to a CAR comprising an extracellular domain with a CHD. is a schematic representation of the pGAR vector.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley DETAILED DESCRIPTION OF THE INVENTION The present ion relates to novel polypeptides comprising a novel truncated hinge domain ("THD") and polynucleotides encoding the same. Some s of the invention relate to a polynucleotide encoding a chimeric antigen or (CAR) or a T cell receptor (TCR) comprising the THD sed herein. The present invention also es vectors (e.g., viral vectors) comprising such polynucleotides and compositions comprising such vectors. The present invention further provides polynucleotides encoding such CARs or TCRs and compositions comprising such polynucleotides. The present invention additionally provides engineered cells (e. g., T cells) sing such polynucleotides and/or transduced with such viral s and compositions comprising such engineered cells. The present invention provides compositions (e.g., ceutical compositions) including a plurality of engineered T cells. The present ion provides methods for manufacturing such engineered T cells and compositions and uses (e.g., in treating a melanoma) of such engineered T cells and compositions. And, the present invention provides a method of inducing an immunity against a tumor comprising stering to a subject an effective amount of a cell comprising a polynucleotide, a vector, or a polypeptide of the present invention. Other aspects of the invention relate to cells comprising the CAR or the TCR and their use in a T cell therapy, e.g., an autologous cell therapy (eACTTM), for the treatment of a patient ing from a cancer.

DEFINITIONS id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74" id="p-74"

[0074] In order for the present invention to be more readily understood, certain terms are ﬁrst deﬁned below. Additional tions for the following terms and other terms are set forth throughout the Speciﬁcation.

As used in this Specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly es otherwise. id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76" id="p-76"

[0076] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive and covers both "or" and "and".

The term "and/or" where used herein is to be taken as speciﬁc disclosure of each of the two specif1ed features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or [Annotation] eadley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley C" is intended to encompass each of the following aspects: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

The terms "e.g.," and "zle." as used herein, are used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the speciﬁcation.

The terms "or more", "at least", "more than", and the like, e. g., "at least one" are understood to include but not be limited to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, l3, l4, ,16,17,18,1920,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64, 67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89, 90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110, 2,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129, 130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148, l49or150,200,300,400,500,600,700,800,900,lOOO,2000,3000,4000,SOOOornunethan the stated value. Also included is any greater number or fraction in between.

Conversely, the term "no more than" includes each value less than the stated value. For example, "no more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92,91,90,89,88,87,86,85,84,83,82,81,80,79,78,77,76,75,74,73,72,71,70,69,68, 67,66,65,64,63,62,61,60,59,58,57,56,55,54,53,52,51,50,49,48,47,46,45,44,43, 42,41,40,39,38,37,36,35,34,33,32,31,30,29,28,27,26,25,24,23,22,21,20,19,18, l7,l6,lS,l4,l3,l2,ll,lO,9,8,7,6,5,4,3,2,l,and()nudeoﬁdes [Usoindudedisany lesser number or fraction in between.

The terms "plurality", "at least two", "two or more", "at least second", and the like, are understood to include but not d to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, l2, l3, l4, 15,16,17,18,1920,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64, 67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89, 90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110, 111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129, 130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148, 149 or150,200,300,400,500,600,700,800,900,lOOO,2000,3000,4000,5000 orrnore Also included is any greater number or fraction in between.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley Throughout the speciﬁcation the word "comprising," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element, integer or step, or group of ts, integers or steps, but not the exclusion of any other element, r or step, or group of elements, integers or steps. It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of’ and/or "consisting essentially of’ are also provided.

Unless speciﬁcally stated or evident from context, as used herein, the term "about" refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the tions of the measurement system. For example, "about" or "comprising essentially of’ can mean within one or more than one standard deviation per the ce in the art. "About" or "comprising essentially of’ can mean a range of up to 10% (i.e., ::10%). Thus, "about" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value.

When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of "about" or "comprising essentially of’ should be assumed to be within an able error range for that particular value or composition.

As bed herein, any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an r), unless otherwise indicated. id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85" id="p-85"

[0085] Units, preﬁxes, and s used herein are provided using their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the s deﬁning the range.

Unless deﬁned ise, all technical and scientiﬁc terms used herein have the same meaning as commonly tood by one of ordinary skill in the art to which this disclosure is related. For example, Juo, "The Concise Dictionary of Biomedicine and Molecular Biology", 2nd ed., (2001), CRC Press, "The nary of Cell & Molecular Biology", 5th ed., (2013), Academic Press, and "The Oxford Dictionary Of Biochemistry And [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley Molecular Biology", Cammack el al. eds., 2nd ed, (2006), Oxford University Press, provide those of skill in the art with a l dictionary for many of the terms used in this disclosure.

"Administering" refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.

Exemplary routes of stration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, eritoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, y by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, hecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, eritoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. In some ments, the formulation is administered via a non-parenteral route, e. g., orally. Other non-parenteral routes include a topical, epidermal or mucosal route of administration, for e, intranasally, vaginally, rectally, sublingually or lly.

Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.

The term "antibody" (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds cally to an antigen. In general, and antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulﬁde bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain nt region is comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, and FR4. The variable s of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the g of the immunoglobulin to [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley host tissues or factors, including various cells of the immune system (e.g., or cells) and the first component (Clq) of the classical complement system.

Antibodies can include, for e, monoclonal antibodies, recombinantly produced antibodies, monospecific dies, multispecif1c antibodies (including bispecif1c antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric dies, immunoglobulins, synthetic dies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, odies, antibody fusions (sometimes referred to herein as "antibody conjugates"), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulf1de-linked Fvs (dev), anti-idiotypic (anti-Id) dies (including, e.g., anti-anti-Id antibodies), minibodies, domain dies, synthetic antibodies imes referred to herein as "antibody mimetics"), and antigen-binding fragments of any of the above. In certain embodiments, antibodies bed herein refer to polyclonal antibody populations.

An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, IgE and IgM. IgG subclasses are also well known to those in the art and e but are not limited to human IgG1, IgG2, IgG3 and IgG4.

"Isotype" refers to the Ab class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and turally occurring Abs, monoclonal and polyclonal Abs, chimeric and humanized Abs, human or nonhuman Abs, wholly synthetic Abs, and single chain Abs. A nonhuman Ab may be humanized by recombinant methods to reduce its immunogenicity in man. Where not expressly stated, and unless the context indicates otherwise, the term "antibody" also includes an antigen-binding fragment or an antigen-binding n of any of the aforementioned globulins, and es a monovalent and a divalent fragment or portion, and a single chain Ab.

An "antigen binding molecule, 77 (L antigen binding portion," or "antibody fragment" refers to any molecule that comprises the n binding parts (e.g., CDRs) of the antibody from which the molecule is derived. An antigen binding molecule can include the antigenic complementarity determining regions (CDRs). Examples of antibody fragments inclutDut are not d to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley scFv antibodies, and multispeciﬁc antibodies formed from antigen binding molecules.

Peptibodies (i.e., Fc fusion les comprising peptide binding s) are r example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some ments, the antigen binding molecule binds to an antigen on a cell involved in a roliferative disease or to a viral or ial antigen. In n embodiments, the antigen binding le binds to BCMA, CLL- l, or FLT3. In further embodiments, the antigen binding molecule is an dy fragment that speciﬁcally binds to the antigen, including one or more of the mentarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of avimers.

As used herein, the term "variable region" L or ‘variable domain" is used interchangeably and are common in the art. The variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the g and speciﬁcity of a particular antibody for its ular antigen. The variability in sequence is concentrated in those regions called complementarity ining regions (CDRs) while the more highly conserved s in the variable domain are called framework regions (FR). Without wishing to be bound by any ular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., man primate) framework regions (FRs).

The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or an antigen-binding molecule thereof. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"

[0094] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or an antigen-binding molecule thereof.

A number of deﬁnitions of the CDRs are commonly in use: Kabat numbering, Chotlniumbering, AbM numbering, or contact numbering. The AbM definition is a [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley compromise between the two used by Oxford Molecular’ s AbM antibody modelling re.

The contact deﬁnition is based on an analysis of the available complex crystal structures.

Table l. CDR Numbering Loop Chothia Contact L l L24--L34 L30--L36 L2 L50--L56 L46--L55 L3 L89--L97 L89--L96 H1 35B H26--H35B H26--H32..34 H30--H35B (Kab at Numbering) H3 l--H35 35 H26--H32 H30--H35 (Chothia Numbering) H2 H50--H65 H50--H58 H52--H56 H47--H58 H3 H95--H102 H95--H102 H95--H102 H93--HlOl id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97" id="p-97"

[0097] The term "Kabat numbering" and like terms are ized in the art and refer to a system of numbering amino acid residues in the heavy and light chain variable s of an antibody, or an antigen-binding molecule thereof. In certain s, the CDRs of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat EA & Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA el al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35, which optionally can include one or two onal amino acids, following 35 (referred to in the Kabat ing scheme as 35A and 35B) (CDRl), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are typically t at amino acid positions 24 to 34 (CDRl), amino acid positiD 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley embodiment, the CDRs of the antibodies described herein have been ined according to the Kabat ing scheme.

In certain aspects, the CDRs of an antibody can be determined according to the a ing scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196: 901-917, ikani B el al., (1997) J Mol Biol 273: 927-948, Chothia C el al., (1992) J Mol Biol 227: 799-817, Tramontano A el al., (1990) J Mol Biol 215(1): 175-82, and US. Patent No. 226). Typically, when using the Kabat numbering convention, the Chothia CDR-Hl loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the a CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the a CDR-Ll loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. The end of the Chothia CDR-HI loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B, if neither 35A nor 35B is present, the loop ends at 32, if only 35A is present, the loop ends at 33, if both 35A and 35B are present, the loop ends at 34). In a speciﬁc embodiment, the CDRs of the antibodies described herein have been determined according to the Chothia numbering scheme. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"

[0099] As used herein, the terms "constant region" and "constant domain" are interchangeable and have a meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector ons, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid ce relative to an immunoglobulin variable domain.

As used herein, the term "heavy chain" when used in nce to an antibody can refer to any distinct type, e.g., alpha (0L), delta (6), epsilon (a), gamma (y) and mu (u), based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of dies, respectively, including subclasses of IgG, e.g., IgG1, Ing, IgG3 and IgG4.

As used herein, the term "light chain" when used in reference to an antibody can rento any distinct type, e. g., kappa (K) or lambda (9») based on the amino acid sequence [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley ofthe constant domains. Light chain amino acid sequences are well known in the art. In speciﬁc embodiments, the light chain is a human light chain.

"Binding afﬁnity" generally refers to the th of the sum total of non- covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an n). Unless indicated otherwise, as used herein, "binding afﬁnity" refers to intrinsic binding afﬁnity which reﬂects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The afﬁnity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Afﬁnity can be measured and/or expressed in a number of ways known in the art, including, but not d to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is ated from the quotient of keg/ken, whereas KA is calculated from the quotient of kon/koff. kon refers to the association rate constant of, e.g., an antibody to an antigen, and koff refers to the dissociation of, e. g., an antibody to an antigen. The km and koff can be determined by techniques known to one of ordinary skill in the art, such as BIACORE® or KinExA. id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103" id="p-103"

[0103] As used herein, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having side chains have been deﬁned in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e. g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, gine, glutamine, serine, ine, ne, ne, tryptophan), nonpolar side chains (e. g., alanine, valine, leucine, isoleucine, e, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e. g., tyrosine, alanine, tryptophan, histidine). In n embodiments, one or more amino acid es within a CDR(s) or within a framework region(s) of an antibody or antigen-binding le thereof can be replaced with an amino acid residue with a similar side chain.

As, used herein, the term "heterologous" means from any source other than naturally occurring sequences. For example, a heterologous sequence included as a part of a costimulatory protein having the amino acid sequence of SEQ ID NO: 1, e.g., the corresponding human costimulatory protein, is amino acids that do not naturally occur as, i.e., do not align with, the wild type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than that of the wild type human costimulatory protein-encoding sequence.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley As used herein, an "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody can speciﬁcally bind. An e can be, for e, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come er from two or more non-contiguous regions of a polypeptide or polypeptides (conformational, non-linear, discontinuous, or non-contiguous epitope). In certain embodiments, the epitope to which an antibody binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide ng assays, and/or mutagenesis mapping (e.g., irected mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giegé R el al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350, McPherson A (1990) Eur J Biochem 189: 1- 23, Chayen NE (1997) Structure 5: 1269-1274, McPherson A (1976) J Biol Chem 251: 6300- 6303). Antibody:antigen crystals may be studied using well known X-ray diffraction techniques and may be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc., see e. g. Meth Enzymol (1985) volumes 114 & 115, eds WyckoffHW el al.,, US. 2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1): 37-60, Bricogne G (1997) Meth Enzymol 276A: 361- 423, ed Carter CW, i P el al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10): 323). Mutagenesis mapping studies may be accomplished using any method known to one of skill in the art. See, e.g., Champe M el al, (1995) J Biol Chem 270: 394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, ing alanine scanning mutagenesis ques.

As used herein, an antigen binding le, an antibody, or an antigen binding molecule thereof "cross-competes" with a reference antibody or an antigen binding molecule thereof if the interaction between an antigen and the first binding molecule, an antibody, or an n binding molecule thereof blocks, limits, ts, or otherwise s the ability of the reference binding molecule, reference antibody, or an antigen g molecule f to interact with the antigen. Cross ition can be complete, e. g., binding of the binding molecule to the antigen tely blocks the y of the reference binding molecule to bind the antigen, or it can be partial, e. g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind the antigen. In certain embodiments, an antigeDnding molecule that cross-competes with a reference antigen binding molecule binds [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley the same or an overlapping epitope as the reference antigen binding molecule. In other embodiments, the antigen binding molecule that cross-competes with a reference antigen binding molecule binds a different epitope as the reference antigen binding molecule.

Numerous types of competitive binding assays can be used to determine if one antigen binding le competes with another, for e: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (Stahli et al., 1983, Methods in Enzymology 9:242-253), solid phase direct biotin-avidin EIA (Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct labeled assay, solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor , solid phase direct label RIA using 1-125 label (Morel et al., 1988, Molec. Immunol. 25 , solid phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82).

As used herein, the terms "immunospecif1cally binds, 77 (4'1mmunospecif1cally recognizes,77 (L specif1cally binds," and "speciﬁcally recognizes" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., epitope or immune x) as such binding is understood by one skilled in the art. For example, a molecule that specifically binds to an antigen may bind to other es or polypeptides, generally with lower affinity as determined by, e. g., immunoassays, BIACORE®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In a speciﬁc embodiment, molecules that specifically bind to an antigen bind to the n with a KA that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than the KA when the molecules bind to another antigen.

In another embodiment, molecules that specifically bind to an antigen bind with a dissociation constant (Kd) of about 1 x 10'7 M. In some embodiments, the antigen binding molecule specif1cally binds an antigen with "high affinity" when the Kd is about 1 x 10'9 M to about 5 x 10'9 M. In some embodiments, the antigen g le specif1cally binds an n with "very high affinity" when the Kd is 1 x 10'10 M to about 5 x 10'10 M. In one ment, the n binding molecule has a Kd of 10'9 M. In one embodiment, the off-rate is less than about 1 x 105. In other embodiments, the antigen binding molecule binds human BCMA with a Kd of between about 1 x 10'7 M and about 1 x 10'13 M. In yet another embodiment, the antigen binding molecule binds human BCMA with a Kd of about 1 x 10'10 M to about 5 x 10'10 M.

[Annotation] Anne.Headley None set by eadley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley In a speciﬁc embodiment, provided herein is an antibody or an antigen binding molecule f that binds to a target human antigen, e.g., human BCMA or human CLL-l, with higher y than to another species of the target antigen, e. g., a non-human BCMA or a non-human CLL-l. In certain embodiments, ed herein is an antibody or an n binding molecule t thereofthat binds to the target human antigen, e.g., human BCMA or human CLL-l, with a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or higher afﬁnity than to another species of the target antigen as measured by, e.g., a radioimmunoassay, surface plasmon resonance, or kinetic exclusion assay. In a speciﬁc embodiment, an antibody or an antigen binding molecule thereof described herein, which binds to a target human antigen, will bind to another species of the target antigen with less than 10%, %, or 20% of the binding of the antibody or an antigen binding molecule thereof to the human antigen as measured by, e.g., a radioimmunoassay, e plasmon resonance, or kinetic exclusion assay.

An "antigen" refers to any molecule that provokes an immune response or is capable of being bound by an dy or an n binding molecule. The immune response may involve either antibody production, or the activation of c immunologically- competent cells, or both. A person of skill in the art would readily understand that any macromolecule, including virtually all ns or peptides, can serve as an antigen. An antigen can be nously sed, zle. expressed by c DNA, or can be recombinantly expressed. An antigen can be speciﬁc to a certain tissue, such as a cancer cell, or it can be broadly expressed. In addition, fragments of larger molecules can act as antigens. In one embodiment, antigens are tumor antigens. In one particular embodiment, the antigen is all or a fragment of BCMA, FLT3, or CLL-l.

The term "neutralizing" refers to an antigen binding le, scFv, antibody, or a nt thereof, that binds to a ligand and prevents or reduces the biological effect of that ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or a fragment thereof, directly blocking a binding site on the ligand or otherwise alters the ligands ability to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments, the antigen binding le, scFv, antibody, or a fragment thereof prevents the protein to which it is bound from performing a biological function.

As used herein, the term "BCMA" refers to B cell maturation antigen, which can include, but is not limited to, native BCMA, an isoform of BCMA, or an interspecies BCMGmolog ofBCMA. BCMA (also known as INFRSF17, CD269, and INFRSF13A) is [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley a member of the tumor necrosis factor (TNF)-receptor superfamily. BCMA is expressed on the e of multiple myeloma cells, while highly restricted to plasma cells and a sub set ofmature B cells in healthy tissue. The amino acid sequence of human BCMA (hBCMA) is provided in NCBI Accession Q02223.2 (GI:313104029). As used herein, BCMA includes human BCMA and non-human BCMA homologs, as well as variants, fragments, or post-transnationally d forms f, including, but not limited to, N— and O-linked glycosylated forms of BCMA. BCMA proteins may r include fragments comprising all or a portion of the extracellular domain ofBCMA (e.g., all or a portion of amino acids 1-54 of .

As used herein, the term "CLL-l" refers to C-type lectin-like molecule-1, which can include, but is not limited to native CLL-l, an isoform of CLL-l, or an interspecies CLL- 1 homolog of CLL-l. CLL-l (also known as C-type lectin domain family 12 member A, CLEC12A, dendritic cell-associated lectin 2, , myeloid inhibitory C-type lectin-like receptor, and MICL) is a cell surface receptor that modulates signaling es and mediates tyrosine phosphorylation of target MAP kinases. CLL-l expression is observed, e. g., in acute myeloid leukemia (AML) cells. The amino acid sequence of human CLL-l (hCLL-l) is provided in UniProtKB/Swiss-Prot Accession No. Q5QGZ9.3 (GI:308153619). As used herein, CLL-l includes human CLL-l and non-human CLL-l homologs, as well as variants, fragments, or post-transnationally modified forms f, ing, but not limited to, N— and O-linked glycosylated forms of CLL-l. id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114" id="p-114"

[0114] As used herein the term "FLT3" refers to Fms-like tyrosine kinase 3 (FLT-3), which can include, but is not limited to native FLT3, an n of FLT3, or an interspecies FLT3 homolog ofFLT3. FLT3 (also known as Cluster of differentiation antigen 135 (CD135), receptor-type tyrosine-protein kinase FLT3, FMS-related tyrosine kinase 3, stem cell tyrosine kinase 1, FL cytokine receptor, growth factor receptor tyrosine kinase type III, STKl, or fetal liver kinase-2 ) is a cytokine receptor which belongs to the receptor tyrosine kinase class III. CD135 is the receptor for the cytokine Flt3 ligand (FLT3L). FLT3 is expressed on the surface of s poietic progenitor cells and on the surface of acute d leukemia (AML) cells. The amino acid sequence of human FLT3 ) is provided in UniProtKB/Swiss-Prot Accession No. P3 6888 (G1: 156630887). As used herein, FLT3 includes human FLT3 and non-human FLT3 homologs, as well as variants, fragments, or post- transnationally modified forms thereof, ing, but not limited to, N— and O-linked glycosylated forms of FLT3.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley The term "autologous" refers to any material derived from the same individual to which it is later to be re-introduced. For example, the engineered autologous cell therapy (eACTTM) method described herein involves collection of lymphocytes from a patient, which are then engineered to express, e. g., a CAR construct, and then administered back to the same patient.

The term "allogeneic" refers to any al derived from one individual which is then introduced to another individual of the same species, e. g., allogeneic T cell transplantation. [01 17] The terms "transduction" and "transduced" refer to the s whereby foreign DNA is uced into a cell via viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, a RNA , an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a viral , a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector, a lentiviral vector, or any combination f. id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118" id="p-118"

[0118] As used herein, the term ated" refers to anything less than the whole. For example, a truncated hinge domain (alternatively referred to herein as "THD") amino acid sequence can include any amino acid sequence shorter than the full length or complete hinge domain ("CHD"). In some embodiments, a THD consists essentially of or consists of amino acids 118-152, 119—152, 120—152, 121—152, 2, 123—152, 124—152, 125—152, 126-152, 127—152, 128-152, 129—152, or 130—152, of SEQ ID NO: 1. In one embodiment, the THD consists essentially of or consists of the amino acid sequence of SEQ ID NO: 3, which ts of amino acids 123 to 152 of SEQ ID NO: 1.

A "cancer" refers to a broad group of various es characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring s and may also metastasize to distant parts ofthe body through the lymphatic system or bloodstream. A "cancer" or "cancer tissue" can include a tumor. Examples of cancers that can be treated by the methods of the present invention include, but are not limited to, cancers of the immune system ing lymphoma, leukemia, myeloma, and other leukocyte malignancies. In some ments, the methods of the present invention can be used to reduce the tumor size of a tumor derived from, for example, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal an, stomach cancer, testicular cancer, e cancer, carcinoma of the fallopian tubes, [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma ofthe vulva, le myeloma, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma ), follicular lymphoma (FL), transformed follicular lymphoma, c marginal zone lymphoma (SMZL), cancer ofthe esophagus, cancer ofthe small intestine, cancer of the endocrine system, cancer of the d gland, cancer of the parathyroid gland, cancer of the l gland, sarcoma of soft , cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid ia, acute lymphoblastic leukemia (ALL) (including non T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or , carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), y CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's a, epidermoid cancer, squamous cell cancer, T-cell ma, environmentally induced cancers including those d by asbestos, other B cell malignancies, and combinations of said cancers. In one particular embodiment, the cancer is multiple a. The particular cancer can be responsive to chemo- or radiation therapy or the cancer can be refractory. A refractory cancer refers to a cancer that is not amendable to surgical intervention and the cancer is either initially unresponsive to chemo- or radiation therapy or the cancer becomes unresponsive over time. id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120" id="p-120"

[0120] An tumor effect" as used herein, refers to a biological effect that can present as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or amelioration of various physiological symptoms associated with the tumor. An anti-tumor effect can also refer to the tion of the occurrence of a tumor, e. g., a vaccine.

A "cytokine," as used herein, refers to a non-antibody protein that is released by one cell in response to contact with a speciﬁc antigen, wherein the cytokine interacts with a second cell to mediate a response in the second cell. A cytokine can be endogenously expressed by a cell or administered to a subject. Cytokines may be ed by immune cells, including macrophages, B cells, T cells, and mast cells to propagate an immune response. Cytokines can induce various responses in the recipient cell. Cytokines can include homeostatic cytokines, chemokines, pro-inﬂammatory cytokines, effectors, and acute-phase proteins. For example, homentic cytokines, including interleukin (1L) 7 and 1L-15, e immune cell survival [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley and proliferation, and pro-inﬂammatory cytokines can promote an inﬂammatory response.

Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL- , lL-12p40, 70, 1L-15, and interferon (IFN) gamma. Examples of pro-inﬂammatory cytokines include, but are not limited to, lL-la, IL-lb, 1L-6, lL-l3, IL-l7a, tumor necrosis factor alpha, TNF-beta, ﬁbroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble ellular adhesion molecule 1 (sICAM-l), soluble vascular adhesion molecule 1 (sVCAM-l), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, me B, soluble Fas ligand (sFasL), and perforin. es of acute phase-proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

"Chemokines" are a type of cytokine that mediates cell chemotaxis, or directional movement. Examples of chemokines include, but are not limited to, 1L-8, lL-l6, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-l or CCL2), MCP-4, macrophage inﬂammatory n 10t(MlP-10t, MlP-la), MlP-lB (MlP-lb), gamma-induced protein 10 (lP-lO), and thymus and activation regulated chemokine (TARC or .

A "therapeutically effective amount, 77 (L effective dose, 77 (L effective amount," or "therapeutically effective " of a therapeutic agent, e. g., engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes e sion evidenced by a decrease in severity of disease symptoms, an increase in ncy and on of disease symptom- free periods, or a prevention of impairment or disability due to the disease afﬂiction. The ability of a therapeutic agent to promote disease regression can be ted using a y ods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

The term "lymphocyte" as used herein includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) cyte that represent a major component ofthe inherent immune system. NK cells reject tumors and cells infected by viruses.

It works h the process of apoptosis or programmed cell death. They were termed "natural killers" because they do not require activation in order to kill cells. T-cells play a major role in cell-nDated-immunity (no dy involvement). Its T-cell receptors (TCR) differentiate [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley themselves from other cyte types. The thymus, a specialized organ of the immune system, is primarily sible for the T cell’s maturation. There are six types of T-cells, namely: Helper T-cells (e. g., CD4+ cells), Cytotoxic T-cells (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cells or killer T cell), Memory T-cells ((i) stem memory TSCM cells, like naive cells, are CD45RO—, CCR7+, +, CD62L+ ectin), CD27+, CD28+ and 1L-7ROL‘l', but they also express large amounts of CD95, 1L- 2RB, CXCR3, and LFA-l, and show numerous functional attributes distinctive of memory cells), (ii) central memory TCM cells express L-selectin and the CCR7, they secrete lL-2, but not lFNy or 1L-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but e effector cytokines like lFNy and lL-4), Regulatory T-cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), Natural Killer T-cells (NKT) and Gamma Delta T-cells. B-cells, on the other hand, play a pal role in humoral immunity (with antibody involvement). It makes antibodies and antigens and performs the role of antigen-presenting cells (APCs) and turns into memory B-cells after activation by antigen interaction. In mammals, immature B-cells are formed in the bone marrow, where its name is derived from.

The term "genetically engineered" or eered" refers to a method of modifying the genome of a cell, including, but not limited to, deleting a coding or non-coding region or a portion thereof or inserting a coding region or a n thereof. In some embodiments, the cell that is modified is a lymphocyte, e. g., a T cell, which can either be obtained from a patient or a donor. The cell can be modiﬁed to express an ous construct, such as, e. g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR), which is orated into the cell's genome.

An "immune response" refers to the action of a cell of the immune system (for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, phils, mast cells, dendritic cells and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues ed with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inﬂammation, normal human cells or tissues.

The term "immunotherapy" refers to the treatment of a subject afﬂicted with, or at risk of cting or suffering a recurrence of, a disease by a method sing inducing, enha SL1 ressin or otherwise an immune Of , modifying response. Examples [Annotation] Anne.Headley None set by eadley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley immunotherapy include, but are not limited to, T cell therapies. T cell therapy can include adoptive T cell therapy, tumor-inﬁltrating cyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACTTM), and allogeneic T cell transplantation.

However, one of skill in the art would recognize that the conditioning methods disclosed herein would enhance the effectiveness of any transplanted T cell therapy. es of T cell therapies are described in U. S. Patent Publication Nos. 154228 and 2002/0006409, US.

Patent No. 5,728,388, and International Publication No.

The T cells of the immunotherapy can come from any source known in the art.

For example, T cells can be entiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained from, e. g., peripheral blood clear cells ), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation and/or sis. onal methods of isolating T cells for a T cell y are disclosed in US. Patent Publication No. 287748, which is herein incorporated by references in its entirety.

The term "engineered Autologous Cell Therapy," which can be abbreviated as "eACTTM," also known as adoptive cell transfer, is a process by which a patient's own T cells are collected and subsequently genetically altered to recognize and target one or more antigens expressed on the cell surface of one or more speciﬁc tumor cells or malignancies. T cells can be engineered to express, for example, chimeric antigen receptors (CAR) or T cell receptor (TCR). CAR positive (+) T cells are engineered to express an extracellular single chain variable fragment (scFv) with specificity for a particular tumor antigen linked to an intracellular signaling part comprising at least one costimulatory domain and at least one activating domain.

The costimulatory domain can be derived from a lly-occurring costimulatory domain, e.g., having the amino acid sequence of SEQ ID NO: 1, or a variant thereof, e. g., a variant having a truncated hinge domain ("THD"), and the activating domain can be derived from, e.g., CD3-zeta. In certain embodiments, the CAR is designed to have two, three, four, or more costimulatory domains. The CAR scFv can be designed to target, for e, CD19, which is a transmembrane protein expressed by cells in the B cell lineage, including all normal B cells and B cell malignances, including but not limited to NHL, CLL, and non-T cell ALL. In some nts, the CAR is engineered such that the costimulatory domain is expressed as a [Annotation] Anne.Headley None set by eadley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley separate polypeptide chain. Example CAR T cell therapies and constructs are described in US.

Patent Publication Nos. 2013/0287748, 2014/0227237, 2014/0099309, and 2014/0050708, and these references are incorporated by reference in their ty.

A "patient" as used herein includes any human who is afﬂicted with a cancer (e.g., a lymphoma or a leukemia). The terms "subject" and "patient" are used interchangeably herein.

As used herein, the term "in vitro cell" refers to any cell which is cultured ex vivo. In particular, an in vitro cell can include a T cell.

The terms de," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by e bonds.

A protein or peptide contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence.

Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used , the term refers to both short chains, which also commonly are ed to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. eptides" e, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of ptides, modiﬁed polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a ation thereof.

"Stimulation," as used herein, refers to a primary response induced by binding of a stimulatory molecule with its cognate ligand, wherein the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, e.g., the T cell receptor CD3 complex, that specifically binds with a cognate stimulatory ligand present on an antigen present cell. A "stimulatory ligand" is a ligand that when present on an antigen presenting cell (e.g., an APC, a dendritic cell, a B-cell, and the like) can specif1cally bind with a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, tion of an immune response, eration, and the like. Stimulatory ligands include, but are not limited to, an anti-CD3 antibody (such as OKT3), an MHC Class I molecule loaded with a peptide, a superagonist anti-CD2 dy, and a superagonist anti-CD28 antibody.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley A "costimulatory signal," as used herein, refers to a , which in combination with a primary signal, such as TCR/CD3 ligation, leads to a T cell response, such as, but not limited to, proliferation and/or upregulation or down regulation of key molecules.

A mulatory ligand" as used herein, includes a molecule on an antigen presenting cell that speciﬁcally binds a cognate co-stimulatory le on a T cell. Binding of the costimulatory ligand provides a signal that mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A ulatory ligand s a signal that is in addition to the primary signal provided by a stimulatory molecule, for instance, by binding of a T cell receptor (TCR)/CD3 complex with a major histocompatibility complex (MHC) molecule loaded with peptide. A co-stimulatory ligand can include, but is not limited to, 3/TR6, 4-lBB ligand, agonist or dy that binds Toll ligand receptor B7-l (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpes virus entry mediator (HVEM), human leukocyte antigen G (HLA-G), 1LT4, globulin-like transcript (1LT) 3, inducible costimulatory ligand (ICOS-L), intercellular on molecule (ICAM), ligand that specifically binds with B7-H3. beta receptor, MHC class I chain-related , lymphotoxin protein A (MICA), MHC class I chain-related protein B (MICB), 0X40 ligand, PD-L2, or programmed death (PD) Ll. A co-stimulatory ligand includes, without limitation, an antibody that speciﬁcally binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, ligand that specifically binds with CD83, lymphocyte function-associated antigen-l (LFA-l), natural killer cell receptor C (NKGZC), 0X40, PD-l, or tumor is factor superfamily member 14 (TNFSF14 or LIGHT).

A "costimulatory molecule" is a cognate g partner on a T cell that specifically binds with a costimulatory ligand, thereby ing a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory les include, but are not limited to, A "costimulatory molecule" is a cognate binding r on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory les include, but are not d to, 4-1BB/CD137, B7-H3, BAFFR, BLAME 8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69DD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley CDl-la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAMl, CRT AM, DAP-lO, DNAMl (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-l, ICAM-l, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, , LAT, LFA-l, LFA-l, LIGHT, LIGHT (tumor necrosis factor superfamily member 14, TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-l (CDl la/CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 ), 0X40, PAG/Cbp, PD-l, PSGLl, SELPLG (CD162), signaling cytic activation molecule, SLAM l, CD150, 1130—3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE/RANKL, VLAl, or VLA-6, or fragments, truncations, or ations thereof.

The terms ing" and "decreasing" are used interchangeably herein and indicate any change that is less than the original. "Reducing" and "decreasing" are relative terms, requiring a comparison between pre- and post- measurements. "Reducing" and "decreasing" include te depletions.

"Treatment" or "treating" of a t refers to any type of intervention or process performed on, or the administration of an active agent to, the t with the objective of reversing, ating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease. In one embodiment, "treatment" or "treating" es a partial remission. In another embodiment, "treatment" or "treating" includes a complete remission.

To calculate percent identity, the sequences being compared are typically aligned in a way that gives the largest match between the sequences. One example of a computer program that can be used to determine percent ty is the GCG program e, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, Genetics Computer Group, University ofWisconsin, Madison, Wis). The computer algorithm GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined. The sequences are aligned for optimal matching of their respective amino acid or nucleotide (the "matched span," as determined by the algorithm). In certain embodiments, a standard comparison matrix (see, f et al., 1978, Atlas of Protein Sequence and ure :345-352 for the PAM 250 comparison matrix, Henikoff et al., 1992, Proc. Natl. Acad. Sci.

U.S.AD:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm. ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley Various aspects of the invention are described in further detail in the following sub sections.

I. Chimeric Antigen Receptors and T Cell Receptors Chimeric antigen receptors (CARs or ) and T cell receptors (TCRs) are genetically engineered receptors. These engineered ors can be readily inserted into and expressed by immune cells, including T cells in accordance with techniques known in the art.

With a CAR, a single receptor can be programmed to both recognize a speciﬁc n and, when bound to that antigen, te the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR can target and kill the tumor cell.

One aspect of the present ion is directed to polynucleotides encoding chimeric antigen receptors (CARs) or T cell receptors (TCRs) comprising a costimulatory domain comprising a novel extracellular domain comprising a truncated hinge domain ("THD"), and engineered T cells comprising a ulatory domain comprising the novel THD. The costimulatory domain can further comprise a transmembrane domain and/or an intracellular . In some embodiments, a CAR or TCR encoded by the polynucleotide of the present invention further comprises an antigen binding molecule that speciﬁcally binds to a target n. In some embodiments, the CAR or TCR d by the polynucleotide further comprises an activating domain. In one particular embodiment, the CAR or TCR encoded by the polynucleotide comprises (i) an antigen binding molecule that speciﬁcally binds to a target antigen, (ii) a costimulatory domain comprising an extracellular domain, a transmembrane domain, and an intracellular , and (iii) an activating domain, wherein the extracellular domain comprises, consists essentially of, or consists of a THD described , e.g., SEQ ID NO: 3. id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143" id="p-143"

[0143] In some embodiments, an orientation of the CARs in accordance with the invention comprises an antigen binding domain (such as scFv) in tandem with a costimulatory domain and an activating . The costimulatory domain can comprise one or more of an extracellular portion, a transmembrane portion, and an intracellular portion. In other embodiments, multiple costimulatory domains can be utilized in tandem.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley LA. Costimulatory Domain. ic n receptors incorporates costimulatory ling) domains to increase their potency. See US. Patent Nos. 7,741,465, and 6,319,494, as well as Krause el al. and Finney el al. (supra), Song el al., Blood 119:696-706 (2012), Kalos el al., Sci Transl. Med. 3:95 (2011), Porter el al., N. Engl. J. Med. 365:725-33 (2011), and Gross el al., Annu. Rev.

Pharmacol. Toxicol. 56:59—83 . The costimulatory protein having the amino acid sequence of SEQ ID NO: 1 is a costimulatory protein found naturally on T-cells. The complete native amino acid sequence of this costimulatory protein is described in NCBI Reference Sequence: 130.1. See Figure 1A. The complete native c acid ce of this costimulatory protein is described in NCBI Reference Sequence: NM_OO6139. 1.

Novel Extracellular Domain: The present sure shows that a novel extracellular domain of a costimulatory protein and comprising a truncated hinge domain ("THD") can improve one or more properties of a CAR or a TCR. In some embodiments, the THD domain is a truncated version of a complete hinge domain ("CHD"). In certain embodiments, the isolated polynucleotide encoding a THD comprises (i) an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 123 to 152 of SEQ ID NO: 1, n the THD domain does not contain amino acids 1 to 122 of SEQ ID NO: 1. id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146" id="p-146"

[0146] In other embodiments, the THD consists essentially of or consists of an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 123 to 152 of SEQ ID NO: 1. In other embodiments, the THD consists essentially of or ts of an amino acid sequence encoded by a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% cal to SEQ ID NO: 3.

In some embodiments, the isolated polynucleotide encoding a THD consists essentially of or consists of (i) an amino acid ce at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 123 to 152 of SEQ ID NO: 1 and (ii) optionally :: one amino acid, :: two amino acids, :: three amino acids, :: four amino acids,nf1ve amino acids, or :: siX amino acids. In some embodiments, the isolated [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley polynucleotide encoding a THD consists essentially of or consists of (i) an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% cal to amino acids 123 to 152 of SEQ ID NO: 1 and (ii) optionally one or two amino acids, one to three amino acids, one to four amino acids, one to ﬁve amino acids, or one to siX amino acids.

The one to siX amino acids that can be added or deleted from the amino acid sequence in the THD can be at either the N—terminus, at the C-terminus, or both the N—terminus and the C- terminus.

In some embodiments, the isolated polynucleotide encoding a THD consists essentially of or consists of (i) an amino acid ce at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 123 to 152 of SEQ ID NO: 1 and (ii) one additional N—terminal amino acid, two additional inal amino acids, three additional N—terminal amino acids, four additional inal amino acids, ﬁve additional N- tenninal amino acids, or siX additional N—terminal amino acids.

In some embodiments, the onal amino acids can be N—terminal amino acids. In some embodiments, the additional amino acids can be heterologous. In other embodiments, the additional amino acids are part of the naturally occurring costimulatory protein sequence. id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150" id="p-150"

[0150] In some embodiments, the THD consists essentially of or consists of an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 123 to 152 of SEQ ID NO: 1, amino acids 122 to 152 of SEQ ID NO: 1, amino acids 121 to 152 of SEQ ID NO: 1, amino acids 120 to 152 of SEQ ID NO: 1, amino acids 119 to 152 of SEQ ID NO: 1, amino acids 118 to 152 of SEQ ID NO: 1, or amino acids 117 to 152 ofSEQ IDNO: l.

In other embodiments, the THD consists essentially of or consists of an amino acid ce at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to amino acids 124 to 152 of SEQ ID NO: 1, amino acids 125 to 152 of SEQ ID NO: 1, amino acids 126 to 152 of SEQ ID NO: 1, amino acids 127 to 152 of SEQ ID NO: 1, amino acids 128 to 152 of SEQ ID NO: 1, amino acids 129 to 152 of SEQ ID NO: 1, or amino acids 130 "Dz of SEQ ID NO: 1.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley In some embodiments, the THD does not comprise amino acids 1-116 of SEQ ID NO: 1. In some embodiments, the THD does not comprise amino acids 1-117 of SEQ ID NO: 1. In some embodiments, the THD does not comprise amino acids 1-118 of SEQ ID NO: 1. In some embodiments, the THD does not se amino acids 1-119 of SEQ ID NO: 1. In some embodiments, the THD does not comprise amino acids 1-120 of SEQ ID \10: 1. In some embodiments, the THD does not comprise amino acids 1-121 of SEQ ID \0: 1. In some embodiments, the THD does not comprise amino acids 1-122 of SEQ ID \0: 1. In some embodiments, the THD does not comprise amino acids 1-123 of SEQ ID \0: 1. In some embodiments, the THD does not comprise amino acids 1-124 of SEQ ID \0: 1. In some ments, the THD does not comprise amino acids 1-125 of SEQ ID \0: 1. In some embodiments, the THD does not comprise amino acids 1-126 of SEQ ID \0: 1. In some embodiments, the THD does not comprise amino acids 1-127 of SEQ ID \0: 1. In some embodiments, the THD does not comprise amino acids 1-128 of SEQ ID \0: 1. In some embodiments, the THD does not comprise amino acids 1-129 of SEQ ID NO: 1. id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153" id="p-153"

[0153] The corresponding amino acid sequence of the THD is set forth in SEQ ID NO. 3 LDNEKSNGTI IHVKGKHLCP SPLFPGPSKP. A nucleotide sequence encoding the extracellular portion of THD is set forth in SEQ ID NO. 2 CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTC TGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCA. id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154" id="p-154"

[0154] In certain embodiments, the polynucleotide encoding a costimulatory domain in a CAR or TCR comprises a nucleotide sequence at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to SEQ ID NO: 3, n the nucleotide sequence encodes a THD and wherein the CAR or TCR does not comprise amino acids 1 to 122 of SEQ ID NO: 1.

In one particular ment, the THD consists essentially of or consists of an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid ce of SEQ ID NO: 3. In a speciﬁc embodiment, the cleotide encoding THD consists essentially of or consists of a tide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the THD further comprises some or all of a member of the immunoglobulin family such as IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragment thereof.

In some embodiments, the THD is derived from a human complete hinge domain ("CHD"), e.g., from the costimulatory protein having the amino acid sequence of SEQ ID NO: 1. In other embodiments, the THD is derived from a rodent, murine, or primate (e.g., non-human primate) CHD of a costimulatory protein. In some ments, the THD is derived from a chimeric CHD of a costimulatory protein.

Transmembrane Domain: The costimulatory domain for the CAR or TCR of the invention can further comprise a transmembrane domain and/or an intracellular signaling domain. The embrane domain can be designed to be fused to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in a CAR is used. In some instances, the transmembrane domain can be selected or modiﬁed by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the or x. The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is l, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this ion can be derived from (i.e., comprise) 4-lBB/CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CDl la, CDl lb, CDl lc, CDl ld, CDS, CEACAMl, CRT AM, cytokine or, , DNAMl (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), 1A4, ICAM-l, ICAM-l, Ig alpha (CD79a), IL-2R beta, IL- 2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1,KIRDS2, LAT, LFA-l, LFA-l, a ligand that speciﬁcally binds with CD83,, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-l (LFA-l, CDl-la/CD18), MHC class 1 molecn NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 ), OX-40, PAG/Cbp, [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley programmed death-l (PD-l), PSGLl, SELPLG (CD162), ing Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMFl, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, , , SLP-76, TNF or proteins, TNFR2, INFSF14, a Toll ligand receptor, TRANCE/RANKL, VLAl, or VLA-6, or a fragment, truncation, or a combination thereof.

Optionally, short linkers can form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

In one speciﬁc embodiment, the nucleotide sequence of the ulatory protein’s transmembrane domain is set forth in SEQ ID NO. 4: TTCTGGGTGTTGGTCGTAGTGGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCAC CGTGGCTTTTATAATCTTCTGGGTT In one embodiment, the polynucleotide encoding a transmembrane domain within a ulatory domain comprises a nucleotide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequence of SEQ ID NO: 4.

The amino acid sequence ofthe ulatory protein’s transmembrane domain is set forth in SEQ ID NO. 5: FWVLVVVGGV LACYSLLVTV AFIIFWV.

In one particular embodiment, the transmembrane domain within a costimulatory domain comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 5.

In r ment, the transmembrane domain is derived from (i.e., comprises) CD8. In one ment, the nucleotide sequence of the CD8 extracellular domain and transmembrane domain is set forth in SEQ ID NO: 238 GCTGCAGCATTGAGCAACTCAATAATGTATTTTAGTCACTTTGTACCAGTGTTCTT GCCGGCTAAGCCTACTACCACACCCGCTCCACGGCCACCTACCCCAGCTCCTACC ATCGCTTCACAGCCTCTGTCCCTGCGCCCAGAGGCTTGCCGACCGGCCGCAGGGG GCGCTGTTCATACCAGAGGACTGGATTTCGCCTGCGATATCTATATCTGGGCACC CCTGGCCGGAACCTGCGGCGTACTCCTGCTGTCCCTGGTCATCACGCTCTATTGT AATCACAGGAAC.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley In some embodiments, the polynucleotide encoding a transmembrane domain within a costimulatory domain comprises a nucleotide ce at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequence of the CD8 transmembrane domain.

The amino acid sequence of the CD8 extracellular domain and transmembrane domain is set forth in SEQ ID NO. 239 AAALSNSIMYFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN.

In one particular embodiment, the embrane domain within a costimulatory domain comprises an amino acid ce at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of the CD8 transmembrane domain.

Intracellular (signaling) : The intracellular (signaling) domain of the engineered T cells of the invention can provide ing to an activating domain, which then activates at least one of the normal effector functions of the immune cell. Effector on of a T cell, for example, can be cytolytic activity or helper activity including the ion of cytokines.

In certain embodiments, le intracellular signaling domain e (i.e., comprise), but are not limited to 4-lBB/CDl37, ting NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CDl la, CDl lb, CDl lc, CDl ld, CDS, CEACAMl, CRT AM, cytokine receptor, DAP-lO, DNAMl (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), 1A4, ICAM-l, , Ig alpha (CD79a), IL-2R beta, IL- 2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-l, LFA-l, ligand that speciﬁcally binds with CD83,, LIGHT, LIGHT, LTBR, Ly9 (CD229), Ly108), lymphocyte function-associated antigen-l (LFA-l, CDl-la/CDl8), MHC class Diolecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRFl), OX-40, ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley PAG/Cbp, programmed death-l (PD-l), PSGLl, SELPLG (CD162), Signaling Lymphocytic Activation les (SLAM proteins), SLAM (SLAMFl, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 , SLAMF7, SLP-76, INF receptor proteins, TNFR2, INFSF14, a Toll ligand receptor, TRANCE/RANKL, VLAl, or VLA-6, or a fragment, truncation, or a combination f.

An example of a nucleotide sequence encoding the intracellular signaling domain is set forth in SEQ ID NO. 6: AGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCCACGC GGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGATTTCG CTGCCTATCGGAGC In one embodiment, the polynucleotide encoding an intracellular signaling domain within a costimulatory domain comprises a nucleotide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequence of SEQ ID NO: An example of an intracellular signaling domain is set forth in SEQ ID NO. 7: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.

In one particular embodiment, the intracellular signaling domain within a costimulatory domain comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the ulatory domain ses, consists essentially of, or consists of the extracellular THD, and the costimulatory proteins’ s transmembrane and intracellular domains. For e, a nucleotide sequence encoding a costimulatory domain is set forth in SEQ ID NO. 240: CTTGATAATGAAAAGTCAAACGGAACAATCATTCACGTGAAGGGCAAGCACCTC TGTCCGTCACCCTTGTTCCCTGGTCCATCCAAGCCATTCTGGGTGTTGGTCGTAGT GGGTGGAGTCCTCGCTTGTTACTCTCTGCTCGTCACCGTGGCTTTTATAATCTTCT GGGTTAGATCCAAAAGAAGCCGCCTGCTCCATAGCGATTACATGAATATGACTCC ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley ed set by eadley ACGCCGCCCTGGCCCCACAAGGAAACACTACCAGCCTTACGCACCACCTAGAGA TTTCGCTGCCTATCGGAGC In some embodiments, the polynucleotide encoding a costimulatory domain comprises, consists essentially of, or consists of a nucleotide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide sequence of SEQ ID NO: 240, wherein the ulatory domain does not comprises amino acids 1 to 122 of SEQ ID NO: 1, amino acids 1 to 121 of SEQ ID NO: 1, amino acids 1 to 120 of SEQ ID NO: 1, amino acids 1 to 119 of SEQ ID NO: 1, amino acids 1 to 118 of SEQ ID NO: 1, or amino acids 1 to 118 ofSEQ IDNO: 1.

The corresponding amino acid sequence ofthe costimulatory domain is set forth in SEQ ID NO. 241: LDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVR SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS In some embodiments, the costimulatory domain comprises, consists essentially of, or consists of a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 241, wherein the costimulatory domain does not comprises amino acids 1 to 122 of SEQ ID NO: 1, amino acids 1 to 121 of SEQ ID NO: 1, amino acids 1 to 120 of SEQ ID NO: 1, amino acids 1 to 119 of SEQ ID NO: 1, amino acids 1 to 118 of SEQ ID NO: 1, or amino acids 1 to 118 of SEQ ID NO: 1.

LB. Activating Domain. id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178" id="p-178"

[0178] CD3 is an element of the T cell receptor on native T cells, and has been shown to be an important intracellular activating element in CARs. In one embodiment, the CD3 is CD3 zeta, the nucleotide sequence of which is set forth in SEQ ID NO. 8: AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAAC CAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAACCC CCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCTATTC TGA/ﬁ‘AGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGGTTTGT [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACATGCAAG CCCTGCCACCTAGG In some embodiments, the polynucleotide ng an activating domain comprises a nucleotide sequence at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the nucleotide ce of SEQ ID NO: 8.

The corresponding amino acid of intracellular CD3 zeta is set forth in SEQ ID NO. 9: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP In some embodiments, the activating domain comprises a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence of SEQ ID NO: 9.

In some embodiments, the activating domain comprises an amino acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% cal to the amino acid ce of: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PR (SEQ ID NO: 251).

I. C. Antigen Binding Molecules id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183" id="p-183"

[0183] CARs can be ered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted n. In some embodiments, the antigen binding molecule is an antibody fragment thereof, e.g., one or more single chain antibody fragment ("scFv"). An scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. See US.

Patent Nos. 7,741,465, and 6,319,494 as well as Eshhar el al., Cancer Immunol Immunotherapy (1997) 45: 6. An scFv retains the parent antibody's ability to speciﬁcally interact with targetnigen. scFvs are useful in chimeric antigen receptors because they can be engineered [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley to be expressed as part of a single chain along with the other CAR components. Id. See also Krause el al., J. Exp. Med, Volume 188, No. 4, 1998 26), Finney el al., Journal of Immunology, 1998, 161: 2791—2797. It will be appreciated that the antigen binding molecule is typically contained within the extracellular portion of the CAR such that it is capable of recognizing and binding to the antigen of interest. Bispeciﬁc and multispeciﬁc CARs are contemplated within the scope of the invention, with speciﬁcity to more than one target of interest.

In some embodiments, the polynucleotide encodes a CAR or a TCR comprising a THD of the present invention and an antigen binding molecule that speciﬁcally binds to a target antigen. In some embodiments, the target antigen is a tumor antigen. In some embodiments, the antigen is ed from a associated e antigen, such as 5T4, alphafetoprotein (AFP), B7-1 (CD80), B7-2 , BCMA, B-human chorionic gonadotropin, CA-125, carcinoembryonic antigen (CEA), carcinoembryonic antigen (CEA), CD123, CD133, CD138, CD19, CD20, CD22, CD23, CD24, CD25, CD30, CD33, CD34, CD4, CD40, CD44, CD56, CD8, CLL-l, c-Met, CMV-speciﬁc n, CSPG4, CTLA-4, disialoganglioside GD2, ductal-epithelial mucine, EBV-speciﬁc antigen, EGFR variant III (EGFRvIII), ELFZM, endoglin, ephrin B2, epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule ), lial tumor antigen, ErbB2 neu), ﬁbroblast associated protein (fap), FLT3, folate binding protein, GD2, GD3, glioma-associated antigen, glycosphingolipids, gp3 6, HBV- speciﬁc antigen, eciﬁc antigen, HERl-HERZ, HERZ- HER3 in combination, HERV-K, high molecular weight-melanoma associated antigen (HMW- MAA), HIV-1 envelope glycoprotein gp41, HPV-speciﬁc antigen, human telomerase reverse riptase, IGFI receptor, IGF-II, IL-l 1Ralpha, IL-13R-a2, Inﬂuenza Virus-speciﬁc antigen, CD38, insulin growth factor (IGFl)—l, intestinal carboxyl esterase, kappa chain, LAGA-la, lambda chain, Lassa Virus-speciﬁc antigen, lectin-reactive AFP, lineage-speciﬁc or tissue speciﬁc antigen such as CD3, MAGE, MAGE-Al, major ompatibility complex (MHC) molecule, major histocompatibility complex (MHC) molecule presenting a tumor- speciﬁc peptide epitope, M-CSF, ma-associated antigen, mesothelin, mesothelin, MN- CA IX, MUC-l, mut hsp70-2, mutated p53, mutated p53, d ras, neutrophil elastase, NKGZD, Nkp30, NY-ESO-l, p53, PAP, prostase, prostase speciﬁc antigen (PSA), prostate- carcinoma tumor antigen-1 (PCTA-l), prostate-speciﬁc antigen, prostein, PSMA, RAGE-1, RORl, RUl, RU2 (AS), surface adhesion le, surviving and telomerase, TAG-72, the extra Dain A (EDA) and extra domain B (EDB) nectin and the Al domain oftenascin- [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley C (TnC Al) thyroglobulin, tumor l antigens, vascular endothelial , growth factor receptor-2 (VEGFRZ), Virus-speciﬁc surface antigen such as an HIV-specific antigen (such as HIV gp120), as well as any derivate or variant of these surface markers. In certain embodiments, the antigen binding molecule specifically binds to BCMA. In other embodiments, the antigen g le speciﬁcally binds to CLL-l. In other embodiments, the antigen binding molecule specif1cally binds to FLT3.

In some embodiments, the antigen binding molecule specifically binds BCMA.

In certain embodiments, the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid sequence selected from SEQ ID NOs: 13-20, (b) a VH CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 21-28, (c) a VH CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 29-36, (d) a VL CDRl comprising an amino acid sequence selected from SEQ ID NOs: 37-44, (e) a VL CDR2 sing an amino acid sequence selected from SEQ ID NOs: 45-52, and/or (f) a VL CDR3 comprising an amino acid sequence ed from SEQ ID NOs: 53-60. id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186" id="p-186"

[0186] In one embodiment, the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 13, (b) a VH CDR2 sing an amino acid ce of SEQ ID NO: 21, (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 29, (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 37, (e) a VL CDR2 sing an amino acid sequence of SEQ ID NO: 45, and/or (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 53.

In another ment, the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 14, (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 22, (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 30, (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 38, (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 46, and/or (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 54.

In r embodiment, the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 15, (b) a VH CDR2 comprising an amino acid ce of SEQ ID NO: 23, (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 31, (d) a VL CDRl sing an amino acid sequence of SEQ ID NO: 39, (e) a VL CDR2 sing an amino acid sequence of SEQ ID NO: 47, and/or (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 55.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley In another embodiment, the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 16; (b) a VH CDR2 sing an amino acid sequence of SEQ ID NO: 24; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 32; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 40; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 48; and/or (f) a VL CDR3 sing an amino acid sequence of SEQ ID NO: 56.

In another embodiment; the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 17; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 25; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 33; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 41; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 49; and/or (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 57.

In another embodiment; the antigen binding molecule comprises (a) a VH CDRl sing an amino acid of SEQ ID NO: 18; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 26; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 34; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 42; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 50; and/or (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 58.

In another embodiment; the n binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 19; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 27; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 35; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 43; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 51; and/or (f) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 59. id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193" id="p-193"

[0193] In another embodiment; the n g molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 20; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 28; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 36; (d) a VL CDRl sing an amino acid sequence of SEQ ID NO: 44; (e) a VL CDR2 comprising an amino acid ce of SEQ ID NO: 52; and/or (f) a VL CDR3 sing an amino acid sequence of SEQ ID NO: 60.

In certain embodiments; the antigen binding molecule comprises a VH sing an amino acid sequence selected from the group consisting of SEQ ID NOs: 77-84 and an comprising an amino acid sequence selected from the group consisting of SEQ ID [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley NOS: 85-92. In one embodiment, the antigen binding molecule comprises a VH comprising an amino acid sequence of SEQ ID NO: 77 and a VL comprising an amino acid sequence of SEQ ID NO: 85. In another embodiment, the antigen binding molecule ses a VH comprising an amino acid ce of SEQ ID NO: 78 and a VL comprising an amino acid sequence of SEQ ID NO: 86. In another embodiment, the antigen binding molecule comprises a VH sing an amino acid sequence of SEQ ID NO: 79 and a VL comprising an amino acid sequence of SEQ ID NO: 87. In another embodiment, the antigen binding molecule comprises a VH comprising an amino acid ce of SEQ ID NO: 80 and a VL comprising an amino acid sequence of SEQ ID NO: 88. In r embodiment, the antigen binding molecule comprises a VH comprising an amino acid ce of SEQ ID NO: 81 and a VL comprising an amino acid ce of SEQ ID NO: 89. In another embodiment, the antigen binding molecule comprises a VH comprising an amino acid sequence of SEQ ID NO: 82 and a VL comprising an amino acid sequence of SEQ ID NO: 90. In another embodiment, the antigen binding molecule comprises a VH comprising an amino acid sequence of SEQ ID NO: 83 and a VL comprising an amino acid sequence of SEQ ID NO: 91. In another embodiment, the antigen binding molecule comprises a VH comprising an amino acid ce of SEQ ID NO: 84 and a VL comprising an amino acid ce of SEQ ID NO: 92.

In one particular ment, the polynucleotide of the present invention comprises a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence ed form the group consisting of SEQ ID NOs: 61-68. In another embodiment, the polynucleotide of the t invention comprises a tide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence selected form the group consisting of SEQ ID NOs: 69-76.

Other known anti-BCMA antibodies or antigen binding molecules thereof can be used as antigen binding les of a CAR or TCR comprising a THD of the present invention. Non-limiting examples of such BCMA antibodies or antigen binding molecule thereof include antibodies or antigen binding molecules described in W02015158671A1, published October 22, 2015 and WO2016014565A2, published January 28, 2016.

In some embodiments, the antigen binding le speciﬁcally binds CLL-l.

In cerD embodiments, the antigen binding molecule comprises (a) a VH CDRl comprising [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley an amino acid sequence selected from SEQ ID NOS: 93-96; (b) a VH CDR2 sing an amino acid sequence selected from SEQ ID NOs: 97-100; (c) a VH CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 101-104; (d) a VL CDRl comprising an amino acid sequence selected from SEQ ID NOs: 105-108; (e) a VL CDR2 comprising an amino acid sequence selected from SEQ ID NOs: 109-112; and/or (1) a VL CDR3 comprising an amino acid sequence selected from SEQ ID NOs: 113-116.

In one embodiment, the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 93; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 97; (c) a VH CDR3 sing an amino acid sequence of SEQ ID NO: 101; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 105; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 109; and/or (1) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 113.

In one embodiment; the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 94; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 98; (c) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 102; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 106; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 110; and/or (1) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 114.

In one ment; the antigen binding le comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 95; (b) a VH CDR2 sing an amino acid sequence of SEQ ID NO: 99; (c) a VH CDR3 comprising an amino acid ce of SEQ ID NO: 103; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 107; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 111; and/or (1) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 115. id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201" id="p-201"

[0201] In one ment; the antigen binding molecule comprises (a) a VH CDRl comprising an amino acid of SEQ ID NO: 96; (b) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 100; (c) a VH CDR3 comprising an amino acid ce of SEQ ID NO: 104; (d) a VL CDRl comprising an amino acid sequence of SEQ ID NO: 108; (e) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 112; and/or (1) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 116.

In n embodiments; the antigen binding molecule comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 125- 128 an VL sing an amino acid sequence selected from the group consisting of SEQ ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ID NOs: 129-132. In one embodiment, the n binding molecule comprises a VH comprising an amino acid ce of SEQ ID NO: 125 and a VL comprising an amino acid sequence of SEQ ID NO: 129. In another embodiment, the antigen binding molecule comprises a VH sing an amino acid sequence of SEQ ID NO: 126 and a VL comprising an amino acid sequence of SEQ ID NO: 130. In r embodiment, the antigen binding molecule comprises a VH comprising an amino acid sequence of SEQ ID NO: 127 and a VL comprising an amino acid sequence of SEQ ID NO: 131. In another embodiment, the antigen binding molecule comprises a VH comprising an amino acid sequence of SEQ ID NO: 128 and a VL sing an amino acid sequence of SEQ ID NO: 132. id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203" id="p-203"

[0203] In one particular ment, the polynucleotide of the present invention comprises a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence selected form the group consisting of SEQ ID NOs: 117-120. In another embodiment, the polynucleotide of the present ion ses a nucleotide sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to a nucleotide sequence selected form the group consisting of SEQ ID NOs: 121- id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204" id="p-204"

[0204] Other examples of anti-CLL-l antibodies or antigen binding molecules thereof include antibodies or antigen binding molecules described in W02016014535, published January 28, 2016, and US 2016/0051651 A1, published Feb. 25, 2016.

The antigen binding molecule d by the polynucleotide of the present invention can be single d or double chained. In some embodiments, the antigen binding molecule is single chained. In certain embodiments, the antigen binding le is selected from the group consisting of an scFv, an Fab, an Fab', an Fv, an F(ab')2, a dAb, and any combination thereof. In one particular embodiment, the n binding molecule comprises an scFv.

In certain embodiments, the antigen binding molecule comprises a single chain, wherein the heavy chain variable region and the light chain variable region are connected by a linker. In some embodiments, the VH is located at the N terminus of the linker and the VL is located at the C terminus of the linker. In other embodiments, the VL is located at the N termirDof the linker and the VH is located at the C us of the linker. In some [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley embodiments, the linker comprises at least about 5, at least about 8, at least about 10, at least about 13, at least about 15, at least about 18, at least about 20, at least about 25, at least about , at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100 amino acids. In some embodiments, the linker ses at least about 18 amino acids. In n ments, the linker comprises an amino acid sequence that is at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 12) or the amino acid sequence GGGSGGGGS (SEQ ID NO: 237). In one embodiment, the linker is a w linker. In certain embodiments, the binding molecule comprises a single chain, wherein the heavy chain variable region and the light chain variable region are connected by a linker, wherein the linker comprises the amino acid sequence of SEQ ID NO: 12.

In some embodiments, the antigen binding molecule binds a target antigen (e.g., human BCMA, human FLT3, or human CLL-l) with a KD of less than 1 X 10'6 M, less than 1 X 10'7 M, less than 1 X 10'8 M, or less than 1 X 10'9 M. In one particular embodiment, the antigen binding molecule binds a target antigen (e.g., human BCMA, human FLT3, or human CLL-l) with a K13 of less than 1 X 10'7 M. In another embodiment, the n binding molecule binds a target antigen (e.g., human BCMA, human FLT3, or human CLL-l) with a KD of less than 1 X 10'8 M. In some embodiments, the antigen binding molecule binds a target antigen (e.g., human BCMA, human FLT3, or human CLL-l) with a KD of about 1 X 10'7 M, about 2 X 10'7 M, about 3 x 10'7 M, about 4 x 10'7 M, about 5 x 10'7 M, about 6 x 10'7 M, about 7 x 10'7 M, about 8 X 10'7 M, about 9 X 10'7 M, about 1 X 10'8 M, about 2 X 10'8 M, about 3 X 10'8 M, about 4 x 10'8 M, about 5 x 10'8 M, about 6 x 10'8 M, about 7 x 10'8 M, about 8 x 10'8 M, about 9 x 10'8 M, about 1 X 10'9 M, about 2 X 10'9 M, about 3 X 10'9 M, about 4 X 10'9 M, about 5 X 10'9 M, about 6 x 10'9 M, about 7 x 10'9 M, about 8 x 10'9 M, about 9 x 10'9 M, about 1 x 10'10 M, or about 5 X 10'10 M. In certain embodiments, the K1) is calculated as the quotient of koff/kon, and the kon and koff are determined using a monovalent antibody, such as a Fab fragment, as measured by, e. g., BIAcore® surface plasmon resonance technology. In other embodiments, the K1) is calculated as the quotient of koff/kon, and the km and koff are determined using a nt antibody, such as a Fab fragment, as measured by, e. g., e® surface plasmon resonance technology.

[Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley In some embodiments, the antigen binding molecule binds a target antigen (e.g., human BCMA, human FLT3, or human CLL-l) with an association rate (ken) of less than 1 X '4 M'1 s'l, less than 2 X 10'4 M'1 s'l, less than 3 X 10'4 M'1 s'l, less than 4 X 10'4 M'1 s'l, less than 5 X 10'4 M'1 s'l, less than 6 X 10'4 M'1 s'l, less than 7 X 10'4 M'1 s'l, less than 8 X 10'4 M'1 s'l, less than 9 X 10'4 M'1 s'l, less than 1 X 10'5 M'1 s'l, less than 2 X 10'5 M'1 s'l, less than 3 X '5 M'1 s'l, less than 4 X 10'5 M'1 s'l, less than 5 X 10'5 M'1 s'l, less than 6 X 10'5 M'1 s'l, less than 7 X 10'5 M'1 s'l, less than 8 X 10'5 M'1 s'l, less than 9 X 10'5 M'1 s'l, less than 1 X 10'6 M'1 s'l, less than 2 X 10'6 M'1 s'l, less than 3 X 10'6 M'1 s'l, less than 4 X 10'6 M'1 s'l, less than 5 X '6 M'1 s'l, less than 6 X 10'6 M'1 s'l, less than 7 X 10'6 M'1 s'l, less than 8 X 10'6 M'1 s'l, less than 9 X 10'6 M'1 s'l, or less than 1 X 10'7 M'1 s'l. In certain embodiments, the kon is determined using a monovalent antibody, such as a Fab fragment, as measured by, e.g., BIAcore® surface plasmon resonance technology. In other embodiments, the kon is determined using a bivalent antibody as measured by, e.g., BIAcore® surface n resonance technology.

In some embodiments, the antigen binding molecule binds a target antigen (e.g., human BCMA, human FLT3, or human CLL-l) with an dissociation rate (koff) of less than 1 X '2 s'l, less than 2 X 10'2 s'l, less than 3 X 10'2 s'l, less than 4 X 10'2 s'l, less than 5 X 10'2 s'l, less than 6 X 10'2 s'l, less than 7 X 10'2 s'l, less than 8 X 10'2 s'l, less than 9 X 10'2 s'l, less than 1 X 10'3 s'l, less than 2 X 10'3 s'l, less than 3 X 10'3 s'l, less than 4 X 10'3 s'l, less than 5 X 10'3 s' 1, less than 6 X 10'3 s'l, less than 7 X 10'3 s'l, less than 8 X 10'3 s'l, less than 9 X 10'3 s'l, less than 1 X 10'4 s'l, less than 2 X 10'4 s'l, less than 3 X 10'4 s'l, less than 4 X 10'4 s'l, less than 5 X 10'4 s' 1, less than 6 X 10'4 s'l, less than 7 X 10'4 s'l, less than 8 X 10'4 s'l, less than 9 X 10'4 s'l, less than 1 X 10'4 s'l, or less than 5 X 10'4 s'l~ In certain ments, the koff is determined using a monovalent dy, such as a Fab fragment, as measured by, e.g., BIAcore® surface plasmon resonance technology. In other embodiments, the koff is determined using a bivalent antibody as measured by, e.g., e® surface plasmon nce technology.

In some embodiments, the polynucleotide encodes a TCR, wherein the TCR further comprises a fourth complementarity determining region (CDR4). In certain embodiments, the polynucleotide encodes a TCR, wherein the TCR further comprises a nt region. In some embodiments, the nt region is selected from a constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley LB. Switch Domain It will be appreciated that adverse events may be minimized by transducing the immune cells (containing one or more CARs or TCRs) with a suicide gene. It may also be desired to incorporate an inducible "on" or "accelerator" switch into the immune cells. Suitable techniques e use of inducible caspase-9 (U.S. Appl. 2011/0286980) or a thymidine kinase, before, after or at the same time, as the cells are transduced with the CAR construct of the present ion. Additional methods for introducing suicide genes and/or "on" switches include TALENS, zinc , RNAi, siRNA, shRNA, nse technology, and other techniques known in the art. id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212" id="p-212"

[0212] In ance with the invention, additional on-off or other types of control switch techniques may be incorporated herein. These techniques may employ the use of dimerization domains and optional activators of such domain dimerization. These techniques include, e.g., those described by Wu et al., Science 2014 350 (6258) ing FKBP/Rapalog dimerization systems in certain cells, the contents ofwhich are incorporated by nce herein in their entirety. Additional dimerization technology is described in, e.g., Fegan et al. Chem.

Rev. 2010, 110, 3315—3336 as well as US. Patent Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the contents of which are also incorporated by reference herein in their entirety.

Additional dimerization pairs may include porine-A/cyclophilin, or, en/estrogen receptor (optionally using tamoxifen), glucocorticoids/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. Further examples of dimerization technology can be found in e.g., WO 27261, 2014/0286987, US 2015/0266973, US 2016/0046700, US. Patent No. 8,486,693, US 2014/0171649, and US 2012/0130076, the contents of which are further incorporated by reference herein in their entirety.

I.E. Leader e In some embodiments, the polynucleotide of the present ion s a CAR or a TCR can further comprises a leader peptide (also referred to herein as a "signal peptide"). In certain embodiments, the leader peptide comprises an amino acid ce that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 11). In some Dodiments, the leader peptide comprises the amino acid sequence of SEQ ID NO: 11. ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley In some embodiments, the polynucleotide of the present invention encodes a CAR or a TCR, wherein the CAR or the TCR comprises a leader peptide (P), an n binding le (B), a ulatory protein’s extracellular domain (E), a transmembrane domain (T), a costimulatory region (C), and an activation domain (A), wherein the CAR is conﬁgured according to the following: P-B-E-T-C-A. In some embodiments, the antigen binding molecule ses a VH and a VL, wherein the CAR is conﬁgured according to the ing: P-VH- VL-E-T-C-A or P-VL-VH-E-T-C-A. In some ments, the VH and the VL are connected by a linker (L), wherein the CAR is conﬁgured according to the following, from N—terminus to C-terminus: P-VH-L-VL-E-T-C-A or P-VH-L-VL-E-T-C-A. id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215" id="p-215"

[0215] In some embodiments, the polynucleotide of the present invention encodes a CAR, wherein the CAR comprises an amino acid sequence at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from Table 2. In certain embodiments, the polynucleotide of the present invention encodes a CAR, wherein the CAR comprises an amino acid sequence ed from Table 2.

Table 2. Example CAR ces CAR Amino ACl'd Nucleotlde Sequence.

Construct Sequence ATGGCAC"CCCCGTAACTGCTCTGCTGCr1 A _ GCCGTTGGCATTGCTCCTGCACGCCGCAC HAAR?QVF H .

GCCCGCAGGTGACCCTCAAAGAGTCTGGA VKP"?".F CCCG"GCTCGTAAAACCTACGGAGACCCr1 SLINARMGV GACAC"CACCTGCACAGTCTCCGGCF GKAIﬁWIA GCCTCATCAATGCCAGG r SYRTSIKS TGGATCAGGCAACCGCCCG SKSQVV. 1NMDPV GGAATGGCTCGCACATATTF r1ATYYCARI?GYGG\IG CTGAAAAAAGCTATCGGACF DYHYYGMDVWGQGTTV AGTCGGCTCACGATTAGTAAGGACACATC 1VSSGGGGSGGGGSGG CAAGAGCCAAG"GGTGCTTACGATGACF DIQMTQSPSSJSA ACA"GGACCCTGTGGATACTGCAACCTAr1 _ DRVTITCRASQGI TAC"G"GCTCGAATCCCTGGTTATGGCGG JGWYQQKPGKA?K 1GGGGACTACCACTACTACGGTA."GG JIYASSTJQSGV?SR 1CTGGGGCCAAGGGACCACGGTTACr1 SGSGSG"?FT.TISS 1CAAGCGGAGGGGGAGGGAG"GGGGG EDFA"YYC. {VN 1GGCGGATCTGGCGGAGGAGGCAGCGA"A _ 1FGQGTKVI RA 1CCAGA"GACGCAG"CCCCTAG"""CAC""'1 EVMYP?PY DV?K "CCCTGGGGGATCGGGTTACCAr1 HVKGKinC?S 1ACATGCCGCGCGTCACAGGGTATCCGGA PJF?GPSK?FWV4VVV ATGA"CTGGGATGGTACCAGCAGAAGCCG GGVIACYS.LV"VAFI GGAAAGGC"CCTAAGCGCCTCATCTACGC IFWVRSKRSR..{SDY CAGC"CCACCCTGCAGAGTGGAGTGCCCr1 MNMTPRRPGPTRKHYQ CCCGGTTT"CAGGCAGTGGCTCCGGTACG PYA?PRDFAAYRSRVK AC"CTTACAATTAGCAGCCTGCA FSRSADAPAYQQGQVQ GCCAGAAGATTTTGCAACTTACTACTGTr1 LYN?LNLGRRﬁﬁYDVL [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley ation] Anne.Headley Unmarked set by Anne.Headley TGCAGCATAATAATTTCCCCTGGACCTTr1 DKRRG EWGGKPRR GGTCAGGGCACCAAGG"GGAGATCAAAAG LYVﬁLQKDKM AGCAGCCGCCATCGAAGTAATGTATCCCC AﬂAYS- WKGﬁRRRG CCCCGTACCTTGACAATGAGAAGTCAAAr1 KGHDG ISTATKD GGAACCATTATCCA"1 G"1I'"AAGGGCAAACA TYDA .P ?R CC"C"GCCCTTCTCCACTGTTCCCTGGCC CmAGmAAGCCGmmmmGGGmGCmGGmGGmA GmCGGmGGGGmGCmGGCmmemACr TCTCGTGACCGTCGCCTTTATAAF GGGTCAGATCCAAAAGAAGCCGCCF CATAGCGA"1TACA"1GAATATGAC" CCGCCCTGGCCCCACAAGGAAACA AGCCTI'"ACGCACCACCTAGAGA"1T " GCCTA"CGGAGCCGAGTGAAATTTF ATCAGCTGATGCTCCCGCCTAF GACAGAATCAAC"1TTACAA"1GAGC" C"GGGTCGCAGAGAAGAGTACGACGF GGACAAACGCCGGGGCCGAGATCCr1 TGGGGGGGAAGCCGAGAAGGAAGAAF " CAAGAAGGCCTGTACAACGAGCTTCAAAA AGACAAAATGGCTGAGGCGTACTCTGAGA r1CGGCATGAAGGGCGAGCGGAGACGAGGC AAGGGTCACGATGGCTTGTATCAGGGCCT GAGTACAGCCACAAAGGACACCTA"1GACG CCC"CCACATGCAGGCACTGCCCCCACGC ATGGCAC"CCCCGTAACTGCTCTGCTGCr1 MALPVTA _ GCCGTTGGCATTGCTCCTGCACGCCGCAC HAAR?QVF H .

GCCCGCAAGTTACTTTGAAGGAGTCTGGA VKPT7T.F CC"GTAC"GGTGAAGCCAACCGAGACACr1 SLINARMGV GACACTCACGTGTACAGTGAGTGGTTTT"1 GKAIﬁWIA TCAACGCAAGGATGGGCGTCAGC SYRTSIKS r1GGA"CAGGCAACCCCCTGGCAAGGCTCr1 SKSQVV. 1NMDPV GGAA"GGCTCGCTCACATATTCAGCAATG r1ATYYCARI?GYGG\IG CCGAAAAAAGCTACCGGACAAGCCTGAAA DYHYYGMDVWGQGTTV r1CCCGCCTGACTATTTCCAAGGACACTTC 1VSSGGGGSGGGGSGG CTCAGGTGGTGCTGACCATGACCA DIQMTQSPSSJSA ACATGGACCCGGTGGACACCGCCACCTAT _ DRVTITCRASQGI r1AC"GCGCAAGAA"CCCTGGGTATGGTGG JGWYQQKPGKAPK GAA"GG"GACTACCATTATTATGGGATGG _IYASSTJQSGV?SR A"G"GTGGGGGCAAGGCACAACCGTAACG GSGSGTTFTITISS G"C"CAAGCGGTGGGGGAGGCTCAGGGGG YYC4Q{NN CGGAGGCTCCGGAGGTGGCGGCTCCGACA _WTFGQGTKVEIKRA ""1 CAGA"1GACCCAAAGCCCGTCCAGCCTG SVG" I I {VK r1CCGCCAGCCTGGGAGATAGAGTGACAAT .C?SP.F?G?SKP CACGTGTAGAGCTTCCCAAGGG r1AAGAA VGGVJACYSL A"GA"CTCGGGTGGTATCAGCAGAAGCCC _ 1VAFIIFWV GGCAAAGCCCCCAAAAGGCTTATATATGC DYMNM"1 _ TAGTAGTACAC"GCAGTCTGGAGTTCCTT CCCGATTTTCAGGTAGCGGCTCCGGTACA GAGTTCACCCTCACGATAAGCTCACTCCA GCC"GAGGATTTCGCAACGTACTACTGCC TCCAGCACAACAATTTTCCCTGGACTTTC GGCCAGGGCACCAAGG"GGAGATCAAGAG GGCCGC"GCCCTTGATAATGAAAAGTCAA GHDGJYQG ACGGAACAATCATTCACGTGAAGGGCAAG _ TATKDTYDA4iMQA CACC"CTGTCCGTCACCCTTGTTCCCTGG __?R r1CCA"CCAAGCCATTCTGGGTGTTGGTCG r1AGTGGGTGGAGTCCTCGCTTGTTACTCT [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GTCACCGTGGCTT"TATAA"CF "AGA"CCAAAAGAAGCCGCCF TCCA"AGCGA"TACATGAA"ATGACF CC"GGCCCCACAAGGAAACA CCAGCC"1I'"ACGCACCACC""AGAGA"1"r CTGCCTA"CGGAGCCGAG"GAAA"""F AGA"CAGC"GATGC"CCCGCC"A"CAGCA GGGACAGAATCAAC"TTACAA"GAGC"GA ACC"GGG"CGCAGAGAAGAGTACGACGW'1 T"GGACAAACGCCGGGGCCGAGA"CC"GA G r1GGGGGGGAAGCCGAGAAGGAAGAA"C C"CAAGAAGGCCTG"ACAACGAGC"TCAA AAAGACAAAATGGC"GAGGCGTAC"C"GA GA"CGGCATGAAGGGCGAGCGGAGACGAG GCAAGGGTCACGATGGCTTG"ATCAGGGC C"1 GAGF1ACAGCCACAAAGGACACCTN"GA CGCCC"CCACA"GCAGGCAC"GCCCCCAC GCTAG A"GGCAC"CCCCGTAAC"GC"CTGCTGCr1 MAIPVTA. ?LALLL GCCGT"GGCA""GC"CC"GCACGCCGCAC HAARPQIQ ?SGGGV GCCCGCAGATCCAG"TGG"GGAA"CAGGG VQ?GRSLQJSCVASGF G"GG"GCAGCCGGG"AGGAGCCr1 TFKNYGMiWVRQAPGK GAGAC"G"CA"GCG"GGCGTC"GGC"TCA GLEWVAVIWYDGSNEY CA"TCAAGAAC"ACGGCA"GCAC"GGG"G YG RF"ISRDVS CGACAGGCCCCCGGAAAGGG""TGGAG"G KNVILYLQMNSJRADDr1 GG"CGCCG"GA"CTGG"ACGACGGA"C"A AVYYCARSGIAVAGAF ATGAG"AT"ACGGAGA"CCTG"GAAGGGA DYWGQG"4V"VSSGGG AGG"TCACCA"CTCCCGCGACAA"AGCAA GSGGGGSGGGGS?IV.

AAA"A"GC"C"ACCTGCAAA"GAAC"CAC "QSPDTISIS?G?KA" "CAGGGCGGAF 1ACGGCGG"C"ACTAT JSCRASQSVSSSFJAW r1GCGC"CGC"CAGGGAT"GC"GTGGCCGG YQQKPGQA?S .IYVA CGA"F 1GGGGACAGGG"ACCC SRRAAGIP "GG"GACAGF 1CAAGCGGAGGCGGCGGC GTDFTITISR r1CTGGCGGCGGCGGATC"GGCGGGGGGGG GMFYCQiYGR".

AAG"GAGA""G"G""GACACAG"C"CCCG PGTKVDIKQAAAI ATACCCTG"CAC"G"CACCCGGCGAGAAG YPP?Y.DN?KSVGF GCAACGC"GAG""GCAGAGCAAGCCAG"C HVKGK{.C?SP.F_ AGTC"CC"C""C"""TC"GGCC"GG"A"C V_ AGCAAAAACCAGG"CAGGCACCATC"C"C 4V"VAFIIFWV CTGA"""ACG""GCCAGCAGACGGGCGGC TGGCA""CCCGACAGG""CTC"GGAAGCG GA"C"GGGACCGA""TT""ACCC""GACAAW'1 AGCCGC""GGAGCCCGAAGAC"""GGTAr1 G""""AC"GCCAGCAC"ACGGAAGGACAC C"""CACATT"GGCCCGGGCACGAAAG"C 1AhAAAACGCGCAGCCGCCA""GAAGr1 AA"Gr1ACCCACCACC"1"Ar" 1G AAAAGTCCAA"GG"ACCA""AF 1C AAGGGAAAGCATC"C"GTCCAAGCCCTCr1 GT"CCCCGGCCCC"CCAAACCA""CTGGG r1GC"GG"GG"CGTCGGCGGAG""C"GGCC mGCmAmmCmCmGCmCGTGACmemGCATm CA"CA""""C"GGGTGAGA"CCAAAAGAA "GC"CCA"AGCGA""ACA"GAAr1 CCACGCCGCCC"GGCCCCACAAG GAAACAC"ACCAGCCT"ACGCACCACC"A GAGA"""CGCTGCCTA"CGGAGCCGAG"G AAA""""CTAGATCAGC"GATGC"CCCGC CTA"CAGCAGGGACAGAATCAAC"TTACA [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ATGAGC"GAACCTGGGTCGCAGAGAAGAG TACGACG"TT"GGACAAACGCCGGGGCCG AGATCC"GAG r1GGGGGGGAAGCCGAGAA GGAAGAAF 1CAAGAAGGCCTG"ACAAC GAGC"TCAAAAAGACAAAATGGC"GAGGC GTAC"C"GAGA"CGGCATGAAGGGCGAGC GGAGACGAGGCAAGGGTCACGATGGCTTG r1ATCAGGGCCTGAG"ACAGCCACAAAGGA "GACGCCC"CCACA"GCAGGCAC r1GCCCCCACGC"AG ATGGCAC"CCCCGTAAC"GC"CTGCTGCr1 MAIPVTA. ?LALLL GCCG""GGCA"r 1GCACGCCGCAC HAARPQIQ ?SGGGV GCCCGCAGA"F 1GGAG"CAGGr1 VQ?GRSLQJSCVASGF GG"GGCG"GG""CAGCCCGGACGGTCCCr1 TFKNYGMiWVRQAPGK GCGAC"C"C""GTG"GGCAAGCGGA"T"A VIWYDGSNEY CC""TAAGAAC"A"GGCA"GCAC"GGG"G YGDPVKGRF"ISRDVS AGGCAGGCCCC"GGAAAAGGACTGGAG"G KNVILYLQMNSJRADDr1 GG""GCTGF 1C"GG"ACGACGGG"CCA AVYYCARSGIAVAGAF A"F 1GGCGA"CCTG"GAAGGGA DYWGQG"4V"VSSGGG CGG"T"ACAA"CTCACGCGA"AACTCAAA GSGGGGSGGGGS?IV.

GAACA"GC"G"ACC"GCAAA"GAAC"CTC r1QSPDT.ST.S?G':‘.KAr1 r1GCGCGC"GA"GACAC"GCCG"GTA"TAr1 JSCRASQSVSSSE r1GCGC"CGGAGTGG"A"CGCCGTCGCAGG YQQKPGQAPSIIIYVA AGCAT"TGA""A""GGGGGCAAGGGACCC SRRAAGIPDRFSGSGS I'1CGTGACAG"GAGW'"CCGGAGGGGGAGG"1 GTDF"‘T.TI SR .4. .3 ﬁDJ: r1CTGG"GGAGGCGGCTCTGGTGGGGGAGG GMFYCQiYGR"?F"FG CAGCGAGA"CG""C"GACCCAG"CTCC"G PG"KVDIKRAAAIDN? TG"CAC"G"CCCC"GG"GAAAAG KSNG"IIHVKGK{4C? GCCACACTG"Cr GTCCCAGAG SPJF?G?SKPFWV_ CGTT"CCAG"F 1CC""GCA"GG"A"C VGGVIACYSL AACAAAAACCCGGGCAGG 1CCAAGCT"G IIFWVQSKRS CTGA"CTACG"GGCCAGCCGCCGGGCCGC YMNM"? QPG.

AGGCATCCC"GATAGG""""AGCGGT"C"G QPYA? .3 GGAGCGGGACGGACTTCACCT"GACAA"A KFSRSA TCACGGC"GGAACCCGAAGAC""CGGAAT QLYN? GT"""AT"GCCAGCAC"ACGGAAGAAC"C LDKR CA""CACCTTTGGCCCGGGAACGAAGG"A RKVPQ.

GACA"CAAGAGAGCAGCAGCCC"CGACAA MAﬁAYS CGAGAAA"CCAATGGAACCATTA"CCA"G GKGHDG r1GAAGGGGAAACATC"CTGCCC""CACCA DTYDA I'1"G""""CCC"GGACCCAGCAAGCC""""T""""G GG""C"GG"CG"GGTGGGGGGCG"CC"GG AC"CCC"CC"CGT"ACAG"CGCC mmCAmAAmCmemGGGTmAGAmCCAAAAG CC"GC"CCA"AGCGA"TACA"GA A"ATGAC"CCACGCCGCCC"GGCCCCACA AGGAAACAC"ACCAGCC""ACGCACCACC r1AGAGA"""CGCTGCCTA"CGGAGCCGAG r1GAAA""""C"AGA"CAGC"GATGC"CCC CAGCAGGGACAGAATCAAC"TTA CAA"GAGC"GAACC"GGG"CGCAGAGAAG AGTACGACG""T"GGACAAACGCCGGGGC CGAGA"CC"GAG r1GGGGGGGAAGCCGAG GAA"CC"CAAGAAGGCCTG"ACA ACGAGC"TCAAAAAGACAAAATGGC"GAG GCGTAC"C"GAGA"CGGCATGAAGGGCGA GCGGAGACGAGGCAAGGGTCACGATGGCT TGTATCAGGGCCTGAGTACAGCCACAAAG [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley GACACCTATGACGCCCTCCACA"GCAGGC ACTGCCCCCACGC"AG FS- ATGGCAC"CCCCG"AAC"GCTC"GC"GCr1 .PVTA 21495CARHX GCCGT"GGCA"TGCTCC"GCACGCCGCAC HAARPﬁVQ L GCCCGGAGGTGCAGCTG""GGAG"C"GGG VQ?GGSLQ GGAGGCT"GG"ACAGCC"GGGGGGTCCCr1 TFSSYAMSWVRQA.

GAGAC"C"CC"G"GCAGCCTC"GGA"TCA GLEWVSAISGSGGSTY CC"T"AGCAGC"ATGCCA"GAGCF YADSVKGRFTISRDVS CGCCAGGCF KNTLYLQW HDT GG"C"CAGCTAT"AGTGGF AVYYCARA GCACATAC"ACGCAGACTCCGF WGQGTMV"VS CGGT"CACCA"C"CCAGAGACAAF SGKPGSGﬁGS"KG.

GAACACGC"G"A"CTGCAAATGAACAGCC 4"QSPAT.SLS?GH r1GAGAGCCGAGGACACGGCGG"G"A.C""AC r1JSCRASQSVS r1GCGCAAGAGCCGAGA"GGGAGCCGTATT YQQKPGQAPR.

ATGGGG"CAGGGTACAA"GG"CA SNRA"GI?ARFSGS CCGTC"CCTCAGGG"C"ACATCCGGC"CC G"DF".TISS.._.

GGGAAGCCCGGAAG"GGCGAAGG"AG"AC AVYYC QISW.

AAAGGGGGAAA""G"G""GACACAG"CTC GG"KV CCCTG"C"""G"C"CCAGGGGAA KSNGF {VKGKi AGAGCCACCC"C"CC"GCAGGGCCAG"CA SPJF?G?SKPFWV_ GAGTG""AGCAGG"AC""AGCCTGG"ACC VGGVIACYSL AACC"GGCCAGGC"CCCAGGC"C IIFWV CT CA"1 C""AT GAT GCA"1 CCAACAGGGCCAC YMNM"1 .3 r1GGCA"CCCAGCCAGG""CAG"GGCAG"G QPYA?? GG"C"GGGACAGAC""CAC"C"CACCA"C KFSRSA AGCAGCC"AGAGCC""GAAGA""""GCAGr1 QLYN? I'"""A""""AC""G"CAGCAGAGAA"C""CCTGGC LDKR C"T"TGGCGGAGGGACC RKVPQﬁG GAGA"CAAACGGGCCGC"GCCC"F MAﬁAYS G"CAAACGGAACAA"CAF GKGHDG "GAAGGGCAAGCACC"C"GTCCGF DTYDA 1"GT"CCC"GG"CCA"CCAAGCCAF GG"1 G"1 r" GG"1 CGF1AGT GGG"1 GGAG" CmmGmmACmCmCmGCmCGmCACCGmG mmmAmAAmCmmCmGGGTmAGAmCCAAAAG AAGCCGCC"GC"CCA"AGCGA"TACA"GA A"ATGAC"CCACGCCGCCCTGGCCCCACA AGGAAACAC"ACCAGCC""ACGCACCACC r1AGAGA"""CGCTGCC"A"CGGAGCAGGG r1GAAG""""CCAGA"C"GCAGATGCACCA GCGTA"CAGCAGGGCCAGAACCAACTGTA r1AACGAGC"CAACC"GGGACGCAGGGAAG ACGTTTTGGACAAGCGCAGAGGA CC"GAGA"GGGTGGCAAACCAAG ACGAAAAAACCCCCAGGAGGG"CTC"A"A ATGAGC"GCAGAAGGATAAGA"GGC"GAA GCCTA""C"GAAA"AGGCA"GAAAGGAGA GCGGAGAAGGGGAAAAGGGCACGACGG"T TG"ACCAGGGACTCAGCAC"GC"ACGAAG GA"AC""A"GACGC"CTCCACA"GCAAGC CC"GCCACC"AGGF FS- ATGGCAC"CCCCG"AAC"GCTC"GC"GCT ?V"AL .PLAL. 21495CARLX GCCGT"GGCA""GC"CC"GCACGCCGCAC RP?IV 1QSPA".

H GCCCGGAAA""G"G""GACACAG"C"CCA H _ .SCRASQ GCCACCC"G"C""F 1C"CCAGGGGAAAG JAWYQQKPGQA AGCCACCC"C"CC"GCAGGGCCAGTCAGA PRIIIYDASNRA"GI? GTGTTAGCAGG"AC""AGCCTGGF GSGF 1LTI CAGAAACC"GGCCAGGC"CCCAGGCTCCT SSI. ﬁDtAVYYCQQR [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley TGATGCA"CCAACAGGGCCAC"G ISW?FTFGGGTKVI GCATCCCAGCCAGGTTCAG"GGCAG"GGG RGSTSGSGKPGSG r1C"GGGACAGAC""CACTC"CACCA""CAG TKGﬁVQILﬁSGGG CAGCC"AGAGCC"GAAGA"""TGCAG""F ?GGSLRJSCAASGFTF A""AC"G"CAGCAGAGAA"C"CCTGGCCF SSYAMSWVRQA?GKGL I""1CAC""T""TGGCGGAGGGACCAAGG" EWVSAISGSGGSTYYA GA"CAAACGGGGG"CTACA"CCGGCF DSVKGRFTISRDVSKV GGAAGCCCGGAAG"GGCGAAGG"AGF TLYLQMVSIRA?D"AV AAGGGGGAGGTGCAGCTG""GGAG"CF YYCAQAEMGAVFDIWG GGGAGGCT"GG"ACAGCC"GGGGGGF QG"MVTVSSAAAIDN? TGAGACTC"CC"G"GCAGCCTCF KSNG"IIHVKGK{4C? ACC"T"AGCAGC"ATGCCA"GAGCF SPJF?G?SKPFWV_ CCGCCAGGCF CYSL GGG"C"CAGCTAT"AGTGGF IIFWV TAC"ACGCAGACTCCGF YMNM"? CCGGT"CACCA"C"CCAGAGACAAF QPYA?? AGAACACGC"G"A"CTGCAAATGAACAGC KFSRSA C"GAGAGCCGAGGACACGGCGG"G"AC"A QLYN? C"GCGCAAGAGCCGAGA"GGGAGCCG"AT LDKR "CGACA"ATGGGG"CAGGG"ACAA"GGF RKVPQ.

ACCG"C"CCTCAGCCGC"GCCC"F MAﬁAYS "GAAAAGTCAAACGGAACAATCAF GKGHDG "GAAGGGCAAGCACC"C"G"CCGF DTYDA 1"GT"CCC"GG"CCA"CCAAGCCAF GG"1 G"1 r" GG"1 CGF1AGT GGG"1 GGAG" CmmGmmACmCmCmGCmCGmCACCGmGGCT mmmAmAAmCmmCmGGGTmAGAmCCAAAAG AAGCCGCC"GC"CCA"AGCGA"TACA"GA A"ATGAC"CCACGCCGCCCTGGCCCCACA AGGAAACAC"ACCAGCC""ACGCACCACC r1AGAGA"""CGCTGCC"A"CGGAGCAGGG r1GAAG""""CCAGA"C"GCAGATGCACCA GCGTA"CAGCAGGGCCAGAACCAACTGTA AGC"CAACC"GGGACGCAGGGAAG AGTATGACGTTTTGGACAAGCGCAGAGGA CGGGACCC"GAGA"GGGTGGCAAACCAAG ACGAAAAAACCCCCAGGAGGG"CTC"A"A ATGAGC"GCAGAAGGATAAGA"GGC"GAA GCCTA""C"GAAA"AGGCA"GAAAGGAGA GCGGAGAAGGGGAAAAGGGCACGACGG"T TG"ACCAGGGACTCAGCAC"GC"ACGAAG GA"AC""A"GACGC"CTCCACA"GCAAGC CC"GCCACC"AGG"AA PC- ATGGCAC"CCCCG"AAC"GCTC"GC"GCr1 MAIPVTA. 21497CARHX GGCA"TGC"CC"GCACGCCGCAC VQ L GCCCGCAGG"GCAGCTGG"GGAG"C"GGG VQ?GRSL% GGAGGCG"GG"CCAGCC"GGGAGGTCCCr1 TFSSYGMiWV GAGAC"C"CC"GTGCAGCGTC"GGA"TCA GLEWVAVISY CC"TCAG"AGC"A"GGCA"GCAC"GGG"C YADSVKGRF"ISR CGCCAGGC"CCAGGCAAGGGGCTGGAG"G KNT.YLQWNS.

GG"1 GGCAG"1TA""A"1 CGTA"1 GA"1 GGAAGF1A AVYYCAR DG"1Y ATAAATAC"A"GCAGACTCCG"GAAGGGC YFDIWGRGT CGATTCACCA"C"CCAGAGACAA"TCCAA STSGSGK.

GAACACGC"G"A"C"GCAAA"GAACAGCC CCGAGGACACGGCGG"G"AC"AC 1GCGCCAGAGACGG"AC"TA"CTAGG"GG 1CTCTGGTAC"TCGACT"ATGGGGGAGAG _ GTACCTTGG"CACCGTCF 1CAGGG"CT SGSGr1 "4K ACATCCGGC"CCGGGAAGCCCGGAAG"GG . ﬁDVGVYYCMQ [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by eadley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley CGAAGG"AG"ACAAAGGGGGA"AT"GTGA GLGLP4"FGGG"KVE TGAC"CAGTCTCCAC"C"CCC"GCCCG"C KRAAA.DN?KSNG"II ACCCC"GGAGAGCCGGCCTCCATC"CC"G HVKGK{.C?SP.F?G? CAGG"C"AG"CAGAGCC"CC"GCA"AG"A SKPFWVJVVVGGVJAC ATGG r1ACAACTA"""GGA""GGTACC"G YSLJV"VAFIIFWV CAGAAGCCAGGGCAG"C""CCACAGC"CCT KRS R .

""TGGG""C"AA"CGGGCC"CCG RPG?r1 GGGTCCC"GACAGG""CAG"GGCAG"GGA DFAAY TCAGGCACAGATr" r"I'"ACAC"1 GAAAA"1CAG CAGAG""GGAGGC""GAGGA"G""""GGGG"""""1 A"""AC""GCATGCAGGGAC"CGGCC"CCCr1 C"CAC"TTTGGCGGAGGGACCAAGG""GA ACGGGCCGC"GCCC""GA"AA"G AAAAG"1CAAACGGAACAA"1 CA"1 r" CACG"1 G AAGGGCAAGCACC"C""GTCCG""CACCC"""1 GT"CCC"GG"CCA"CCAAGCCA"TCTGGG 1Gm'"GG"CG"AGTGGG"GGAG"CC"CGCr1 1GmmACmCmCmGCmCGmCACCGmGGCTmm 1AhAA"C""C"GGGT"AGA"CCAAAAGAA GCCGCC"GC"CCA"AGCGA"TACA"GAAr1 ATGAC"CCACGCCGCCCTGGCCCCACAAG GAAACACF1ACCAGCC"1I'"ACGCACCACCF1A GAGA"""CGCTGCC"A"CGGAGCAGGG"G AAG""""CCAGA"C"GCAGATGCACCAGC GTA"CAGCAGGGCCAGAACCAACTGTA"A ACGAGC"CAACC"GGGACGCAGGGAAGAG TATGACGTTTTGGACAAGCGCAGAGGACG GGACCC"GAGA"GGGTGGCAAACCAAGAC GAAAAAACCCCCAGGAGGG"CTC"A"AAT GAGC"GCAGAAGGATAAGA"GGC"GAAGC CTA""C"GAAA"AGGCA"GAAAGGAGAGC GGAGAAGGGGAAAAGGGCACGACGG"TTG 1ACCAGGGACTCAGCAC"GC"ACGAAGGA ""A"GACGCTC"CCACA"GCAAGCCC 1AGG""AA PC- 1CCCCG"AAC"GC"CTGC"GCT .PVTALL _ 21497CARHX 1TGGCA""GC"CC"GCACGCCGCAC DIVWF L "AT"1 GT GAT GAC"1 CAGTCF1CCA ' 1GCCCG"CACCCC"GGAGAGCC 1CCATC"CC"GCAGG"C"AGTCAGA ?Q " GCA"AG""AATGG ACTA"1 ""GGTACC"GCAGAAGCCAGGGCA "CCACAGC"CCTGATC"A"F 1" 1CGGGCC"CCGGGGTCCC"GACAGG 1CAG"GGCAG"GGATCAGGCACAGATW'1 "ACAC"1 GAAAA"1CAGCAGAG"GGAGGC"G _ AGGA"GTTGGGG"T"A""AC"GCATGCAG SGFTFSSYGMHWVRQA GGAC"CGGCCTCCC"C"CAC"TTTGGCGG ?GKGLEWVAVISYDGS AGGGACCAAGG""GAGA"CAAACGGGGGr1 VKYYADSVKGRF"ISR CTACA"CCGGC"CCGGGAAGCCCGGAAGr1 DNSKNr1 GGCGAAGG"AG"ACAAAGGGGCAGGTGCA EDTAVY GCTGG"GGAG"C"GGGGGAGGCG"GG"CC GLWYFD GGAGG"CCCTGAGACTC"CC"Gr1 SSAAA.

GCAGCGTC"GGA"TCACC"TCAG"AGC"A {VKGK{ _ r1GGCA"GCAC"GGG"CCGCCAGGC"CCAG SKPFWV. JAC GCAAGGGGCTGGAG"GGG"GGCAG"TA"A VAFIIFWVRS r1CGTA"GA"GGAAG"AATAAATAC"AT"GC KRSR..{SDYMNM"PR AGACTCCG"GAAGGGCCGATTCACCAT"Cr1 RPGP"RKHYQPYAPPR CCAGAGACAATTCCAAGAACACGC"G"Ar1 DFAAYRSRVKFSRSAD [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by eadley C"GCAAA"GAACAGCC"GAGAGCCGAGGA APAYQQGQNQLYNH CACGGCGGTGTAC"AC"GCGCCAGAGACG .GRRﬁﬁYDVLDKR G"AC""A"CTAGG"GG"C"C"GGTACT"C DPEWGGKPRRKVPQ GACT"ATGGGGGAGAGG"ACC""GGTCAC .YV?.QKDKMAﬁAYS.

CG"C"CC"CAGCCGC"GCCC""GATAA"G IGWKGERRRGKGHDG AAAAG"CAAACGGAACAA"CA"F 1G YQGJSTATKDTYDA4i AAGCACC"C"G"CCGF 1" MQAJ??R GT"CCC"GG"CCA"CCAAGCCA"TCTGGG 1GW'"GG"CG"AGTGGG"GGAG"CC"CGCr1 1GmmACmCmCmGCmCGmCACCGmGGCTmm 1AfﬁAA"C""C"GGGT"AGA"CCAAAAGAA GCCGCC"GC"CCA"AGCGA"TACA"GAAr1 ATGAC"CCACGCCGCCCTGGCCCCACAAG GAAACAC"ACCAGCC"""ACGCACCACCF1A GAGA"""CGCTGCC"A"CGGAGCAGGG"G AAG""""CCAGA"C"GCAGATGCACCAGC GTA"CAGCAGGGCCAGAACCAACTGTA"A ACGAGC"CAACC"GGGACGCAGGGAAGAG TATGACGTTTTGGACAAGCGCAGAGGACG GGACCC"GAGA"GGGTGGCAAACCAAGAC ACCCCCAGGAGGG"CTC"A"AAT GAGC"GCAGAAGGATAAGA"GGC"GAAGC CTA""C"GAAA"AGGCA"GAAAGGAGAGC GGAGAAGGGGAAAAGGGCACGACGG"TTG 1ACCAGGGACTCAGCAC"GC"ACGAAGGA "GACGCTC"CCACA"GCAAGCCC 1GCCACC""AGG""AA AJ- ATGGCAC"CCCCGTAAC"GC"CTGC"GCr1 MAIPVTA..LPLA. 21508CARHX GCCG""GGCA""GC"CC"GCACGCCGCAC HAARPQVQIVQSGAH L GCCCGCAGGTGCAGCTGG"GCAG"C"GGG KK?GASVKVSCKASGY GC"GAGG"GAAGAAGCC"GGGGCCTCAGr1 r1F"SYYVI-IWVRQA?GQ GAAGG"T"CC"GCAAGGCATC"GGA"ACA GLEWMGIINPGGGS"S CC"TCACCAGC"ACTATA"GCAC"GGG"G YAQKFQGRVTM"RD"S CGACAGGCCCC"GGACAAGGGCT"GAG"G "S"VYWH.SSLQS?D" GA"GGGAA"AA"CAACCC"GG"GG"GG"A AVYYCA ESWPWDVWG GCACAAGC"ACGCACAGAAG""CCAGGGC QG"TV"VSSGS"SGSG AGAGTCACCA"GACCAGGGACACG"CCAC KPGSGﬁGS"KG.

GAGCACAG"C"ACA"GGAGC"GAGCAGCC QSPATISVS_ r1GAGATCTGAGGACACGGCGG"GTAC"AC SCRASQSVS r1GCGCCAGAGAGAG"TGGCCAATGGACGr1 QKPGQAPR ATGGGGCCAGGGAACAACTG"CACCG""Cr1 QA"GI_ CCTCAGGG"CTACA"CCGGC"CCGGGAAG ?F"LTISS CCCGGAAG"GGCGAAGGTAG"ACAAAGGG AAY.

AG"GA"GACGCAG"CTCCAGCCA KVEIKQAAA CCCTG"CTG"G"C"CCAGGGGAAAGAGCC G"IIHVKGK{ ACCC"CTCC"GCAGGGCCAG"CAGAG"Gr1 F?G?SKPFWV_ r1AGCAGCAAC""AGCCTGG"ACCAGCAGA VIACYSL.VF AACC"GGCCAGGC"CCCAGGC"CCTC I'"C VRSKRSQ . r1ATGGTGCA"CCACCAGGGCCAC"GG"Ar1 r D RPG?r1 CCCAGCCAGG""CAG"GGCAG"GGGTC"G _D DFAAY GGACAGAG"1 r" CAC"1 C"1 CACCA"1CAGCAGC TC"GAAGA""T"GCAGT""A§"A C"GTCAGCAG"ACGCCGCC"ACCC"AC"T T"GGCGGAGGGACCAAGGT"GAGA"CAAA CGGGCCGC"GCCCT"GA"AA"GAAAAG"C AAACGGAACAATCA""CACG"GAAGGGCA AGCACC"C"GTCCG"CACCC""GT"CCCr1 GG"CCA"CCAAGCCA"TCTGGG"G""GGr1 CG"AGTGGGTGGAG"CCTCGC""G""ACr1 [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley C"C"GC"CGTCACCGTGGCTT"TATAA"C GGGT""AGA""CCAAAAGAAGCCGCC" GCTCCA"AGCGA"TACATGAA"ATGAC"C GCCCTGGCCCCACAAGGAAACAC ""ACCAGCC"""ACGCACCACC""AGAGA"""""1 CGCTGCC""A""CGGAGCAGGG"GAAGW'VV1 CCAGA"C"GCAGATGCACCAGCGTA"CAG CAGGGCCAGAACCAACTGTA"AACGAGCr1 CAACC"GGGACGCAGGGAAGAGTATGACG TTTTGGACAAGCGCAGAGGACGGGACCCr1 GAGA"GGGTGGCAAACCAAGACGAAAAAA CCCCCAGGAGGG"CTC"A"AATGAGC"GC AGAAGGATAAGA"GGC"GAAGCCTA""Cr1 GAAA"AGGCA"GAAAGGAGAGCGGAGAAG GGGAAAAGGGCACGACGG"TTG"ACCAGG GCAC"GC"ACGAAGGAF " GACGC"C"CCACA"GCAAGCCCF TAGGF1AA AJ- ATGGCAC"CCCCG"AAC"GCTCF " AL .PLAL. 21508CARLX GCCG""GGCA""GCTCC"GCACGCCGCAC HAARPI 1QSPA".

H GCCCGGAAA"AGTGA"GACGCAG"C"CCA SVSPGHRA".SCRASQ GCCACCC"G"C"G"G"C"CCAGGGGAAAG SVSSNJAWYQQKPGQA CC"C"CC"GCAGGGCCAGTCAGA PRJLIYGAS"%A"GI? G"G""AGCAGCAAC""AGCCTGG"ACCAG ARFSGSGSG"EF"LTI CAGAAACC"GGCCAGGC"CCCAGGC"CCT SS.QS?DFAVYYCQQY CA"C"ATGGTGCA"CCACCAGGGCCAC"G AAYPTFGGG"KVEIKQ G"A"CCCAGCCAGG""CAG"GGCAG"GGG GS"SGSGKPGSGEGSr1 TC"GGGACAGAG""CAC"C"CACCA"CAG KGQVQIVQSGA?VKK? CAGCC"GCAG"C"GAAGAT"TTGCAGT"F GASVKVSCKASGY"Fr1 GTCAGCAG"ACGCCGCC"ACCCF SYYWiWVRQA?GQGLE AC"TT"GGCGGAGGGACCAAGG"F WMGIINPGGGS"SYAQ CAAACGGGGG"C"ACA"CCGGCF KFQGQVTMTQD"S"Sr1 AGCCCGGAAG"GGCGAAGG"AGF VYM?.SSLRS?DTAVY GGGCAGGTGCAGCTGG"GCAG"C"GGGGC YCAQESWPMDVWGQGr1 r1GAGG"GAAGAAGCCTGGGGCC"CAGTGA TVTVSSAAA.DN?KSN AGGTT"CCTGCAAGGCATC"GGA"ACACC 1II-IVKGK-IIC?SP. r1TCACCAGC"ACTATA"GCAC"GGG"GCG ?G?SKPFWV4VVVGG ACAGGCCCC"GGACAAGGGCT"GAG"GGA .ACYSLIV"VAFIIF I'1GGGAA""AA""CAACCC"GG""GG""GG""AGC RSKRS R .

ACAAGC"ACGCACAGAAG""CCAGGGCAG D RPGDF1 CA"GACCAGGGACACG"CCACGA _D DFAAY GCACAG"C"ACA"GGAGC"GAGCAGCC"G AGATCTGAGGACACGGCGG"GTACF 1G CGCCAGAGAGAG""GGCCAA"GGACGr GGGGCCAGGGAACAACTG"CACCG"Cr TCAGCCGC"GCCC""GATAA"GAAAAG"C AAACGGAACAATCA""CACG"GAAGGGCA AGCACC"C"GTCCG"CACCC""GT"CCCr1 GG"CCA"CCAAGCCA"TCTGGG"G""GGr1 CG"AGTGGG"GGAG"CC"CGC""G""ACr1 CmCmGcmCGmCACCGmGGCTmmmAmAAmC I""1C"GGGT""AGA""CCAAAAGAAGCCGCC" "AGCGA"TACA"GAA"ATGAC"C CACGCCGCCCTGGCCCCACAAGGAAACAC ""ACCAGCC""""ACGCACCACC""AGAGA"""""1 CGCTGCC""A""CGGAGCAGGG"GAAG'W'VV1 CCAGA"C"GCAGATGCACCAGCGTA"CAG CAGGGCCAGAACCAACTGTA"AACGAGCr1 CAACC"GGGACGCAGGGAAGAGTATGACG [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] eadley Unmarked set by eadley TTTTGGACAAGCGCAGAGGACGGGACCC"1 GAGA"GGGTGGCAAACCAAGACGAAAAAA CCCCCAGGAGGG"CTC"A"AATGAGC"GC AGAAGGATAAGA"GGC'11GAAGCCTA'11'11C'11 GAAA"AGGCA"GAAAGGAGAGCGGAGAAG GGGAAAAGGGCACGACGG"TTG"ACCAGG GACTCAGCAC"GC"ACGAAGGAF 1 GACGC"C"CCACA"GCAAGCCCF TAGGF1AA NM- ATGGCAC"CCCCG"AAC"GCTCr 1 1AL ?IALL. 21517CARHX GCCG""GGCA"TGCTCC"GCACGCCGCAC _ .Q ?SGPG.

L GCCCGCAGCTGCAGC"GCAGGAG"CGGGC H _ VSGG CCAGGACF1GG'11 GAAGCCF1TCGGAGACCC'11 S SYYWGWI RQP .3 GTCCC"CACC"GCAC"G"C"CTGGF 1 GKGLEWIGSISYSGS"1 CCATCAGCAGF1AGTAGTF1ACF1AC" YY‘\I P S .4KS RVT I SVD"1 TGGA"CCGCCAGCCCCCAGGGAAGGGGC"1 LKJSSVTAAD GGAG"GGA"TGGGAG"A"C"CCTAF 1G TAVYYCARGRGYA"SJ GGAGCACC"AC"ACAACCCG"CCCF DIWGQG"MV"VSSG AGTCGAGTCACCA"A"CCG"AGACACG"C 1SGSGKPGSG CCAG""C"CCC"GAAGC"GAG"T HIV."QSPAT _ C"G"GACCGCCGCAGACACGGCGG"G"AC HQA".SCRASQSVSS TAC"GCGCCAGAGGCAGGGGATA"GCAAC JAWYQQKPGQAP CAGCTTAGCC""CGA"A"CTGGGG"C "GI_ G'11ACAA'11GG'11CACCG"C'11CCTCAGGG'11C'11 GSG'11DF'11 ACAF1CCGGC'11CCGGGAAGCCCGGAAGF1GG .2 DFAVYYC CGAAGG'11AG"ACAAAGGGGGAAA""1G'11G"1 _ FGGG"KVI I'1GACACAG"CTCCAGCCACCCTG'11C'11'11'11G ?KSNGF I'1C"1CCAGGGGAAAGAGCCACCC"C'11CC'11G ?SP.F_ CAGGGCCAG"CAGAGTG"1'11AGCAGC'11AC"1 JVVVGGV I'1AGCCTGG'11ACCAACAGAAACC"GGCCAG 1VAFIIFWV GC"1CCCAGGC"CCTCAF1C'11ATGATGCA'11C .4 {S DYMNM"1 _ CAACAGGGCCAC"GGCAF1CCCAGCCAGG'11 YA? _ I'1CAG"GGCAG"GGGTC"1GGG.ACAGAC"1"1C QSRVKFSRSA AC"C"CACCA"CAGCAGCC"AGAGCC"GA. QGQVQLYN? AGAT"""GCAG"""AT"AC"G"CAGCAGA EYDVLDKR GACACG"CTGGCC"CC"AC"T"TGGCGGA GKPRRKVPQ.

AAGG""GAGA"CAAACGGGCCGC QKDKMAﬁAYS 1GATAA"GAAAAG"CAAACGGAA ERRRGKGHDG 1"CACG"GAAGGGCAAGCACC"C TATKDTYDA 1GTCCG"CACCC""GT"CCC"GG"CCA"C ?R CAAGCCA"TCTGGG"G""GG"CG"AGTGG mCCmCGCmmemACmCmCmGCmC GmCACCGmGGCTmmmAmAAmCmmCr I'1AGA"1CCAAAAGAAGCCGCC"GC" 1A GCGA"TACA"GAA"ATGAC"CCACGCCGC CCTGGCCCCACAAGGAAACA 1ACCAGCC I'1'11ACGCACCACC"1.AGAGA"1"1"1CGCTGCC"1 A"CGGAGCAGGG"G.AAG"1"1"1"1CCAGA"C"1 GCAGATGCACCAGCGTA"CAGCAGGGCCA GAACCAACTGTA"AACGAGC"CAACC"GG GACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCC"GAGA"GGG TGGCAAACCAAGACGAAAAAACCCCCAGG AGGG"CTC"A"AATGAGC"GCAGAAGGAT AAGA"GGC"GAAGCCTA""CF CA"GAAAGGAGAGCGGAGAA GGCACGACGG"TTG"ACCAGGGACTCAGC AC"GC"ACGAAGGA"AC""A"GACGCTCT CCACA"GCAAGCCC"GCCACCTAGGTAA [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley NM- C"CCCCG"AAC"GCTCTGC"GCT ?V"AL .PLAL. 21517CARLX GCCGTTGGCA""GC"CC"GCACGCCGCAC RP?IV 1QSPA".

H GCCCGGAAA""G"G""GACACAG"C"CCA _ ?%A".SCRASQ GCCACCC"G"C""F 1C"CCAGGGGAAAG JAWYQQKPGQA AGCCACCC"C"CC"GCAGGGCCAGTCAGA _ .IYDASNRA"GI? GTG""AGCAGC"AC""AGCCTGG"ACCAA ARFSGSGSG"DF"4TI CAGAAACC"GGCCAGGC"CCCAGGC"CCT VYYCQQR CA""C""ATGATGCA"CCAACAGGGCCAC"G .3 ?T FGGGT KVEI K GCA"CCCAGCCAGG""CAG"GGCAG"GGG RGS"SGSGKPGSGEGS TC"GGGACAGAC""CAC"C"CACCA"CAG TKGQ.Q.Q?SGPGIVK CAGCC"AGAGCC"GAAGAT"T"GCAG""F _ ?".S.TCTVSGGSI AT""AC"1 G"1 CAGCAGAGACACG"1CTGGCC" S S YYWGWI RQ P .3 GK CC"AC"T"TGGCGGAGGGACCAAGGF SISYSGS"YY GA"CAAACGGGGG"CTACA"CCGGCF VPSJKSQVTISVD"SK CCGGAAG"GGCGAAGG"AGF VQFSLKJSSVTAADTA AAGGGGCAGCTGCAGC"GCAGGAGF VYYCARGRGYATSJAF CCCAGGACTGG"GAAGCC"F DIWGQG"MVTVSSAAA "GTCCCTCACC"GCAC"Gr . ?KSNG"IIHVKGK "CCATCAGCAG"AGTAGTr { ?SP.F?G ?FW CTGGA"CCGCCAGCCCCCAGG VJVVVGGVIACYS.

"GGAG"GGA"TGGGAG"A"Cr r1VAFIIFWVQSKRS GGGAGCACC"AC"ACAACCCGF 4{SDYMNM"?R GAGTCGAGTCACCA"A"CCG"AGACACGF YA??? CCAAGAACCAG""C"CCC"GAAGC"GAGF RSRVKFSRSA TC"G"GACCGCCGCAGACACGGCGGr QGQVQLYN? CGCCAGAGGCAGGGGATA"GCAA EYDVLDKR CCAGC""AGCC""CGA"A"CTGGGG"CAG GKPRRKVPQ.

GG"ACAA"GGTCACCG"C"CCTCAGCCGC QKDKMAﬁAYS r1GCCC""GATAA"GAAAAGTCAAACGGAA ERRRGKGHDG ""CACG"GAAGGGCAAGCACC"C TATKDTYDA r1G"CCG"CACCC""GT"CCC"GG"CCA""C ?R CAAGCCA"TCTGGG"G""GG"CG"AGTGG GmGGAGmCCmCGCmmemACmCmCmGCmC GmCACCGmGGCTmmmAmAAmCmmCmGGGT CAAAAGAAGCCGCC"GCr 1A GCGA"TACA"GAA"ATGAC"CCACGCCGC CCTGGCCCCACAAGGAAACAC"ACCAGCC I'1"ACGCACCACC""AGAGA"""1""CGCTGCC" A"CGGAGCAGGG"GAAG""""CCAGA"Cr1 GCAGATGCACCAGCGTA"CAGCAGGGCCA GAACCAACTGTA"AACGAGCr 1GG GACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCC"GAGA"GGG TGGCAAACCAAGACGAAAAAACCCCCAGG AGGG"CTC"A"AATGAGC"GCAGAAGGAT AAGA"GGC"GAAGCCTA""C"GAAN CA"GAAAGGAGAGCGGAGAAGGGGAAAAG GGCACGACGG"TTG"ACCAGGGACTCAGC AC"GC"ACGAAGGAF ""A"GACGCTC" CCACA"GCAAGCCC"GCCACCTAGG"AA ATGGCAC"CCCCGTAAC"GC"CTGC"GCr1 MAIPVTAL ?LA.L.

GCCGT"GGCA"TGC"CC"GCACGCCGCAC HAARPﬁVQ ﬁSGGG.

AGGTGCAGCTGG"GGAG"C"GGG VQ?GGS.R.SCAASGF GGAGGCT"GG"ACAGCC"GGGGGGTCCCr1 TFSSYSMNWVRQA?GK GAGAC"C"CC"G"GCAGCCTC"GGA"TCA GLEWVS"ISSSSSTIY CC""CAG"AGC"ATAGCA"GAAC"GGG"C YADSVKGRFTISRDNA CGCCAGGC"CCAGGGAAGGGGCTGGAG"G KNSLYIQMNSIRA?DT GG""TCAACCA""AGTAGr 1AGTAG"A AVYYCARGSQEHLIFD CCA"ATAC"ACGCAGACTCTG"GAAGGGC YWGQG"LVTVSSGSTS [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley CGAT"CACCA"C"CCAGAGACAA"GCCAA GSGKPGSGﬁGS"KG.

GAAC"CACTG"A"C"GCAAATGAACAGCC V4"QSPAT.SLS_ 1GAGAGC"GAGGACACGGCGG"G"AC"AC A"JSCRASQSVS 1GCGCCAGAGG""C"CAGGAGCACC"GAT WYQQKPGQAPR 1TTCGA""AT"GGGGACAGGG"ACA""GG ASNRA"GI_ 1CACCG"CTCC"CAGGG"C"ACA""CCGGC SG"DF".TISS ._ 1CCGGGAAGCCCGGAAG"GGCGAAGG"AG FAVYYC QFYY. 1ACAAAGGGGGAAA""G"G""GACACAGr1 GGG"KV CTCCAGCCACCCTG"C"""G"C"CCAGGG EKSNGF GAAAGAGCCACCC"C"CC"GCAGGGCCAG r1CAGAGTG""AGCAGG"AC""AGCCTGGr1 ACCAACAGAAACC"GGCCAGGC"CCCAGG C"CCTCA"C"ATGATGCA"CCAACAGGGC CAC"GGCA"CCCAGCCAGG""CAG"GGCA "GGGACAGAC""CAC"C"CACC 1CAGCAGCC"AGAGCC"GAAG I'"""""""GC AG"1 r" I"AT I'"AC r" G"1 CAGCAGAGA"1 r" C""ACT ACCC""GGAC"T"TGGCGGAGGGACCAAG G1"GAGA"CAAACGGGCCGC"GCCC""GA TAA"1 GAAAAG"1CAAACGGAACAA"1 CA"1 r" C ACG"GAAGGGCAAGCACC"C"GTCCG"CA KDTYDA CCC""GT"CCC"GG"CCA"CCAAGCC r1T CTGGG"G""GG"CG"AGTGGG"GGAG"CC mCGCmmGmmACmCmCmGCmCGmCACCGmG GCTmmmAmAAmCmmCmGGGTmAGAmCCAA AAGAAGCCGCC"GC"CCA"AGCGA"TACA r1GAA"ATGAC"CCACGCCGCCCTGGCCCC ACAAGGAAACAC"ACCAGCC""ACGCACC ACC"AGAGA"""CGCTGCC"A"CGGAGCA GGG"GAAG""""CCAGA"C"GCAGATGCA CCAGCGTA"CAGCAGGGCCAGAACCAACT GTA"AACGAGC"CAACC"GGGACGCAGGG AAGAGTATGACGTTTTGGACAAGCGCAGA GACCC"GAGA"GGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGG"CTCr1 A"AATGAGC"GCAGAAGGATAAGA"GGCr1 GAAGCCTA""C"GAAA"AGGCA"GAAAGG GAGAAGGGGAAAAGGGCACGACG Gr 1ACCAGGGACTCAGCAC"GC"ACG 1AC""A"GACGC"CTCCACA"GCA AGCCC"GCCACC"AGGTAA TS- Ar "AAC"GCTC"GC"GCT MAL?V"AL .PLAL. 21522CARLX GGCA""GC"CC"GCACGCCGCAC HAARP?IV 1QSPA".

H GCCCGGAAA""G"G""GACACAG"C"CCA SLS?G?%A"ISCRASQ GCCACCC"G"C""F 1C"CCAGGGGAAAG SVS JAWYQQKPGQA CC"C"CC"GCAGGGCCAGTCAGA PRIIIYDASNRA"GI? GTG""AGCAGG"AC""AGCCTGG"ACCAA ARFSGSGSG"DF"4TI CC"GGCCAGGC"CCCAGGC"CCT SSI. ﬁDtAVYYCQQR CA"C"ATGATGCA"CCAACAGGGCCAC"G FYY?WTFGGGTKVEIK GCA"CCCAGCCAGG""CAG"GGCAG"GGG RGSTSGSGKPGSGEGS r1C"GGGACAGAC""CAC"C"CACCA""CAG TKGﬁVQIVﬁSGGGIVQ CAGCC"AGAGCC"GAAG "TGCAG"""""1 ?GGS.R.SCAASGFTF AT"AC""G"CAGCAGAGA""C"ACTACCCr1 SSYSMNWVRQA?GKGL "T"TGGCGGAGGGACCAAGG""GA EWVS"ISSSSSTIYYA GA"CAAACGGGGG"CTACA"CCGGC"CCG DSVKGRFTISRDNAKN GGAAGCCCGGAAG"GGCGAAGG"AGF SLYIQMNSIRA?D"AV GAGGTGCAGCTGG"GGAGTC"GG YYCARGSQEHLIFDYW GGGAGGCT"GG"ACAGCCTGGGGGG"CCC GQG"LVTVSSAAA4DN TGAGACTC"CC"G"GCAGCCTC"GGA"TC EKSNGTIIiVKGKiLC [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley ACC""CAGTAGC"ATAGCA"GAACTGGGr1 G?SKPFWVLV CCGCCAGGC"CCAGGGAAGGGGCTGGAGr1 VVGGVIACYSL.V"VA GGG"""""1 CAACCA"""AGTAG""AG""AGTAG"1 FI I FWV RS KRS R . .HS ACCA"ATAC"ACGCAGACTCTG"GAAGGG ?RRPG?"QKH CCGA""CACCA"C"CCAGAGACAA"GCCA YQPYA??RDFAAYQSR AGAAC"CAC"G"A"C"GCAAATGAACAGC VKFSRSA C"GAGAGC"GAGGACACGGCGG"G"AC"A NQLYN? C"GCGCCAGAGG""C"CAGGAGCACC"GA VLDKR I'"""T""CGA""""A""""GGGGACAGGG""A.CA."""1G RRKVPQ. 1CACCG"CTCC"CAGCCGC"GCCCT"GA KMAﬁAYS 1AA"GAAAAG"CAAACGGAACAATCAﬁ"C RGKGHDG ACG"GAAGGGCAAGCACC"C"G"CCG"CA KDTYDA CCC""GT"CCC"GG"CCA"CCAAGCC r1T G""GG"CG"AGTGGG"GGAG"CC mCGCmmGmmACmCmCmGCmCGmCACCGmG GCTmmmAmAAmCmmCmGGGTmAGAmCCAA AAGAAGCCGCC"GC"CCA"AGCGA"TACA r1GAA"ATGAC""CCACGCCGCCCTGGCCCC AAACAC"ACCAGCC""ACGCACC ACC"AGAGA"""CGCTGCC"A"CGGAGCA GGG"GAAG""""CCAGA"C"GCAGATGCA CCAGCGTA"CAGCAGGGCCAGAACCAACT GTA"AACGAGC"CAACC"GGGACGCAGGG AAGAGTATGACGTTTTGGACAAGCGCAGA GACCC"GAGA"GGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGG"CTCr1 A"AATGAGC"GCAGAAGGATAAGA"GGCr1 GAAGCCTA""C"GAAA"AGGCA"GAAAGG GAGAAGGGGAAAAGGGCACGACG Gr 1ACCAGGGACTCAGCAC"GC"ACG 1AC""A"GACGC"CTCCACA"GCA AGCCC"GCCACCTAGGTAA RY- Ar 1CCCCGTAAC"GCTC"GC"GCr1 MAIPVTAL 21527CARHX GCCGT"GGCA""GCTCC"GCACGCCGCAC HAARPQVQ L GCCCGCAGGTGCAGCTGG"GGAG"C"GGG VQ?GRSLR GGAGGCG"GG"CCAGCC"GGGAGGTCCCr1 TFSSYGMHWVRQA.

GAGAC"C"CC"GTGCAGCGTC"GGA"TCA VISY CC"TCAG"AGC"A"GGCA"GCAC"GGG"C YADSVKGRF"ISR CGCCAGGC"CCAGGCAAGGGGCTGGAG"G KNTLYLQMNSIQA?DT GG"GGCAG"TA"A"CGTA"GA"GGAAG"A AVYYCARTDFWSGSPP ATAAATAC"A"GCAGACTCCG"GAAGGGC GJDYWGQGTJVTVSSG CGATTCACCA"C"CCAGAGACAA"TCCAA S"SGSGKPGSGEGS"K GAACACGC"G"A"CTGCAAATGAACAGCC GDIQJTQSPSSVSASV r1GAGAGCCGAGGACACGGCGG"G"AC""AC GDRV"I"CRASQGISS r1GCGCCAGAAC"GAC""CTGGAGCGGATC WJAWYQQKPGKAPKJL CCC"CCAGGC""AGA""ACTGGGGACAGG I QSGVPSRFS GTACAT"GG"CACCG"C"CCTCAGGG"Cr1 GSGSG"DF".TISS.Q ACA"CCGGC"CCGGGAAGCCCGGAAG"GG D.3DFA"YYCQQIYTFP CGAAGG"AG"ACAAAGGGGGACA"CCAGr1 F"FGGG"KVEIKRAAA 1GACCCAG"C"CCATC""CCGTG"C"GCA VG"IIHVKGK 1CTGTAGGAGACAGAG"CACCATCAC""G HIC?SP.F?GPSKPFW 1CGGGCGAG"CAGGGTA""AGCAGC"GGr1 VJVVVGGVIACYSL 1AGCC"GG"A"CAGCAGAAACCAGGGAAA r1VAFIIFWVQSKRS GCCCC"AAGC"CCTGA"C"ATGGTGCA"C _ _ RPG CAG"""GCAAAG"GGGG"CCCATCAAGGr1 r1CAGCGGCAG"GGA"C"GGGACAGAT""C AC"C"CACCA"CAGCAGCC"GCAGCC"GA 7 AG I""""TGCAAC""‘A"""AC"‘Gr‘CAGCAGA I RGR r1A"ACACCTTCCC"""CAC"T"TGGCGGA RRKNPQﬁG [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley ed set by Anne.Headley GGGACCAAGGTTGAGA"CAAACGGGCCGC QKDKMAﬁAYSﬁIGMKG "GCCCT"GATAA"GAAAAGTCAAACGGAA ERRRGKGHDG.YQG CAATCA""CACG"GAAGGGCAAGCACC"C TATKDTYDALiMQA.

"GTCCG"CACCC""GT"CCC"GG"CCA"C ?R CAAGCCA"TCTGGG"G""GG"CG"AGTGG GmGGAGHCCNCGCHHGHHACHCHCHGCHC GmCACCGNGGCTHHHAHAAHCNHCHGGGT "AGA"CCAAAAGAAGCCGCC"GCF "A GCGA"TACA"GAA"ATGAC"CCACGCCGC CCTGGCCCCACAAGGAAACAC"ACCAGCC ""ACGCACCACC"AGAGA"""CGCTGCC" A"CGGAGCAGGG"GAAG""""CCAGA"C" GCAGATGCACCAGCGTA"CAGCAGGGCCA GAACCAACTGTA"AACGAGCF ‘GG GACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCC"GAGA"GGG TGGCAAACCAAGACGAAAAAACCCCCAGG AGGG"CTC"A"AATGAGC"GCAGAAGGAT AAGA"GGC"GAAGCCTA""C"GAMN CA"GAAAGGAGAGCGGAGAAGGGGAAAAG GGCACGACGG"TTG"ACCAGGGACTCAGC AC"GC"ACGAAGGAF ‘"A"GACGCTC" CCACA"GCAAGCCC"GCCACCTAGG"AA RY- ATGGCAC"CCCCGTAAC"GC"CTGC"GCr1 MALPV"AL..PLALLL ARLX GCCGT"GGCAT"GC"CC"GCACGCCGCAC DIQ4"QSPSSV H GCCCGGACA"CCAG""GACCCAG"C"CCA DRV"I"CRASQ TC""CCG"G"C"GCA"C"GTAGGAGACAG JAWYQQKPGKA AG"CACCATCACT"G"CGGGCGAGTCAGG _ JQSGVP GTA""AGCAGC"GG""AGCC"GG"A"CAG 1DFTJTI CAGAAACCAGGGAAAGCCCC"AAGC"CCT '. I GA"C"ATGGTGCA"CCAG"""GCAAAG"G YTFPFTFGGG"KVEIK GGG"CCCATCAAGG""CAGCGGCAG"GGA SGKPGSGEGS r1C"GGGACAGAT""CAC"C"CACCA"CAG TKGQVQ.V?SGGGVVQ CAGCC"GCAGCC"GAAG I'"""""TGCAAC"""1 ?GRSLRJSCAASGFTF A""AC"G"CAGCAGA"A"ACACCTTCCCF SSYGMHWVRQA?GKGL I'1"CAC""T""TGGCGGAGGGACCAAGG" EWVAVISYDGSVKYYA GA"CAAACGGGGG"C"ACA"CCGGCr DSVKGRF"ISRDNSKN GGAAGCCCGGAAG"GGCGAAGG"AGr r1.YIQMNSRA'TDTAV AAGGGGCAGGTGCAGCTGG"GGAG"CF YYCARTDFWSGSPPGL GGGAGGCG"GG"CCAGCCTGGGAGGr DYWGQG"4VTVSSAAA TGAGACTC"CC"G"GCAGCGTC"GGH' . II{VKGK ACC"TCAG"AGC"A"GGCA"GCAC"GGGF { G?SKPFW CCGCCAGGC"CCAGGCAAGGGGCTGGAGF VJVVVGGVIACYSL GGG"GGCAG"TA"A"CGTA"GA"GGAAGr1 r1VAFIIFWVRSKRS AATAAATAC"A"GCAGACTCCG"GAAGGG 4{SDYMNM"?R CCGATTCACCA"C"CCAGAGACAA"TCCA RKHYQPYA??? AGAACACGC"G"A"CTGCAAATGAACAGC RSRVKFSRSA C"GAGAGCCGAGGACACGGCGG"G"ACTA QGQVQLYN? C"GCGCCAGAAC"GAC""C"GGAGCGGAT EYDVLDKR CCCCTCCAGGC""AGA""ACTGGGGACAG GKPRRKVPQﬁG .

GG"ACA"""GGTCACCGF1CTCCTCAGCCGC QKDKMA ﬁAYS *.I GVIKG r1GCCCT"GATAA"GAAAAG"CAAACGGAA ERRRGKGHDGIYQG CAATCA""CACG"GAAGGGCAAGCACC"C TATKDTYDAuiMQA r1G"CCG"CACCC""GT"CCC"GG"CCA"C ?R CAAGCCA"TCTGGG"G""GG"CG"AGTGG mCCmCGCmmemACmCmCmGCmC G"GGCT"""A"AA"CT"C"GGGT TAGA"CCAAAAGAAGCCGCC"GC"CCA"A GCGA"TACA"GAA"ATGAC"CCACGCCGC [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley CCTGGCCCCACAAGGAAACACTACCAGCC T"ACGCACCACC"AGAGA"""CGCTGCCr1 A"CGGAGCAGGG"GAAG""""CCAGA"Cr1 GCAGATGCACCAGCGTA"CAGCAGGGCCA GAACCAACTGTA"AACGAGC"CAACC"GG GACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCC"GAGA"GGG TGGCAAACCAAGACGAAAAAACCCCCAGG AGGG"CTC"A"AATGAGC"GCAGAAGGAT AAGA"GGC"GAAGCCTA""CF CA"GAAAGGAGAGCGGAGAA GGCACGACGG"TTG"ACCAGGGACTCAGC AC"GC"ACGAAGGAF ""A"GACGCTC" CCACA"GCAAGCCC"GCCACCTAGG"AA PP- ATGGCAC"CCCCGTAACF "CTGC"GCF MAIPVTAL.LPLA.

ARHX GCCGT"GGCA"TGC"CC"GCACGCCGCAC HAARPQVQIVQSGAH L GCCCGCAGGTGCAGCTGG"GCAG"C"GGG KK?GSSVKVSCKASGG GC"GAGG"GAAGAAGCC"GGG"CCTCGGr1 r1FSSYAISWVRQA?GQ GAAGG"C"CC"GCAAGGC"TC"GGAGGCA GII?IFGTAN CC"TCAGCAGC"ATGCTA"CAGCF YAQKFQGRVTITADES CGACAGGCCCC"GGACAAGGGC"F "STAYW?.SSLQS?D" GA"GGGAGGGA"CA"CCC"A"C"F AVYYCAQ"PEYSSSIW ACTACGCACAGAAG"F HYYYGVDVWGQGTTVr1 AGAGTCACGA""ACCGCGGAC VSSGSTSGSGK?GSGE GAGCACAGCC"ACA"GGAGCF GS"KG 4 "GAGATCTGAGGACACGGCGG"GF AVSLG "GCGCCAGAAC"CC"GAATAC"CCF SV.

CA"ATGGCAC"ATTACTACGGCA"GGACG QK. __ r1A"GGGGCCAGGGAACAACTG"CACCGTC RESGV. r1CCTCAGGG"C"ACATCCGGC"CCGGGAA SS GCCCGGAAG"GGCGAAGGTAG"ACAAAGG AHF_ GGGACATCG"GA"GACCCAGTCTCCAGAC r1KVEIKQAAA r1CCCTGGCTG"G"CTC"GGGCGAGAGGGC 1IIHVKGKH.

CACCA"CAAC"GCAAG"CCAGCCAGAGTG _ _ ?SKPFWV_ 1ACAGC"CCAACAA"AAGAACTAC 1GGTACCAGCAGAAACCAGGACA 1AAGC"GCTCA"""AC"GGGCAr1 1ACCCGGGAA"CCGGGG"CCC"GACCGA 1GGCAGCGGG"C"GGGACAGATFV1 1CACCA"CAGCAGCC"GCAGGC"G AAGAT G"1 GGCAG"1 r""A" I'"ACr" G"1 CAGCAG I'1"CGCCCACAC"CC""""""CAC""T""TGGCGG AGGGACCAAGG""GAGA"CAAACGGGCCG C" 1 r" GATAA"1 GAAAAG"1CAAACGGA ""CACG"GAAGGGCAAGCACC" C"GTCCG"CACCC""GT"CCC"GG"CCAr1 CCAAGCCA"TCTGGG"G""GG"CG"AGTG GmCCmCGCmmemACmCmCmGCm CGmCACCGmGGCTmmmAmAAmCmmCmGGG T"AGA"CCAAAAGAAGCCGCC"GCr " AGCGA"TACA"GAA"ATGAC"CCACGCCG CCCTGGCCCCACAAGGAAACAC"ACCAGC C""ACGCACCACC"AGAGA"""CGCTGCC r CAGGG"GAAG""""CCAGA"C r1GCAGATGCACCAGCGTA"CAGCAGGGCC AGAACCAACTGTA"AACGAGC"CAACC"G GGACGCAGGGAAGAGTATGACGTTTTGGA CAAGCGCAGAGGACGGGACCC"GAGATGG GTGGCAAACCAAGACGAAAAAACCCCCAG [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GAGGG"CTC"A"AATGAGC"GCAGAAGGA GGC"GAAGCCTA"F GCA"GAAAGGAGAGCGGAGAA GGGCACGACGG"T"GF CAC"1 GCF1ACGAAGGAF1ACF TCCACA"GCAAGCCC"GCCACCF PP- ATGGCAC"CCCCGF "GCTCF .ALL 21528CARLX GGCAT"GCTCC"GCACGCCGCAC H GCCCGGACATCGTGA"GACCCAGTC"CCA GAC"CCC"GGC"G"G"C"C"GGGCGAGAG GGCCACCA"CAAC"GCAAG"CCAGCCAGA GT G"1 " "ACAGC"1 CCAACAAF1AAGAAC TAC"F 1GGTACCAGCAGAAACCAGG 1GCTCA""""""AC""GGG YYCQQFAH"1 ?FTFGGG 1CCGGGG"CCC"GAC EIKRGSTSGSGKP 1GGCAGCGGG"C"GGGACAGA GSGEGSTKGQVQLVQS 1CACCA"CAGCAGCC"GCAGG GAEVKK?GSSVKVSCK TGTGGCAG"T"A""AC"Gr SSYAISWVRQ CAG""CGCCCACAC"CC"""CAC"TF EWMGGII?IF CGGAGGGACCAAGG""GAGA"CAAACGGG QKFQGRVTIT GG"C"ACA"CCGGC"CCGGGAAGCCCGGA ADES"STAYM7.SSLR AG"GGCGAAGG"AG"ACAAAGGGGCAGGT ? 1AVYYCAR"PEYS GCAGCTGG"GCAG"C"GGGGC"GAGG"GA DVWGQG AGAAGCCTGGG"CC"CGGTGAAGGTC"CC .DN?KS TGCAAGGC"TC"GGAGGCACC"TCAGCAG 4C?SP C"ATGCTA"CAGC"GGG"GCGACAGGCCC _ _ JVVVG AAGGGC""GAG"GGA"GGGAGGG IV"VAFII A"CA"CCC"A"C"""GG"ACAGCAAACTA R.

CGCACAGAAG""CCAGGGCAGAGTCACGA _ ?" I'"""ACCGCGGACGAA"CCACGAGCACAGCC r1ACA"GGAGC"GAGCAGCC"GAGATCTGA GGACACGGCGG"G"ACTAC"GCGCCAGAA C"CC"GAATAC"CC"CCAGCA"ATGGCAC r1ATTAC"ACGGCA"GGACG"A"GGGGCCA GGGAACAACTG"CACCG"C"CCTCAGCCG C"GCCC""GATAA"GAAAAGTCAAACGGA ACAATCA""CACG"GAAGGGCAAGCACCr1 C"GTCCG"CACCC""GT"CCC"GG"CCAr1 CCAAGCCA"TCTGGG"G""GG"CG"AGTG GGHGGAGmccmCGcmmGHHAcmcr 1Gcm CGHCACCGHGGCTHHHAHAAmcmr "GGG T"AGA"CCAAAAGAAGCCGCCr1 " AGCGA"TACA"GAA"ATGAC"CCACGCCG CCCTGGCCCCACAAGGAAACAC"ACCAGC CACCACC"AGAGA"""CGCTGCC r1A"CGGAGCAGGG"GAAG""""CCAGA"C r1GCAGATGCACCAGCGTA"CAGCAGGGCC AGAACCAACTGTA"AACGAGC"CAACC"G GGACGCAGGGAAGAGTATGACGTTTTGGA CAAGCGCAGAGGACGGGACCC"GAGA"GG GTGGCAAACCAAGACGAAAAAACCCCCAG GAGGG"CTC"A"AATGAGC"GCAGAAGGA TAAGA"GGC"GAAGCCTA""CF GCA"GAAAGGAGAGCGGAGAA GGGCACGACGG"TTG"ACCAGGGACTCAG CAC"GC"ACGAAGGA"AC""A"GACGCTC TCCACA"GCAAGCCC"GCCACCTAGGTAA [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley RD- C"CCCCGTAAC"GCTCTGC"GCr1 MAIPVTAL 2153OCARHX GCCGT"GGCA"TGCTCC"GCACGCCGCAC HAARPQVQ L GCCCGCAGGTGCAGCTGG"GGAG"C"GGG LR GGAGGCG"GG"CCAGCC"GGGAGGTCCCr1 TFSSYGMHWVRQA.

GAGAC"C"CC"GTGCAGCGTC"GGA"TCA GLEWVAVISYDGSVKY CC""TCAG"AGC""A""GGCA"GCAC"GGG""C YA DSVKGRF"1 I S R DNS CGCCAGGC"CCAGGCAAGGGGCTGGAG"G KN"LYLQMNS ?D" GG"GGCAG"TA"A"CGTA"GA"GGAAG"A AVYYCVKGP.Q???YD ATAAATAC"A"GCAGACTCCG"GAAGGGC YGWDVWGQG""V"VSS CGAT"CACCA"C"CCAGAGACAA"TCCAA GS"SGSGKPGSGEGSr1 GAACACGC"G"A"C"GCAAA"GAACAGCC KGEIVM"QS?ATJSVS r1GAGAGCCGAGGACACGGCGG"G"ACTAC PG?RAT.SC%ASQSVS r1GCG"CAAGGGGCCGTTGCAGGAGCCGCC SNJAWYQQK?GQAPR ATACGA"TA"GGAA"GGACG"ATGGGGCC LIYSAS"%A"GI_ AGGGAACAACTG"CACCGTC"CC"CAGGG SGSGSGFTTFr1 1CTACA"CCGGC"CCGGGAAGCCCGGAAG QSEDFAVYYC 1GGCGAAGG"AG"ACAAAGGGGGAAA"AG P4"FGGG"KVI 1GA"GACGCAG"CTCCAGCCACCCTG"CT A.DN?KSVGF 1G"C"CCAGGGGAAAGAGCCACCC"CTC K{.C?SP.F_ 1GCAGGGCCAG"CAGAG"G""AGCAGCA WVJVVVGGV AC""AGCCTGG"ACCAGCAGAAACC"GGC IFWV CAGGC"CCCAGGC"CCTCA"C"ATAGCGC CAGGGCCAC"GG"A"CCCAGCCA GG""CAG"GGCAG"GGGTC"GGGACAGAG I""1CAC"C"CACCA"CAGCAGCC"GCAGTC mGAAGATmmmGCAGmmmAmmACmGmCAGC AGCACCACG"CTGGCC"C"CAC"T"TGGC GGAGGGACCAAGG""GAGA"CAAACGGGC CGC"GCCC""GATAA"GAAAAG"CAAACG TCA"1 r"CACG"1GAAGGGCAAGCAC C"C"GTCCG"CACCC""GT"CCC"GG"CC A"CCAAGCCA"TCTGGG"G""GG"CG"AG TGGGmGGAGmCCmCGCmmemACmCmCmG CmCGmCACCGmGGCTmmmAmAAmCmmCmG GGT"AGA"CCAAAAGAAGCCGCC"GC"CC A"AGCGA"TACA"GAA"ATGAC"CCACGC CGCCCTGGCCCCACAAGGAAACAC"ACCA GCC""ACGCACCACC"AGAGA"""CGCTG CC"A"CGGAGCAGGG"GAAG""""CCAGA r1C"GCAGATGCACCAGCGTA"CAGCAGGG CCAGAACCAACTGTA"AACGAGCF r1GGGACGCAGGGAAGAGTATGACGTTTTG CGCAGAGGACGGGACCC"GAGAr1 CAAACCAAGACGAAAAAACCCCC AGGAGGG"CTC"A"AATGAGC"GCAGAAG GATAAGA"GGC"GAAGCCTA"F AGGCA"GAAAGGAGAGCGGAGAA AAGGGCACGACGG"TTGF AGCAC"GC"ACGAAGGAF TCTCCACA"GCAAGCCCF " RD- ATGGCAC"CCCCG"AACF "CTGCF MALPV"AL..PLAL. 2153OCARLX GCCGTTGGCA""GCTCC"GCACGCCGCAC IVM"QSPA"_ H GCCCGGAAA"AGTGA"GACGCAG"C"CCA SVSPGjRA".SCRASQ GCCACCC"G"C"G"G"C"CCAGGGGAAAG AWYQQKPGQA AGCCACCC"C"CC"GCAGGGCCAGTCAGA SAS"%A"GI? GTGT"AGCAGCAACT"AGCCTGG"ACCAG ARFSGSGSG"?F"ITI CC"GGCCAGGC"CCCAGGCTCCT SS.QS?DFAVYYCQQ{ CATC"ATAGCGCA"CCACCAGGGCCACTG HVWPLTFGGGTKVEIK [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley G"ATCCCAGCCAGGTTCAG"GGCAG"GGG SGKPGSGEGS TC"GGGACAGAG"TCACTC"CACCA"CAG TKGQVQ.V?SGGGVVQ CAGCC"GCAGTC"GAAGAT"T"GCAG"TF ?GRSLR4SCAASGFTF A""AC"G"CAGCAGCACCACG"CTGGCCF SSYGMHWVRQA?GKGL C"CAC"T"TGGCGGAGGGACCAAGGF EWVAVISYDGSVKYYA GA"CAAACGGGGG"CTACA"CCGGCF DSVKGRF"ISRDNSKN CCGGAAG"GGCGAAGG"AGF .YLQMNSIQA?DTAV AAGGGGCAGGTGCAGCTGG"GGAG"CF YCVKGP.Q?P GGGAGGCG"GG"CCAGCCTGGGAGGF DVWGQG""VT TGAGACTC"CC"G"GCAGCGTC"GGH' .DN?KSNGF ACC"TCAG"AGC"A"GGCA"GCAC"GGGF {.C?SP.F_ CCGCCAGGC"CCAGGCAAGGGGCTGGAGF VJVVVGGV GGG"GGCAG"TA"A"CGTA"GA"GGAAGr1 V"VAFIIFWV AATAAATAC"A"GCAGACTCCG"GAAGGG CCGAT"CACCA"C"CCAGAGACAA"TCCA AGAACACGC"G"A"C"GCAAA"GAACAGC C"GAGAGCCGAGGACACGGCGG"G"ACTA CAAGGGGCCGTTGCAGGAGCCGC CATACGAT"A"GGAA"GGACG"ATGGGGC CAGGGAACAACTGTCACCG"C"CC"CAGC CGC"GCCC""GATAA"GAAAAGTCAAACG GAACAATCA"1 r"CACG"1GAAGGGCAAGCAC CCG"CACCC""GT"CCC"GG"CC GCCA"TCTGGG"G""GG"CG"AG TGGGmGGAGmCCmCGCmmemACmCmCmG CmCGmCACCGmGGCTmmmAmAAmCmmCmG A"CCAAAAGAAGCCGCC"GC"CC A"AGCGA"TACA"GAA"ATGAC"CCACGC CGCCCTGGCCCCACAAGGAAACAC"ACCA GCC""ACGCACCACC"AGAGA"""CGCTG CC"A"CGGAGCAGGG"GAAG""""CCAGA r1C"GCAGATGCACCAGCGTA"CAGCAGGG CCAGAACCAACTGTA"AACGAGC"CAACC r1GGGACGCAGGGAAGAGTATGACGTTTTG GACAAGCGCAGAGGACGGGACCC"GAGAr1 GGGTGGCAAACCAAGACGAAAAAACCCCC AGGAGGG"CTC"A"AATGAGC"GCAGAAG GATAAGA"GGC"GAAGCCTA"F AGGCA"GAAAGGAGAGCGGAGAA AAGGGCACGACGG"TTGF AGCAC"GC"ACGAAGGAF TCTCCACA"GCAAGCCCF " Clone 24Cl ATGGCAC"CCCCGTAACF "CTGCTGCF MAIPV"AL ?IALL.

THD CAR GCCGT"GGCA"TGCTCC"GCACGCCGCAC HAAR?QVQ ?SGPG.

DNA HXL GCCCGCAGGTCCAACTGCAAGAAAGCGGA VK?S?". ."C"VSGG CCCGGAC"GG"GAAGCC"TC"GAGACACr1 SISSYYWSWIRQPPGK TAG"C"GACG"GCACGG"CAGTGGCGGCr1 GLEWIGYIYYSGS"NY CCA"C"CCTCC"A""A""GG"CA"GGA"A NPSLKSRV"ISVDF CGACAACCCCCAGG"AAGGGCCTGGAA"G NQFSLK GAT"GGC"A"A"C"AC"A""CAGGAAGCA VYYCVS.

CGAAC"ACAATCCCAGCC"GAAG"CCCGA GFDYWGQG"LVTVSSG G"1 GACAA"1T r" CAG"AGA""ACCAGF1AAAAA GGGS GGGGS GGGGS D I CCAG"r 1C""AAAC"G"CAAGCG"GA QLTQSPSSJSASVGDR CAGC"GCCGACACCGC"G"G"AT"AC"GC VSF"CQASQDINNF4N G"C"CAC"GG"G"A""G"GGAGGGGA"TG GKAPKJLIYD T"A"AGCGGG""CG I'"""A""""GGGGACAGG ASN.?"GV?SRFSGSG GAACCC"GGTGACTG"A"C""CCGGCGGC FTISS.QP?D GGCGGC"CAGGGGG"GGCGG"AGTGGCGG IA"YYCQQYGN4PFTF [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley G""CCGA"A"TCAAC"GACACAAr1 EIKRAAAJDN CCCCCAGC"CAC"CAGCGCCAGCGTGGGG I 1IIHVKGK-LJC GACAGGG""AGC"""ACC"G"CAAGCCTC _ _ FWV4V "CAGGA"A"AAA"AACTT"CF .V"VA A"CAACAGAAGCCF RSKRSRuuis C"CCTGA"CTA"G r _RRPG?"RKH AAC"GGCGF CF"TAGCGGC" __RDFAAY C"GGCAGF 1ACAGACT"CACTTT"ACC r ""CAGCCGGAGGACA"CGC CACATA" " G"1 F1ACGGAAAC"1 "GCCC"F """TGGAGGCGGCACCAAA G"TGAAA"CAAAAGGGCCGC"GCCC"GGA GAAAAGAGCAA"1GGGACI'"ATAA""AC A"GT"AAAGGAAAACACC"G"GTCCA""Cr1 KDTYDA CCCC"GT"CCC"GGACCG"CAAAGCC "" mmGGGmGCmCGmGGmmGTCGGmGGCGmmC mCGCCmGmmAmAGCmmGCmGGmGACAGmA GCCT"CA""A"C"T""GGGTGAGA"CCAA AAGAAGCCGCC"GC"CCA"AGCGA"TACA r1GAA"ATGAC"CCACGCCGCCCTGGCCCC ACAAGGAAACAC"ACCAGCCT"ACGCACC ACC"AGAGA"""CGCTGCC"A"CGGAGCA GGG"GAAG""""CCAGA"C"GCAGATGCA CCAGCGTA"CAGCAGGGCCAGAACCAACT GTA"AACGAGC"CAACC"GGGACGCAGGG AAGAGTATGACGTTTTGGACAAGCGCAGA GGACGGGACCC"GAGA"GGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGG"CTCr1 A"AATGAGC"GCAGAAGGATAAGA"GGCr1 GAAGCCTA""C"GAAA"AGGCA"GAAAGG AGAGCGGAGAAGGGGAAAAGGGCACGACG Gr 1ACCAGGGACTCAGCAC"GC"ACG 1AC""A"GACGCTCTCCACA"GCA AGCCC"GCCACC"AGG"AA (CARl.l) CAGGTCCAACTGCAAGAAAGCGGACCCGG .Q?SGPGLVK?S? Clone 24Cl GAAGCC"TC"GAGACAC"TAG"C ISITCTVSGGSISSY THD CAR r1GACG"GCACGG"CAG"GGCGGC"CCA"C WSWIRQPPGKGLEWI DNA HXL r1CCTCC"A""A""GG"CA"GGA"ACGACA YIYYSGS"NYNPSLK ACCCCCAGG"AAGGGCCTGGAA"GGAT"G RV"ISVD"SKNQFSL "PTA" C""AC""A"1 r" CAGGAAGCACGAAC .4 S SVTAADTAVYYCV 1ACAATCCCAGCC"GAAG"CCCGAG"GAC JVYCGGDCYSGFDYW 1CAG"AGA"ACCAG"AAAAACCAGr1 GQG"LVTVSSGGGGSG "C""""AAAC"1 G"1 CAAGCG"1 GACAGC"1 GGGS GGGGS DI QLTQS ACCGC"G"G"AT"AC"GCG"C"C PSSJSASVGDRVSF"C 1G"A""G"GGAGGGGA"TG""A"A QASQDINNF.

GCGGG""CG I'"""A""""GGGGACAGGGAACC PGKAPK C"GGTGACTG"A"C""CCGGCGGCGGCGG "GV?SRFSGSGSGF C"CAGGGGG"GGCGG"AGTGGCGGF 1FTISS.Q?':‘.DIA"YY G""CCGA"A""CAAC"GACACAAF JPF"FGGG"K AGC"CAC"CAGCGCCAGCGTGGGG I .DN?KSVG GG""AGC"""ACC"G"CAAGCCTCF 1IIHVKGKHIC?SP.F A"A"AAA"AACTT"CF ?GPSKPFWV4VVVGGV CAGAAGCCF JACYSLLV"VAFIIFW GA"CTA"G r "GGAAACF VRSKRSR..{SDYMNM GCGF ""CACGCF"TAGCGGC"CF r1PRRPGP"RKHYQPYA AG"GG"ACAGACT"CACTTT"ACCA"CF ?PRDFAAYRSRVKFSR CW'"CAGCCGGAGGACA"CGCCACAT SADAPAYQQGQVQLYN A""AC"G"CAACAG"ACGGAAAC"TGCCC ?LNLGRRﬁﬁYDVLDKR I'"""""AC""""""TGGAGGCGGCACCAAAG" RGRDPEMGGKPRRKNP [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley AA"CAAAAGGGCCGC"GCCCTGGA"AACG ﬂGLYNﬁLQKDKMAEA AAAAGAGCAA"GGGAC"ATAA"ACA"GTr1 ﬁIGMKGﬁRRRGKGH AAAGGAAAACACC"G"GTCCA""C"CCCCr1 .YQGISTATKDTYD GT"CCC"GGACCG"CAAAGCCAF1FVV1GGG {MQA .P ?R r1GC"CG"GG""GTCGG"GGCG""C"CGCC memAmAGCmmGCmGGmGACAGmAGCCTm CA""A"C"T""GGGTGAGA"CCAAAAGAA GCCGCC"GC""CCA"AGCGA"TACA"GAAr1 ATGAC"CCACGCCGCCCTGGCCCCACAAG GAAACAC"ACCAGCCT"ACGCACCACC"A GAGA"""CGCTGCC"A"CGGAGCAGGG"G AAG""""CCAGA"C"GCAGATGCACCAGC GTA"CAGCAGGGCCAGAACCAACTGTA"A "CAACC"GGGACGCAGGGAAGAG GTTTTGGACAAGCGCAGAGGACG GGACCC"GAGA"GGGTGGCAAACCAAGAC GAAAAAACCCCCAGGAGGG"CTC"A"AAT CAGAAGGATAAGA"GGC"GAAGC CTA""C"GAAA"AGGCA"GAAAGGAGAGC GGAGAAGGGGAAAAGGGCACGACGG"TTG 1ACCAGGGACTCAGCAC"GC"ACGAAGGA 1AC""A"GACGCTC"CCACA"GCAAGCCC 1GCCACC"AGG (CARl.2) ATGGCAC"CCCCGTAAC"GC"CTGCTGCr1 MAIPV"AL ?IALL.

Clone 24Cl GCCG""GGCA"TGC"CC"GCACGCCGCAC HAAR?QVQ ?SGPG.

CHD CAR GCCCGCAGGTGCAGCTGCAGGAA"CCGGA VK?S?". ."C"VSGG DNA HXL CCGGGGC"GG"GAAGCCCAGCGAGACTCr1 SISSYYWSWIRQPPGK GAG"C"CACG"G"ACAG"TTC"GGAGGF GLEWIGYIYYSGS"NY GCA"""AGCTCC""AC"A"""GGTCA" NPSLKS RV"I SVD"SK AGGCAGCCCCCCGGGAAGGGA"T NQFSLKJSSVTAADTA C"A"A"""AC"ACAGF VYYCVSJVYCGGDCYS ACAACCCC"CAC"GAAGF GFDYWGQG"LVTVSSG G"""ACAA""CAGCG""GACACC" GGGSGGGGSGGGGS DI "CAG""CAG"""GAAA"TG"CF QLTQSPSSJSASVGDR CAGCAGC"GA"ACAGCCG"C"ATF VSF"CQASQDINNF4N G""TC"C"GG"C"A""GCGG"GGGGA"TG WYQQKPGKAPKIIIYD I'"""ACAG""GGC""""""GAC""A""""GGGGGCAGG ASN.?"GV?SRFSGSG G"ACTC"GGT"ACAG""TC""CCGGGGGG SG"DF"FTISS ?D GGAGGC"C"GGGGGCGGAGGCTCAGG"GG IA"YYCQQYGN__ 1F 1GGAGGCAGCGACA"CCAG""GACACAGA GGG"KVEIK%AAAIEV G"CCGCC"CCG"CGGG MYP??Y. H 1I 1G"CA"T"ACC"G"CAGGCCTC IHVKGKi 1CAGGATA""AA"AACTT"C"GAA""GGT ?SK.

A"CAGCAAAAGCCCGGAAAGGCACCCAAG C"1 GTTGA"""""ACGACGCCAG""AACC" GACAGGCG"GCCCTCCCGG"""AGF GCGGAAGCGG"ACGGATTT"ACCTF A"CAGC"C"C"CCAACCCGAAGACAF ]\Zx(:(:r1zx(:r1zxr1r1(3r1(:]x]x(:]x]xr1]xr TGCC""""ACAT"TGGCGGCGGCACC G"GGAGA""AAGCGGGCGGCAGC"A"F GG"GA"G"A"CCACCGCC""ACC"GG .

ACGAAAAGAGTAACGG"ACCATCA"F ISTATKDTYDA_ "GAAAGG"AAACACC"G"G"CC"F ??R CC"C""CCCCGGGCCA"CAAAGCCC"F 1"C"TG"GG"CGTGGGAGGCG"GCF 1GmmAmmCmCmGCmCGTmACCGmGGC 1A"CA""T"""GGGTTAGA"CCAAAA GAAGCCGCC"GC"CCA"AGCGA"TACA"G [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] eadley ed set by Anne.Headley AATATGAC"CCACGCCGCCCTGGCCCCAC AAGGAAACACTACCAGCCT"ACGCACCAC C"AGAGA"""CGCTGCC"A"CGGAGCAGG G"GAAG""""CCAGA"C"GCAGATGCACC AGCGTA"CAGCAGGGCCAGAACCAACTGT A"AACGAGC"CAACC"GGGACGCAGGGAA GAGTATGACGTTTTGGACAAGCGCAGAGG ACGGGACCC"GAGA"GGGTGGCAAACCAA GACGAAAAAACCCCCAGGAGGG"CTC"Ar1 AATGAGC"GCAGAAGGATAAGA"GGC"GA AGCCTA""C"GAAA"AGGCA"GAAAGGAG AGCGGAGAAGGGGAAAAGGGCACGACGGr1 TTG"ACCAGGGACTCAGCAC"GC"ACGAA GGA"AC""A"GACGCTCTCCACA"GCAAG CCC"GCCACC"AGG"AA (CARl.2) CAGGTGCAGC"GCAGGAA"CCGGACCGGG .Q?SGPGLVK?S? Clone 24Cl GCTGG"GAAGCCCAGCGAGACTC"GAG"C ISITCTVSGGSISSY CHD CAR TCACG"G""ACAG""T""C""GGAGG""AGCA"""1 WSWIRQPPGKGLEWI DNA HXL AGCTCC"AC"A""GGTCAr A YIYYSGS"NYNPSLK GCCCCCCGGGAAGGGA"T 1CG D"SKNQFSL GC"A"A"""AC"ACAGF JSSVTAADTAVYYCV "ACAACCCC"CAC"GAAGF JVYCGGDCYSGFDYW AA"CAGCG""GACACCr GQG"LVTVSSGGGGSG I'1CAG""""""GAAA""TG""C" GGGSGGGGS DIQLTQS GC"GA"ACAGCCG"C"ATF PSSJSASVGDRVSFF "C"GG"C"A""GCGG"GGGGAF 1ACA QASQDINNF.

G"GGC"""GAC"A""GGGGGCAGGG"ACT PGKAPK.

C"GGT"ACAG""TC""CCGGGGGGGGAGG r1GV?SRF C"C"GGGGGCGGAGGCTCAGG"GG"GGAG S GCAGCGACA"CCAG""GACACAGAGCCCG CQQYGN..

AG""CCT"G"CCGCC"CCG"CGGGGATAG VEIKQAAAII __ AG"G"CA"T"ACC"G"CAGGCCTC"CAGG Y. H 1IIHVKG ATA""AA"AACTT"C"GAA""GGTA"CAG _ ?F CAAAAGCCCGGAAAGGCACCCAAGC"GTT GA"""ACGACGCCAG"AACC"GGAGACAG GCG"GCCCTCCCGG"""AG"GGTAGCGGA AGCGG"ACGGATTT"ACCT""ACTAF C"C"C"CCAACCCGAAGACA""GCAACCF ACI'"A"1 r" G"1 CAACAA""A"1 GGAAACCT GC C" I'1""ACA.T""TGGCGGCGGCACCAAGG""GGA ""AAGCGGGCGGCAGC"A""GAGG"GA r1G"A""CCACCGCC""ACC"GGATAACGAA AAGAGTAACGG""ACCAT CA"1 r"CACG"1GAA ACACC""G"G"CC""C"CCCC"Cr1 "1CCCCGGGCCA"CAAAGCCC""""C""GGG"""1 C"TG"GG"CGTGGGAGGCG"GC""GC""G mmAmmCmCmGCmCGTmACCGmGGCGTmmA mCAmmmmmmGGGTmAGAmCCAAAAGAAGC C"CCA"AGCGA"TACA"GAACAT GAC"CCACGCCGCCC"GGCCCCACAAGGA AACACI'"ACCAGCC"1I'"ACGCACCACC""AGA GA"""CGCTGCC"A"CGGAGCAGGG"GAA CAGA"C"GCAGATGCACCAGCGT A"CAGCAGGGCCAGAACCAACTGTA"AAC GAGC"CAACC"GGGACGCAGGGAAGAGTA TGACGTTTTGGACAAGCGCAGAGGACGGG ACCC"GAGATGGGTGGCAAACCAAGACGA AAAAACCCCCAGGAGGG"CTC"ATAATGA GCTGCAGAAGGATAAGA"GGC"GAAGCCT [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley A""C"GAAATAGGCA"GAAAGGAGAGCGG AGAAGGGGAAAAGGGCACGACGGTTTG"A CCAGGGACTCAGCAC"GC"ACGAAGGA"A C""A"GACGC"CTCCACA"GCAAGCCC"G CCACC"AGG (CARl.3) C"CCCCGTAAC"GC"CTGCTGCr1 MAIPV"AL _.ALL Clone 24Cl GCCG""GGCA""GC"CC"GCACGCCGCAC HAAR?QVQ CD8 CAR AGGTGCAA"TGCAAGAG"CCGGC VK?S?".

DNA HXL CCCGGAC"CG""AAACCCAG"GAGACGCr1 SISSYYWSWIRQPPGK TAGCC"GACC"GTACCG"C"CAGGGGGCA GLEWIGYIYYSGS"NY 1C""A""AC"GGAGC"GGA"C NPSLKSRV"ISVDF AAAAGGCCT"GAA"G NQFSLK 1ACA"C"AC"AC"C"GGCTCAA VYYCVS. 1AA"CCA"CCC"GAAG"CCCGC GFDYWGQG"LVTVSSG 1CTC"G"GGACACCAGCAAGAA GGGSGGGGSGGGGS DI "CAC"GAAG""GF 1"A QLTQSPSSJSASVGDR CCGCGGCCGACACCGCCGr1 1GT VSF"CQASQDINNF_ G"G"C"C"TG"GTAC"GF 1G WYQQKPGKAPK.

C"A""CCGGG""CGAC"ACF ASN.?"GV?SRF GGAC"C"GGTAACCG"G"CC"CAGGCGGC FTISS _ "CAGGAGGAGGCGGCAGTGGAGG IA"YYCQQYGN__ TGGCGGC"CCGACA"CCAGC"GACACAAr1 EIK%AAA CACCATC""CCC"""CAGCT"CAGTCGGG SIMYFSiFV?VF__ GACAGAG"G"CC""CACA"GCCAGGCCAG PT""_ CCAGGATA"CAA"AACT"CCF 1GGr1 ASQ? ACCAACAGAAACCCGGAAAG GGAV C""CCTGA""C""A""GATGC""" IWA. _ GACCGGCG"GCCC"CCAGGF CAGGA"1CAGGCAC"1 GAC"1 r" " A"ATCCAG"C""CAGCCCGAAGACAF ""AC"GCCAACAA"ACGGGAACC I""1CCC""""""ACA""""CGGAGGCGGCACCAAG G"GGAAA"CAAAAGGGC"GCAGCAT"GAG CAAC"CAA"AA"GTA"""TAGTCAC""TG r1ACCAG"G"TC""GCCGGCTAAGCC"ACr1 ACCACACCCGC"CCACGGCCACCTACCCC AGC"CC"ACCA"CGC""CACAGCCTC"Gr1 1GCGCCCAGAGGC""GCCGACCGGCC GCAGGGGGCGC"G"TCA"ACCAGAGGACr1 ""TCGCC"GCGA"A"CTATATCTGGG CACCCC"GGCCGGAACC"GCGGCG"AC"C 1G"CCC"GG"CA"CACGCTC"A.T"G 1CACAGGAACAGA"CCAAAAGAAGCC 1GC"CCA"AGCGA""ACATGAATA"G AC"CCACGCCGCCCTGGCCCCACAAGGAA ACACTACCAGCCT"ACGCACCACC"AGAG A"""CGCTGCC"A"CGGAGCAGGG"GAAG I'"""""""CCAGA"C"GCAGATGCACCAGCGTA r1CAGCAGGGCCAGAACCAACTGTA"AACG AGC"CAACC"GGGACGCAGGGAAGAGTAT GACGTTTTGGACAAGCGCAGAGGACGGGA CCC"GAGA"GGGTGGCAAACCAAGACGAA AAAACCCCCAGGAGGG"CTC"A"AATGAG C"GCAGAAGGATAAGA"GGC"GAAGCCTA I""1C"GAAA""AGGCA""GAAAGGAGAGCGGA GAAGGGGAAAAGGGCACGACGG"TTG"AC CAGGGACTCAGCAC"GC"ACGAAGGACAC I'"""A""GACGCTCTCCACA"GCAAGCCC""GC CACCTAGGTAA [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley ed set by Anne.Headley (CARl.3) CAGGTGCAA"TGCAAGAG"CCGGCCCCGG .Q?SGPGLVK?S? Clone 24Cl ACTCG""AAACCCAGTGAGACGCTTAGCC ISITCTVSGGSISSY CD8 CAR r1GACC"GTACCG""C"CAGGGGGCAGCA"C WSWIRQPPGKGLEWI DNA HXL ""A""AC"GGAGC"GGA"CAGGCA YIYYSGS"NYNPSLK GCC"CCAGGAAAAGGCCTF r 1G QV"ISVD"SKNQFSL GG"ACA""C"AC"AC"C"GGCTCAACAAAr1 JSSVTAADTAVYYCV 1A"AA""CCA"CCC"GAAG"CCCGCG"GAC JVYCGGDCYSGFDYW "G"GGACACCAGCAAGAATCAGr1 GQG"LVTVSSGGGGSG "T"CAC""GAAG""G" ""ACCGCG GGGSGGGGS DIQLTQS GCCGACACCGCCGr1 1GTG"G"C PSSJSASVGDRVSF"C ""C""TG""GTAC""G""GGCGGCGAC""GC""A"""1 QASQDINNF.

CCGGG""CGAC"AC"GGGGCCAAGGGACr1 PGKAPK.

C"GGTAACCG"G"CC"CAGGCGGCGGCGG r1GV?SRF G"CAGGAGGAGGCGGCAGTGGAGGTGGCG r1FTISS _ GC"CCGACA"CCAGC"GACACAA"CACCA CQQYGN..

TC""CCC"""CAGCT"CAGTCGGGGACAG CAG"C""CAGCCCGAAGACA"TGCAACCr1 _ ?G?"RK A""AC"GCCAACAA"ACGGGAACC""CCC _?RDFAAYRS CA""""CGGAGGCGGCACCAAGG""GGA AA"CAAAAGGGC"GCAGCAT"GAGCAACr1 CAA"AA"GTA"""TAGTCAC""TG"ACCA IT'IONIOKIO G"G"TC""GCCGGCTAAGCC"AC"ACCAC ACCCGC"CCACGGCCACCTACCCCAGC"C C"ACCA"CGC""CACAGCCTC"G"CCC"G CGCCCAGAGGC""GCCGACCGGCCGCAGG GGGCGC"G""TCA""ACCAGAGGAC""GGA"""1 TCGCC"GCGA"A"CTATATCTGGGCACCC C"GGCCGGAACC""GCGGCG"AC"CCTGCr1 G"CCC"GG"CA"CACGCTC"AT"GTAA"C ACAGGAACAGA"CCAAAAGAAGCCGCC"G C"CCA"AGCGA""ACATGAATA"GAC"CC ACGCCGCCCTGGCCCCACAAGGAAACACT ACCAGCCT"ACGCACCACC"AGAGA"""C GCTGCC"A"CGGAGCAGGG"GAAG""""C CAGA"C"GCAGATGCACCAGCGTA"CAGC AGGGCCAGAACCAACTGTA"AACGAGC"C AACC"GGGACGCAGGGAAGAGTATGACGT CAAGCGCAGAGGACGGGACCC"G AGA"GGGTGGCAAACCAAGACGAAAAAAC CCCCAGGAGGG"CTC"A"AATGAGC"GCA GAAGGATAAGA"GGC"GAAGCCTA""CF AAA"AGGCA"GAAAGGAGAGCGGAGAA GGAAAAGGGCACGACGG"TTGF ACTCAGCAC"GC"ACGAAGGA"ACF ACGC"CTCCACA"GCAAGCCC"GCCACCr (CARl.4) ATGGCAC"CCCCGTAAC"GCTCTGCr MALPV"AL..PLALL.

Clone 24Cl GCCG"TGGCAT"GCTCC"GCACGCCGCAC HAARPDIQ4"QSPSSJ THD CAR GCCCGGA"ATCCAGC"CACGCAA"CCCCC SASVGDRVSF"CQASQ DNA LXH TCAAGCT"GAG"GCC"CCG"GGGCGACCG JNWYQQKPGKA GGTG"CC"TCACATG"CAGGCAAGCCAAG _ ‘GVD ACATAAA"AAT"TCC"GAA"TGG"ACCAA DF"FTI CAAAAACCCGGCAAGGCTCCCAAACTCCT '. 1YYCQQY [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GAT"TATGATGCC"CCAA"C"GGAGACCG GNL?FTFGGG"KVEIK GGG"CCCTTCTAG r1TCAGCGGAAG"GGC RGGGGSGGGGSGGGGS AGCGGCACAGACT""ACA"T"ACTA"CTC SGPGIVK?S? I""1C"C"GCAACCAGAGGACA"CGCCACAT ISITCTVSGGSISSY AC"AT""GCCAGCAA"ACGGCAA"CTGCCC WSWI RQ .3PGKGLEWI I""1CACCW'"CGGAGGCGGAACCAAGG""AGA YIYYSGS"NYNPSLK AA""AAAAGGGGCGG"GGAGGC"CCGGAG QV"ISVD"SKNQFSL GGGGGGGCTC"GGCGGAGGGGGC"CCCAA ADTAVYYCV 1GCAGGAG"CAGGGCC"GGACT JVYCGGDCYSGFDYW 1"" CAGAAACT"1 r" G"1 CAC"1 GA " LVTVS SAAAJ DN 1G"CCGGCGGAAGCA"T"CC I 1IIHVKGK-LJC 1GGTCC"GGA""AGACAGCC _ _ _ ?SKPFWV4V ACCCGGCAAAGGACTGGAA"GGAT"GGAr1 .ACYSL.V"VA ATA"1 "AC"1 C"1 GGAT C""ACAAAC"A"1 RS KRS R . 1CAAA"CCAGGG"CAC"A" _R ?" "Gr 1CAAAGAAT CAG""""C"1 _ _ R 1GAAGC"GAGCTCAG"CAC"GC"GCC GACACCGCAG"1 G""ACI"AT r" G"1 G"1 GAGC C"1 GG"CTAC"GCGGCGGAGA"TGC"ACAGCG G"""CGA""AC"GGGGCCAGGGCACCCTG GmmAGmmCCGCGGCmGCmCmmGA I'"AACGAGAAG"1 CCAACGG"ACGA"1TA"1 CC ACG""AAGGG"AAGCACC"T"GCCC"AGC KDTYDA CCGC"GT"CCCAGGCCCCAG"AAGCCC"T r1TGGG"CC"CG""G"GGTAGG"GGGG"AC r1CGCC"GC"AC"CCC"GC"CG"CAC"G"C GCAT"CA"CA"C"TC"GGGTCAGA"CCAA AAGAAGCCGCC"GC"CCA"AGCGA""ACA r1GAA"ATGAC"CCACGCCGCCCTGGCCCC ACAAGGAAACAC"ACCAGCCT"ACGCACC ACC"AGAGA"""CGCTGCC"A"CGGAGCA AG""""CCAGA"C"GCAGATGCA CCAGCGTA"CAGCAGGGCCAGAACCAACT GTA"AACGAGC"CAACC"GGGACGCAGGG AAGAGTATGACGTTTTGGACAAGCGCAGA GGACGGGACCC"GAGA"GGGTGGCAAACC AAGACGAAAAAACCCCCAGGAGGG"CTCr1 A"AATGAGC"GCAGAAGGATAAGA"GGCr1 GAAGCCTA""C"GAAA"AGGCA"GAAAGG AGAGCGGAGAAGGGGAAAAGGGCACGACG G"r 1ACCAGGGACTCAGCAC"GC"ACG 1AC""A"GACGCTCTCCACA"GCA 1GCCACCTAGGTAA (CARl.4) 1ATCCAGC"CACGCAA"CCCCCTCAAG DIQLTQSPSSLSASVG Clone 24Cl 1GAG"GCC"CCG"GGGCGACCGGG"Gr1 DRVSF"CQASQDINNF THD CAR 1TCACA"G"CAGGCAAGCCAAGAC "A JNWYQQKPGKAPK.

DNA LXH 1GAA""GG"ACCAACAAAA YDASN.?"GVPSRF "CCTGA."""""1 DF"FTISS_ 1GGAGACCGGGG"C I 1YYCQQYGNJ.

"AG ""CAGCGGAAG"GGCAGCGG TFGGG"KVEIKRGGGG CT""ACA"T"ACTA"CTC""C"C SGGGGSGGGGSQVQJQ r1GCAACCAGAGGACA"CGCCACATAC"AT ?SGPGIVK?S?TISIT r1GCCAGCAA"ACGGCAA"CTGCCC""CAC CTVSGGSISSYYWSWI C""CGGAGGCGGAACCAAGG"AGAAAC"A QQPPGKGLEWIGYIYY GCGG"GGAGGCF1CCGGAGGGGGG YNPSLKS RVF1I GGCTC"GGCGGAGGGGGC"CCCAAG"ACA NQFSLKJS A""GCAGGAG"CAGGGCC"GGACTCG"GA TAADTAVYYCVSJVYC AGCC""CAGAAACTTTG"CAC"GACA"GT GGDCYSGFDYWGQG"L ACAG"GTCCGGCGGAAGCATT"CCAG"TA VTVSSAAALDNEKSNG [Annotation] eadley None set by Anne.Headley ation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley CTA"TGGTCC"GGA"TAGACAGCCACCCG 1IIHVKGK-LJCPSPLF GCAAAGGACTGGAA"GGATTGGA"ATA"C ?G?SKPFWV4VVVGGV "AC"ACTC"GGATC"ACAAAC"AF 1CC .ACYSL.V"VAFIIFW CAGCC"CAAA"CCAGGG"CAC"AF 1G QSKRSR..{SDYMNM "GGA"ACA"CAAAGAATCAG""CF "r D QQPG?"RKHYQPYA AAGC"GAGCTCAG"CAC"GC"GCCGAC _ _D RDFAAYRSRVKFSR CGCAG"G"AC"AT"G"G"GAGCC"GGr1 DAPAYQQGQNQLYN AC"GCGGCGGAGA"TGC"ACAGCGG""F HIVIG ﬁﬁYDVLDKR GA""AC"GGGGCCAGGGCACCCTGG""AC R CG""AG""CCGCGGC"GC"C""GA"AACG .G .

AGAAG"CCAACGG""ACGA""TA""CCACG"""1 ﬁIGWKGﬁRRRGKGH AAGGG"AAGCACC"T"GCCC"AGCCCGCr1 TATKDTYD GT"CCCAGGCCCCAG"AAGCCC"T"TGGG .{MQA.??R r1CC"CG"""G"GGTAGG"GGGG"AC"CGCC C""CCC""GC"CG"CAC"G"CGCATr1 CA"CA"C"TC"GGGTCAGA"CCAAAAGAA GCCGCC"GC"CCA"AGCGA""ACA"GAAr1 ATGAC"CCACGCCGCCCTGGCCCCACAAG GAAACAC"ACCAGCCT"ACGCACCACC"A GAGA"""CGCTGCC"A"CGGAGCAGGG"G AAG""""CCAGA"C"GCAGATGCACCAGC GTA"CAGCAGGGCCAGAACCAACTGTA"A ACGAGC"CAACC"GGGACGCAGGGAAGAG TATGACGTTTTGGACAAGCGCAGAGGACG GGACCC"GAGA"GGGTGGCAAACCAAGAC GAAAAAACCCCCAGGAGGG"CTC"A"AAT GAGC"GCAGAAGGATAAGA"GGC"GAAGC CTA""C"GAAA"AGGCA"GAAAGGAGAGC GGAGAAGGGGAAAAGGGCACGACGG"TTG 1ACCAGGGACTCAGCAC"GC"ACGAAGGA 1AC"""A""GACGCTC"CCACA"GCAAGCCC C"AGG (CARl.5) ATGGCAC"CCCCGTAAC"GC"CTGC"GCT MAL?VTAL..PLALL.

Clone 24Cl GCCG""GGCAT"GC"CC"GCACGCCGCAC HAARPDIQ4"QSPSSJ CHD CAR GCCCG 1ATCCAGC"GACCCAG""C""CCA DRVSF"CQASQ DNA LXH TCC"CF 1GAG"GCC"CCG"GGG"GACCG CG"C"CF"TCAC""GCCAAGCCAGCCAAG ACA"CAACAAC"""C"GAA"F 1ACCAG CAGAAACCAGGCAAAGCACCAAAGC"CCr1 CGACGCC"CCAACC"GGAAACCG GGG"GCCCAGCAGG""TAGCGGGAGCGGr1 r1C"GGCACGGATT""ACG"TCACCA"C""C C"C"C"GCAGCCCGAGGA"A"AGCr1 1" A"1 I'"AC"1 G"1 CAGCAG""ACGGGAAT C"1 GCCA ?PGKGL I'1""AC""T""""GGGGG"GGAAC""AAGG""GGA YIYYSGS"NYNPSLK AA"CAAAAGGGGCGGCGGGGGAAGCGGGG RV"ISVD"SKNQFSL GCGGGGGC"CAGGTGGCGGAGGGAGCCAG JSSVTAADTAVYYCV G"GCAAC"CCAGGAAAGTGGCCCAGGA"T JVYCGGDCYSGFDYW GG"GAAGCCCAGCGAGACCCTTTCCCT"A VSSAAAIEV C""G"AC"G""AGCGGAGGCAGCA"AAGC DN?KSNG"I AGC"AC"A""GGTCC"GGA"CAGACAGCC HZQMNMQKH2m2H {VKGK{JC?SPLF?G ACCAGGGAAAGGGCT"GAA"GGAT"GGCr1 VJVVVGGVJA ACA""F 1A"TCCGGGTCCACCAAC"AC CYSLJV"VAFIIFWV AACCCA"CCC"CAAG"CCCGCG"GACAAr1 SKRSR..{SDYMNM"_ r1TCCG"CGACACAAGCAAGAACCAG"""Cr1 RRPG?"RKHYQPYA?.

AAC""AGTAGCG"CAC"GC"GCA RDFAAYRSRVKFSRSA GA"ACAGCAG""GTAC"AT"GTG"CAGCCr1 DAPAYQQGQVQLYNj. r1G"C"AC""G"GGCGGCGACTGC"ACAG"G NLGRRﬁﬁYDVLDKRRG GCr 1GA"TACTGGGGACAGGGCACGC"C RDPEWGGKPRRKNPQE [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley G"GACAG"G"CCAGCGCTGCGGC"AF GLYNELQKDKMAEAYS GG"AA"G"A"CCGCCACCGTA"Cr ﬁIGMKGﬁRRRGKGHDG ACGAGAAGTC"AATGGGACAA"CAF .YQGISTATKDTYDAL G"GAAGGGGAAGCACCF1Gr1GTCCA" {MQAJP ?R CC"GT""CCGGG"CCCAG"AAACCCF GGG"GC"TG""G"CGT"GGCGGGGF GCC"GC"A""CCC"GC"GGTGACCG"C GTmmAmmAmmmTCmGGGTmAGAmCCAAAA GAAGCCGCC"GC"CCA"AGCGA"TACA"G AC"CCACGCCGCCCTGGCCCCAC AAGGAAACAC"ACCAGCC""ACGCACCAC C"AGAGA"""CGCTGCC"A"CGGAGCAGG G"GAAG""""CCAGA"C"GCAGATGCACC AGCGTA"CAGCAGGGCCAGAACCAACTGT AGC"CAACC"GGGACGCAGGGAA GACGTTTTGGACAAGCGCAGAGG ACGGGACCC"GAGA"GGGTGGCAAACCAA AAAACCCCCAGGAGGG"CTC"Ar1 AATGAGC"GCAGAAGGATAAGA"GGC"GA AGCCTA""C"GAAA"AGGCA"GAAAGGAG AGCGGAGAAGGGGAAAAGGGCACGACGGr1 TTG"ACCAGGGACTCAGCAC"GC"ACGAA GGA"AC""A"GACGCTCTCCACAr CCC"GCCACCF (CARl.5) GA"ATCCAGCF 1C"CCATCC"C DIQLTQSPSSLSASVG Clone 24Cl T""GAG"GCCF 1GACCGCG"Cr1 DRVSF"CQASQDINNF CHD CAR C"""CAC"F r1C JNWYQQKPGKAPK.

DNA LXH AACAAC""F 1ACCAGCAGAA YDASN ﬁ"GVPSRF ACCAGGCAAAGCACCAAAGC"CC"CA.""Cr1 1FTISS ACGACGCC"CCAACC"GGAAACCGGGG"G I 1YYCQQYGNJ.

CCCAGCAGG""TAGCGGGAGCGG""C"GG TFGGG"KVEIKRGGGG CACGGATT""ACG"TCACCA"C"CC"C"C SGGGGSGGGGSQVQJQ 1GCAGCCCGAGGA"A"AGC"AC""A""AC ?SGPG.VK?S?T.S.T 1G"CAGCAG"ACGGGAATC"GCCA"""AC CTVSGGSISSYYWSWI 1Tm'"GGGGG"GGAAC"AAGG"GGAAA"CA RQ?PGKGLEWIGYIYY AAAGGGGCGGCGGGGGAAGCGGGGGCGGG S GSF1NYNP S LKS RV" I GGTGGCGGAGGGAGCCAGG"GCA SVD"SKNQFSLKJS AC"CCAGGAAAGTGGCCCAGGA"TGG"GA TAADTAVYYCVS AGCCCAGCGAGACCCTTTCCCT"AC"""Gr1 GGDCYSGFDYWGQG".

AC"1 AGCGGAGGCAGCA""AAGCAGC""A VFW/S SAAAI EVMYP P _ C"A""GGTCC"GGA"CAGACAGCCACCAG Y.DN?KSNG"II{VKG GGCT"GAA"GGAT"GGC""ACA"""1 K{4C?SPLF_ r1AC"A""TCCGGGTCCACCAAC"ACAACCC WVJVVVGGV A"CCC"CAAG"CCCGCG"GACAA""TCCG V"VAFIIFWV r1CGACACAAGCAAGAACCAG""C"CCC"G DYMNMF_ AAAC"1I'"AGTAGCG"1 CAC"1 GC"1 1AC .3 _ AGCAG"GTAC"A""GTG"CAGCC""G"Cr1 AC"G"GGCGGCGAC"GC""ACAG""GGC"""""1 GA""ACTGGGGACAGGGCACGC"CG"GAC CAGCGC"GCGGC"A"CGAGG"AA r1G"A""CCGCCACCG"A"C"GGACAACGAG AAG"1 C""AAT GGGACAA"1 CA"1 " " GAA GGGGAAGCACC"G"GTCCA"CCCCCC"GT I""1CCGGG"CCCAG""AAACCC""TCTGGG""G C"TG""G"CGT"GGCGGGG"GC"GGCC"G C"A""CCC"GC"GGTGACCG"CGCGT""A I'"""A""""""TC"GGGT""AGA""CCAAAAGAAGC CGCC"GC"CCA"AGCGA"TACA"GAA5AT GAC"CCACGCCGCCCTGGCCCCACAAGGA [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley AACACTACCAGCCT"ACGCACCACC"AGA GA"""CGCTGCC"A"CGGAGCAGGG"GAA G""""CCAGA"C"GCAGATGCACCAGCGT A"CAGCAGGGCCAGAACCAACTGTA"AAC AACC"GGGACGCAGGGAAGAGTA TGACGTTTTGGACAAGCGCAGAGGACGGG ACCC"GAGA"GGGTGGCAAACCAAGACGA CCCCAGGAGGG"CTC"A"AATGA GC"GCAGAAGGATAAGA"GGC"GAAGCCT A""C"GAAA"AGGCA"GAAAGGAGAGCGG AGAAGGGGAAAAGGGCACGACGG"TTG"A ACTCAGCAC"GC"ACGAAGGA"A C""A"GACGC"CTCCACA"GCAAGCCC"G CCACC"AGG (CARl.6) A"GGCAC"CCCCGTAAC"GCTCTGCTGCr1 MAL?VTAL..PLALL.

Clone 24Cl GCCG""GGCA"TGC"CC"GCACGCCGCAC HAARPDIQ4"QSPSSJ CD8 CAR GCCCGGACA""CAA""GACCCAG"CCCCr1 DRVSF"CQASQ DNA LXH AGCAGF CAAG"G"GGGAGATAG GG""G" "TCACC"G""CAGGC" ACAACT"CC"CAA""GGr CAGAAGCCAGGGAAGGCACCAAAGCF CA"A"ATGACGC""CAAACC""GAAACCG GAG"ACCTAGCCGC""CAGCGGAAGCGGA r1CAGGGAC"GAC""CACTTT"ACCA"CTC I'1"CAC"GCAGCCCGAAGACA"CGCCACA" AC""AC"1GCCAGCAG"ACGGAAAC"TGCC" ?PGKGLEWI I'1""ACA.T"'"""GGGGGCGGCACCAAAG" IYYSGS"NYNPSLK G I'1""AAGCGAGGGGGAGGCGGC"CAGGAG r1ISVD"SKNQFSL GCGG"GGC"CCGGAGGCGGGGG""CCCAG JSSVTAADTAVYYCV G"CCAGC"CCAGGAA"CCGGCCCAGG"CT JVYCGGDCYSGFDYW GG""AAGCCCAG"GAAACT""G"CCC"CA 14V"VSSAAA_JSN CG"G"AC"G"GAGCGGTGG""CAATC"CC (DQMNMQKIi—J H K ,_< "21 U) {FV_ _?AK "CA"AC"A"F "C"r 1ACGGCAACC _ ?%?. _ ?TI "CC"GGAAAGGGCCF 1GGATCGGCr1 _.S.R?H ?AA AC"A"AG"GGCTCCAC"AA""AC {TRGJ DIY AACCCW'"CCC"CAAG"CCAGAG"CACCAr1 __ S I""1CCG"GGACACA"C""AAGAACCAGW'"CA G"C"GAAG"TG"CCAGCGF"ACAGCCGCA GACACAGCCGW'" "Gr "C"1 r1G""TAC"GCGG GcmmCGAmmACr "G GTCACAG"C"CF"CCGCGGCCGCCCF 1C mAACAGmAmmAmGmACTTmmCmCAmr "G . _ _ "G""CC""CCCGC"AAGCCAACr1 _ DKMAﬁAYS AC"ACCCCGGCCCCACGGCCGCCTACCCC I RGKG 1GCACCCACAA"AGCCAG"CAGCC"""GA 1GAGACC"GAGGCT"G"CGGCCGGCr1 1GGGGGTGCAG"GCACACACGAGG"Cr1 mmeGCmr "A"ACATC"GGG CCCC"Cr 1G"GGGG"GC"G CT"C"GAGCF 1CACGCTC"A""""G CAACCA"CGCAACAGA"CCAAAAGAAGCC GCC"GC"CCA"AGCGA""ACATGAATA"G AC"CCACGCCGCCCTGGCCCCACAAGGAA ACACTACCAGCCT"ACGCACCACC"AGAG A"""CGCTGCC"A"CGGAGCAGGG"GAAG I'"""""""CCAGA"C"GCAGATGCACCAGCGTA r1CAGCAGGGCCAGAACCAACTGTA"AACG AGC"CAACC"GGGACGCAGGGAAGAGTAT [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley GACGTTTTGGACAAGCGCAGAGGACGGGA CCC"GAGA"GGGTGGCAAACCAAGACGAA AAAACCCCCAGGAGGG"CTC"A"AATGAG C"GCAGAAGGATAAGA"GGC"GAAGCCTA I""1C"GAAA""AGGCA""GAAAGGAGAGCGGA GAAGGGGAAAAGGGCACGACGG"TTG"AC CAGGGACTCAGCAC"GC"ACGAAGGACAC I'"""A""GACGC"CTCCACA"GCAAGCCC""GC CACC"AGG""AA (CARl.6) GACA"TCAA""GACCCAG"CCCCTAGCAG DIQLTQSPSSLSASVG Clone 24Cl r1C"C"CAGCAAGTG"GGGAGATAGGG"Gr1 DRVSF"CQASQDINNF CD8 CAR CA"TCACC"G"CAGGC"TCACAGGAC r1C KPGKAPK.

DNA LXH AACAACT"CC"CAA""GG"A"CAGCAGAA YDASN.?"GVPSRF GCCAGGGAAGGCACCAAAGC"GCTCPFAr1 DF"FTISS ATGACGC""CAAACC""GAAACCGGAG"A I 1YYCQQYGN4_ CCTAGCCGC""CAGCGGAAGCGGA"CAGG TFGGG"KVEIKRGGGG GAC"GAC""CACTTT"ACCA"CTC""CAC SGGGGSGGGGSQVQJQ r1GCAGCCCGAAGACA"CGCCACA"AC"AC ?SGPGIVK?S?TISIT r1GCCAGCAG"ACGGAAAC"TGCC"""""""AC CTVSGGSISSYYWSWI AT""GGGGGCGGCACCAAAG"GGAG ""A RQ?PGKGLEWIGYIYY AGCGAGGGGGAGGCGGC"CAGGAGGCGG"1 SGSF1NYNPSLKS RVF1I GGC"CCGGAGGCGGGGG""CCCAGG"CCA SVD"SKNQFSLKJSSV 1CCAGGAA"CCGGCCCAGG"CTGG""A TAADTAVYYCVSJVYC ""G"CCC"CACG"G" GGDCYSGFDYWGQG"4 ""CAATC"CC"CA"A AAJSNSIMYF 1ACGGCAACC""CC""G m -IFV_ _ ?AKP""T"1 _ GAAAGGGCCF 1GGATCGGC"A"A"C y ? __ _ ?.

I'"AC""A""AG""GGCTCCAC""AA""""ACAACCC m ' _ ?AAGGAV I""1CCC""CAAG"CCAGAG"CACCA""""""CCG H R _ DIYIWA__ r1GGACACA"C"AAGAACCAG""CAG"C"G >G) ISIVIT AAGF1TG"CCAGCGF1F1ACAGCCGCAGACAC RSK RS R . .HS AGCCGmmmATmACmGTGmeCmCmmGr S _Q%?G?mQK AC"GCGGGGGAGAC"G""A"AGCGGCF 'U _RDFAAYRS GA"F "GGGGCCAGGGCACC"F AGF ""CCGCGGCCGCCC"CF GmAmr 1GmACTTmmCmCAmmmr IT'IONIOKIOA G"G"r ""CCCGC"AAGCCAACF CCCGGCCCCACGGCCGCCTAC CCACAAF1AGCCAG"CAGCCW'" AGACC"GAGGCT"G"CGGCCGGCF GGGTGCAG"GCACACACGAGG"CF T"GC"F "A"ACATC"GGGCCCCF Cr "G"GGGG"GC"GCT"CF GAGCF 1CACGCTC"A""GCAACC A"CGCAACAGA"CCAAAAGAAGCCGCC"G AGCGA""ACATGAATA"GAC"CC CCCTGGCCCCACAAGGAAACACT CT"ACGCACCACC"AGAGA"""C GCTGCC"A"CGGAGCAGGG"GAAG""""C CAGA"C"GCAGATGCACCAGCGTA"CAGC AGGGCCAGAACCAACTGTA"AACGAGC"C AACC"GGGACGCAGGGAAGAGTATGACGT TTTGGACAAGCGCAGAGGACGGGACCC"G AGA"GGGTGGCAAACCAAGACGAAAAAAC CCCCAGGAGGG"CTC"A"AATGAGC"GCA GAAGGATAAGA"GGC"GAAGCCTA"F AAA"AGGCA"GAAAGGAGAGCGGAGAA GGAAAAGGGCACGACGG"TTGF ACTCAGCAC"GCTACGAAGGAF [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley 1CTCCACATGCAAGCCCTGCCACCF (CAR2.l) ATGGCAC"CCCCGTAAC"GC"CTGC"GCr1 _ _.ALL Clone 24C8 GCCG""GGCA"TGCTCC"GCACGCCGCAC _ ?SGPG THD CAR GCCCGCAGGTACAGCTGCAGGAAF DNA HXL CCCGGAC"TG"CAAGCCAAGF I""1C"C""TACA""GTACCG""GA AAGCAG"1 GGAGGC"1 " ACGGCAGCACCCA SKN GGAG"GGA"TGGA"ACA"F TAVYYCASJVYCGGDC GATC"ACACAC"A"AATCCAF WGQG"LVTVS "CCCGGG"CACCA"TAGCATF SGGGGSGGGGSGGGGS "AAGAA"C"G""CAGTC"CAGGCr 1CCT DIQLTQSPSSJSASVG C"GC"GCCGACACAGCCG"G"AC DRVSF"CQASQDINNF r1AC"GCGCC"CC""GG"""AC"GCGGAGG JNWYQQKPGKAPKJLI CGACTG""A"AGCGGC"""GA"TA""GGG YDASN ."GV?SRFSG GGCAGGGGACCC"CGTAACCG"GAGC"CT SGSGr1 1FTISSJQ? GGAGGGGG"GGGAGCGGGGGAGGAGG"TC EDIAF JPF AGGGGGGGGCGGC"CCGA"A"CCAGC"CA r1FGGG"KVI 4 GCCCCTC"AGTC"C"C"GCC"CA DNEKSWG"IIHVKGK{ G"GGGGGA"CGGG"CAG""T"AC""G"CA .C?SP.F?G?SKPFWV AGCTTCACAGGA"A"CAACAAC""CC"TA JVVVGGVIACYSL A""GG"A"CAGCAGAAGCCAGGAAAAGCA VAFIIFWVRSKRS CCCAAGC"GCTCA"C"A"GA"GCC"CAAA _R I'"""""GGAGACGGG"G""""CCCAGTCG I'"""CT C"GGG"CAGGG"CCGGGACCGACT""ACG I'"""""ACGA""CTCC"C""C""GCAGCCCGAAGA CA"1 CGCCACA""ACI'"A"1 r" G"1 CAACAG""ACG GCAAC"TGCC"""CACAT"TGGGGGCGGG AC"AAGG""GAAA"CAAGAGGGCCGC"GC ACr" GGACAA"1 GAGAAG"1 CCAACGGCACCA ACG"GAAGGGCAAGCACC"G"GC DA CC"AG"CC"C"G""CCCAGGCCCA"CCAA R AccmmmmTGGGmmcmTGmmeGGTCGGGG GGG"GC"GGCC"GC"A""C"C"GC"GGTC GCCT"CA"AA""""C"GGGT"AG A"CCAAAAGAAGCCGCC"GC"CCA"AGCG A"TACA"GAA"ATGAC"CCACGCCGCCCT GGCCCCACAAGGAAACAC"ACCAGCC""A CGCACCACC"AGAGA"""CGCTGCC"Ar GGAGCAGGG"1GAAG"1 r" r" r" CCAGA"1 C" GATGCACCAGCGTA"CAGCAGGGCCAGAA GTA"AACGAGC"CAACC"GGGAC GCAGGGAAGAGTATGACGTTTTGGACAAG CGCAGAGGACGGGACCC"GAGA"GGGTGG CAAACCAAGACGAAAAAACCCCCAGGAGG G"CTC"A"AATGAGC"GCAGAAGGATAAG A"GGC"GAAGCCTA""C"GAAA"AGGCAr1 GAAAGGAGAGCGGAGAAGGGGAAAAGGGC ACGACGG"TTG"ACCAGGGACTCAGCACr1 GC"ACGAAGGA"AC""A"GACGC"C"CCA CA"GCAAGCCCF 1AGGTAA (CAR2.l) CAGGTACAGCTGCAGGAA"CTGGGCCCGG IQTSGPGIVKPSQ Clone 24C8 AC"TG"CAAGCCAAG"CA """C"C .4TCTVSGGSISSG THD CAR GTACCG"GAGCGGCGGAAG"A"A GFYWSWIRQHPGKG.* DNA HXL AGCAG""GGAGGC"" "GGTC""""GGA"1 WI GYI {HSGSF .35 ACGGCAGCACCCA 1GGAGr1 _ RVTISIDF 4F GGATTGGA"ACA"F 1CAGGATCr1 JSSVTAADTAVYY ACACACTA"AATCCAF 1AAG"CCCG JVYCGGDCYSGFD [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GG"CACCA"TAGCATTGA"ACGTC"AAGA YWGQG"LVTVSSGGGG A"CTG""TCAGTC""CAGGC"G"CCTCCG"C SGGGGSGGGGS DIQLT AC"GC"GCCGACACAGCCG"G"AC"AC"G QSPSSJSASVGDRVSF CGCC"CC""GG"""AC"GCGGAGGCGACT r1CQASQDINNF.

G""A"AGCGGC"""GA"TA""GGGGGCAG QKPGKAPK GGGACCC"CGTAACCG"GAGC"CTGGAGG ?"GV?SRFSGSGSGF GGG"GGGAGCGGGGGAGGAGG"TCAGGGG "FTISS.Q??DIAF GGGGCGGC"CCGA"A"CCAGC"CAC"CAA JPF"FGGG AGCCCCTC"AGTC"C"C"GCC"CAG"GGG I .DN?KS GGA"CGGG"CAG""T"AC""G"CAAGCTT GK-LJC?SP CACAGGA"A"CAACAAC""CC"TAA""GG _ _ ?SKPFWV4VVVG r1A"CAGCAGAAGCCAGGAAAAGCACCCAA .ACYSLIV"VAFII GC"GCTCA"C"A"GA"GCC"CAAA"""GG FWV R.

AGACGGG"G"""CCCAGTCG I'"""CTC""GGG " _ .3" r1CAGGG"CCGGGACCGACT""ACG"""AC YA?_ GA"CTCC"C"C"GCAGCCCGAAGACA"CG CCACA""AC""A"""1 G"1 CAACAGF1ACGGCAAC YN' . r1TGCC"""CACAT"TGGGGGCGGGAC"AA KR GG""GAAA"CAAGAGGGCCGC"GCAC"GG ACAA"GAGAAG"CCAACGGCACCATC r1C ﬁAYS CACG"GAAGGGCAAGCACC"G"GCCC"AG GH "1CC"C"G""""CCCAGGCCCA"CCAAACCW'1 I'"""TGGGW'"C""TG""""G""GGTCGGGGGGG""G "GC"A"""C"C"GC"GGTCACGGr1 GGCCT"CA"AA""""C"GGGT"AGA"CCA AAAGAAGCCGCC"GC"CCA"AGCGA"TAC A"GAA"ATGAC"CCACGCCGCCCTGGCCC GAAACACF1ACCAGCCF1F1ACGCAC CACC"AGAGA"""CGCTGCC"A"CGGAGC AAG""""CCAGA"C"GCAGATGC ACCAGCGTA"CAGCAGGGCCAGAACCAAC TGTA"AACGAGC"CAACC"GGGACGCAGG TATGACGTTTTGGACAAGCGCAG AGGACGGGACCC"GAGA"GGGTGGCAAAC CAAGACGAAAAAACCCCCAGGAGGG"CTC r1PFAATGAGC"GCAGAAGGATAAGA"GGC r1GAAGCCTA""C"GAAA"AGGCA"GAAAG GAGAGCGGAGAAGGGGAAAAGGGCACGAC "ACCAGGGACTCAGCAC"GC"AC GAAGGA"AC""A"GACGCTC"CCACA"GC AAGCCC"GCCACC"AGG 2) ATGGCAC"CCCCG"AAC"GC"CTGCTGCr1 _ _.ALL.

Clone 24C8 GCCGT"GGCA"TGCTCC"GCACGCCGCAC _ HSGPG.

CHD CAR GCCCGCAGGTGCAGC"GCAGGAAAGCGGr1 DNA HXL CCGGGAC""G"CAAGCCG"CCCAAACGCr1 GAG"C"GACG"GTAC"G"C"CTGGTGGCr1 CTAmmmCmmCCGGGGGCmmmmATmGGTCm 1GGA"CAGACAACACCC"GGCAAAGGGCr1 SKN 1GG 1AGGG"A"A""CACCAC"C"G TAVYYCASJVYCGGDC 1CAC"ACAACCCA"CA""GAAA YSGFDYWGQG"LVTVS 1CCAGAG"GAC"A"C"CAATCGACACA"C SGGGGSGGGGSGGGGS CAAGAACC""T"CAGCC"GAGG""G"CAr1 DIQLTQSPSSJSASVG CAG""ACCGCCGC""GACACCGCGG"G"Ar1 DRVSF"CQASQDINNF r1AT"GCGCC"C"C"CG"G"AC"GCGG"GG JNWYQQKPGKAPKJLI CGA""G"TA"AG"GGC"""GAC"AC"GGG ﬁ"GV?SRFSG GGCAGGGGACA""GGT"ACCG""TCAAGr1 1FTISSJQP GGAGGCGGTGGG"CTGGCGGGGGCGG"AG I 1YYCQQYGNJPF CGGAGG"GGGGGGAGCGACA"ACAGC"TA TFGGG"KVEIK%AAAI CGCAGAGCCCC"CCAGCCTT"CAGCC"CC EVMYP ?PY .DN'QKSNG [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley G"GGGGGATAGGG"G"CCTT"ACCTGCCA 1IIHVKGK-LJCPSPLF GGCTTCCCAGGACA"AAACAACTTCC"CA ?G?SKPFWV4VVVGGV A""GGTA"CAGCAAAAGCCCGGGAAAGCA .ACYSL.V"VAFIIFW CCAAAGC"GCTCA"C"ACGA"GCCAGCAA QSKRSR..{SDYMNM CC"GGAAACCGGAG"GCCGTCTCGC"""Cr1 D_RRPG?"QKHYQPYA G"GGCAG"GGGACCGATTTCACr1 YRSRVKFSRD T""ACAA"CTCAAG"""GCAGCCAGAAGA DAPAYQQGQNQLYN CA""GCAACATAC"AC"G"CAACAG"ACG H V.G ﬁﬁYDVLDKR GCAA"CTCCCC"T"ACAT""GGGGGGGGA R AC"AAAG"GGAGA""AAGCGCGCTGCAGC .G .

CA""GAAG"TA"G"ATCCGCCCCCG"A"C ﬁIGWKGﬁRRRGKGH TGGA"AACGAGAAA"C"AA"GG"ACCA"A .YQGISTATKDTYD A"ACA"G"GAAGGGGAAGCACC"CTGTCC ??R A"CACCGC"GT"CCCCGGCCC""CAAAAC CmmTCmGGGmACmCGmmeCGmGGGmGGA GmmcmGGccchmAmAGmcchmGGmGAC CG"GGCGT""A""CA"C""C"GGGTAAGAr1 GAAGCCGCC"GC"CCA"AGCGAr1 TACA"GAA"ATGAC"CCACGCCGCCCTGG CCCCACAAGGAAACAC"ACCAGCCT"ACG C"AGAGA"""CGCTGCC"A"CGG AGCAGGG"GAAG""""CCAGA"C"GCAGA TGCACCAGCGTA"CAGCAGGGCCAGAACC AACTGTA"AACGAGC"CAACC"GGGACGC AGGGAAGAGTATGACGTTTTGGACAAGCG ACGGGACCC"GAGA"GGGTGGCA AACCAAGACGAAAAAACCCCCAGGAGGGr1 CTC"A"AATGAGC"GCAGAAGGATAAGAr1 GGC"GAAGCCTA""C"GAAA"AGGCA"GA AAGGAGAGCGGAGAAGGGGAAAAGGGCAC GACGG"TTG"ACCAGGGACTCAGCAC"GC r1ACGAAGGA"AC""A"GACGCTC"CCACA r1GCAAGCCC"GCCACC"AGGTAA (CAR2.2) CAGGTGCAGC"GCAGGAAAGCGG"CCGGG .QTSGPGIVKPSQ Clone 24C8 AC""G"CAAGCCG"CCCAAACGC"GAG"C .4TCTVSGGSISSG CHD CAR "GACG"GTAC"G"C"C"GGTGGC"CTA"F GFYWSWIRQHPGKG.* DNA HXL I'1C""""CCGGGGGC""1"""AT"GGTC""""GGA" WI GYI {HSGSF .35 CAGACAACACCC"GGCAAAGGGCF JKSRV"ISIDF 4F GG "A"A""CACCAC"CF VTAADTAVYY ACF CCCA"CA""GAAAF CAS_JVYCGGDCYSGFD "C"CAATCGACACAF YWGQG"LVTVSSGGGG 1CAGCC"GAGG""""G""CA" SGGGGSGGGGS DIQLT ACCGCCGC"GACACCGCGG"G"AF QSPSSJSASVGDRVSF CGCC"C"C"CG"G"AC"GCGGF r1CQASQDINNF. 1TA"AG"GGC"""GAC"AC"GGGGGCAG QKPGKAPK.

""GGT"ACCG""TCAAG"GGAGG .T"GV?SRF 1CTGGCGGGGGCGG"AGCGGAG DF"FTISS 1GGGGGGAGCGACA"ACAGC"TACGCAG YYCQQYGN. 1CCAGCC"""CAGCC"CCG"GGG r1KVEIK GGA"AGGG"G"CC"""ACCTGCCAGGCTT H CCCAGGACA"AAACAACTTCC"CAA""GG KGK{.C?S_ _ TA"CAGCAAAAGCCCGGGAAAGCACCAAA ?FWV_JVVVGGV_ GC"GCTCA"C"ACGA"GCCAGCAACC"GG JLV"VAFIIFWV AAACCGGAG"GCCGTCTCGC""C"C"GGA SR . u-IS 1 _ 1GGCAG"GGGACCGATTTCAC"T""AC GP"RKHYQPYA?_ AA"CTCAAG"""GCAGCCAGAAGACA§"G AAYRSRVKFSRS CAACATAC"AC"G"CAACAG"ACGGCAAT AYQQGQVQLYN' CTCCCCTT"ACAT"TGGGGGGGGAAC"AA QRﬁﬁYDVLDKR AG"GGAGA""AAGCGCGCTGCAGCCAﬁ"G EMGGKPRRKNPQH [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley AAG"TA"GTATCCGCCCCCGTAF1CTGGAT I LQKDKMAﬁAYS ﬁIG AAATC"AATGG"ACCA"AA"ACA GERRRGKGHDGJYQ r1GTGAAGGGGAAGCACC"CTGTCCA"CAC JSTATKDTYDALiMQ CGC"GT"CCCCGGCCC"""CAAAACC""""TC .4}? ?R mGGGmAcmCGmmeCGmGGGmGGAGmmCm GGCC"GC"A"AG"C"GC"GG"GACCG"GG CGT""A"CA"C""C"GGGTAAGA"CCAAA AGAAGCCGCC"GC"CCA"AGCGA"TACAr1 GAA"ATGAC"CCACGCCGCCCTGGCCCCA AACAC"ACCAGCCT"ACGCACCA CC"AGAGA"""CGCTGCC"A"CGGAGCAG GG"GAAG""""CCAGA"C"GCAGATGCAC CAGCGTA"CAGCAGGGCCAGAACCAACTG TA"AACGAGC"CAACC"GGGACGCAGGGA AGAGTATGACGTTTTGGACAAGCGCAGAG ACCC"GAGA"GGGTGGCAAACCA AGACGAAAAAACCCCCAGGAGGG"CTC"A r1AATGAGC"GCAGAAGGATAAGA"GGC"G AAGCCTA""C"GAAA"AGGCA"GAAAGGA GAAGGGGAAAAGGGCACGACGG 1ACCAGGGACTCAGCAC"GC"ACGA 1AC""A"GACGCTCTCCACA"GCAA 1GCCACC"AGG (CAR2.3) 1CCCCGTAAC"GCTCTGCTGCr1 _ ?.ALL.

Clone 24C8 GCCG""GGCA""GC"CC"GCACGCCGCAC _ ?SGPG.

CD8 CAR GCCCGCAGGTGCAG""GCAGGAAAGCGGG DNA HXL CCTGGCC""G"GAAACCAAGCCAGACACr1 GAGCC"GACA"GCAC"G"G"CCGGCGGGr1 CCATA"C""CCGGGGG""T"TATTGGTCC _ r1GGA"ACGCCAGCATCCCGGGAAAGGACr1 SKN r1GAA"GGA""GGA"A"A"CCACCA""CCG TAVYYCAS.

GAAGCACCCAC"ACAA"CCAAGCCF WGQG"LVTVS GG"GACAA"C"CCATCGACA SGGGGSGGGGSGGGGS AAAGAA"C""""""CCC"GCGG"T DIQLTQSPSSJSASVG CAG"AAC"GCCGCCGA"ACCGC"GF CQASQDINNF r1AC"GTGCCAGCC"CG"CTAT"GCG JNWYQQKPGKAPKIII AGA"TG""A""C"GGG""CGA""Ar YDASN.?"GV?SRFSG GTCAAGGCACAC"GG"AAC"G"CAGCAGC DF"FTISSJQ_ GGAGGCGGCGG""CCGGGGGCGGGGGCAG I 1YYCQQYGN.

TGGAGGGGGCGGA"C"GACAT"CAGC"F 1FGGG"KVEIK%AAA CGCAG"CCCCA"C""CAC""AGCGCCA MYFSiFV?VF__ TCGGG"CAGC""CACG"Gr1 " CAGGATA"1CAACAACT r" r" C" 1ACCAGCAGAAGCCAGGCAAG ""GCTGA""TACGATGC"F 1CGAGACGGGAG"GCC"AGCCG "GCCCG"C"TTC"GCCF 1ACCACCACCCCCGCCCCACGACCACC TAC"CCAGCCCCCACCA"CGCC"CCCAGC CCC"CAGCCTGAGGCCAGAGGC""G"CGC GCGGGGGGCGC"GTCCA"ACCAG AGGAC"CGACTTCGCCTGCGATA"T"ATA TA"GGGCCCCCCTCGCCGGCACC"GCGGA [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by eadley ation] Anne.Headley Unmarked set by Anne.Headley G"C""GCTCCTGAGCC"TGTGA"CACGCT T"A" r" GTAACCA"1 CGGAA""AGA"1 CCAAAA GAAGCCGCC"GC"CCA"AGCGA"TACATG AATA"GAC"CCACGCCGCCCTGGCCCCAC AAGGAAACACTACCAGCC""ACGCACCAC C"AGAGA"""CGCTGCC"A"CGGAGCAGG """"CCAGA"C"GCAGATGCACC AGCGTA"CAGCAGGGCCAGAACCAACTGT A"AACGAGC"CAACC"GGGACGCAGGGAA GAGTATGACGTTTTGGACAAGCGCAGAGG ACGGGACCC"GAGA"GGGTGGCAAACCAA GACGAAAAAACCCCCAGGAGGG"CTC"Ar1 AATGAGC"GCAGAAGGATAAGA"GGC"GA AGCCTA""C"GAAA"AGGCA"GAAAGGAG AGCGGAGAAGGGGAAAAGGGCACGACGGr1 TTG"ACCAGGGACTCAGCAC"GC"ACGAA GGA"AC""A"GACGCTCTCCACA"GCAAG CCC"GCCACC"AGG"AA 3) CAGGTGCAG""GCAGGAAAGCGGGCCTGG IQTSGPGIVKPSQ Clone 24C8 CC""G"GAAACCAAGCCAGACAC"GAGCC .4TCTVSGGSISSG CD8 CAR r1GACA"GCAC"G"G"CCGGCGGG"CCATA GFYWSWIRQHPGKG.* DNA HXL I'1C""""CCGGGGG""""T""TATTGGTCC""GGA"1 WI GYI {HSGSF .35 ACGCCAGCATCCCGGGAAAGGAC""GAAr1 JKSRV"ISIDF 4F 1A" 1CCACCA""CCGGAAGC SLRJSSVTAADTAVYY 1ACAA"CCAAGCC""AAA"CCCG CAS_JVYCGGDCYSGFD 1C"CCATCGACACC"CAAAGA YWGQG"LVTVSSGGGG 1CCC""GCGG""T 1A SGGGGSGGGGS DIQLT "ACCGC"GF 1G QSPSSJSASVG 1CG"CTAT"GCGGCGGAGA"T r1CQASQDINNF. 1GGG""CGA""A""GGGGTCAA QKPGKAPK.

GGCACAC"GG"AAC"G"CAGCAGCGGAGG SRF CGGCGG""CCGGGGGCGGGGGCAGTGGAG DF"FTISS GGGGCGGA"C"GACAT"CAGCF"ACGCAG YYCQQYGN. _ r1CCCCA"C""CAC""AGCGCCA r EIKQAAA CGATCGGG"CAGC""CACG"GF ?VF..

G"1 CAGGATA"1CAACAACT r" r" C" r1ACCAGCAGAAGCCAGGCAAG _ _.

G""GCTGA""TACGATGC""CF _ DIYIWA AGACGGGAG" 1AGCCGC""""CTCCGGG _ _ IS .VIT AGCGGCAGCGGCACAGACF1FV1ACC""T"AC . _ RS R .H GA"T" GCCAGAGGA""A""AG _ ?G .3" RK CAACF 1G"CAGCAG"A"GGCAAC _ AYRS Cr 1ACC""CGG"GG"GGCACAAA GGF 1AAAAGAGCCGCAGCG""Gr1 CCAACF 1AAmGTATmTTmCmCAmmmm G"GCCCG"C"TTC"GCC"GCCAAACC"AC CACCACCCCCGCCCCACGACCACCTAC"C CAGCCCCCACCA"CGCC"CCCAGCCCC"C AGCCTGAGGCCAGAGGC""G"CGCCC"GC r1GCGGGGGGCGC"G"CCA"ACCAGAGGAC "CGAC"TCGCC"GCGATA"TF 1PFGG GCCCCCC"CGCC "GCGGAG""C"""1 GCTCC"GAGCC"F "CACGCT""""A"""1 GTAACCA"CGGAAF 1CCAAAAGAAGC CGCC"GC"CCAF ""ACATGAATA" ACGCCGCCCTGGCCCCACAAGGA AACACTACCAGCC""ACGCACCACC"AGA GA"""CGCTGCC"A"CGGAGCAGGG"GAA GT"""CCAGATC"GCAGATGCACCAGCGT ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ATCAGCAGGGCCAGAACCAACTGTATAAC GAGC"CAACCTGGGACGCAGGGAAGAGTA TGACGTTTTGGACAAGCGCAGAGGACGGG ACCC"GAGA"GGGTGGCAAACCAAGACGA AAAAACCCCCAGGAGGG"CTC"A"AATGA GC"GCAGAAGGATAAGA"GGC"GAAGCCT A""C"GAAA"AGGCA"GAAAGGAGAGCGG AGAAGGGGAAAAGGGCACGACGG"TTG"A CCAGGGACTCAGCAC"GC"ACGAAGGA"A C""A"GACGC"CTCCACA"GCAAGCCC"G CCACC"AGG (CAR3.l) A"GGCAC"CCCCGTAAC"GC"CTGCTGCr1 MA.?VTA.

Clone GCCG""GGCA"TGCTCC"GCACGCCGCAC VQ 20C5.l THD GCCCGCAGG"CCAACTGG"GCAG"CCGGA ?GASVK CAR DNA. GCCGAAG"CAAGAAACCAGG"GCCTCCGF "?LSW{WVRQA.

HXL TAAAG"GAG""GCAAAG"CTCF .ﬁWMGGh CCGAGCTCTCTA"GC YAQKFQG CGGCAGGCCCCCGGCAAGGGAF 13""AYMT.

GATGGGCGGG"TCGATCC"GAGGACGGAG AVYYCAT AGAC"A"C"ACGC"CAAAAA""CCAGGGA FDYWGQG"LV"VS CGAG"GAC"G"GACCGAAGACAC"AG"AC GGSGGGGSGGGGS "GCC"ACA"GGAAC"""CCTC"C MTQSPSSJSASVG r1GCGA"CAGAAGA"ACCGCAG"G"AC"AC TI"CRASQSISSY.

"GTGC"AC"GAA"C"AGGGGCA"F 1G YQQKPGKAPKJLISGA GCCC"ACT"CGA""ACTGGGG"CAGGGAA SSJKSGV?SRFSGSGS C"CTGG"GACTG"C"CCAGCGG"GGAGGr1 G"DF".TISS.P??DF GGCAGCGG"GG"GGCGGAAGCGGGGGGGG A"YYCQQSYS"PI"FG CGGC"C"GA"A""CAGA"GAC"CAA"C"C QG"R.?IKRAAAIDN? C"C"G"CCGC""CCG"GGGCG " KSVG"IIHVKGK{4C? AGAG"GACCAT"AC""G"AGGGCGTCCCA SPJF?G?SKPFWV_ GmCAAmCmCCAGmTAmmmGAAmmGGmAmC VGGV AGCAGAAGCCCGGGAAAGCACC"AAGC"G IIFWV r1r 1(3]X(3(I(3(3(3(3(Ir"r"(IF"ZX(3(I(IF"(EZXZXCSZXCS bedIJD4r"_ I'1GGGG""ACCTTCACGGW'"CAGCGGAAGCG QPYA? _ GAACCGAT""CACCC"GACTA"C KFSRSA AGCAGCC"GCCACC"GAGGAC""TGCAAC QLYN? r1TAC"AC""GCCAACAG"CATACAGCAC"C LDKRRG CGATCAC"TTCGGCCAGGGCACCCGGC"C RKVPQ.

GAAA"CAAGCGCGC"GC"GC"""GGACAA MAﬁAYS r1GAGAAG"CAAACGGCACCA"CA"ACA"G GKGHDG I'1""AAAGG""AAACATC""G""GTCCC""CCCCG DTYDA CCCGGCCC""CCAAACCG"TC"G GG""C"GG"GG"GG"CGGAGGCG"AC"CG C""GC"A"AG"C"GC"GGTAAC"G"CGCC T"CA"CA"C""""GGGTGAGA"CCAAAAG AAGCCGCC"GC"CCA"AGCGA""ACA"GA A"ATGAC"CCACGCCGCCCTGGCCCCACA AGGAAACAC"ACCAGCCT"ACGCACCACC r1AGAGA"""CGCTGCC"A"CGGAGCAGGG r1GAAG""""CCAGA"C"GCAGATGCACCA GCGTA"CAGCAGGGCCAGAACCAACTGTA r1AACGAGC"CAACC"GGGACGCAGGGAAG AGTATGACGTTTTGGACAAGCGCAGAGGA CGGGACCC"GAGA"GGGTGGCAAACCAAG ACGAAAAAACCCCCAGGAGGG"CTC"A"A "GCAGAAGGATAAGA"GGC"GAA GCCTAT"C"GAAA"AGGCA"GAAAGGAGA GCGGAGAAGGGGAAAAGGGCACGACGG"T TGTACCAGGGACTCAGCAC"GCTACGAAG [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley 1ACTTATGACGCTCTCCACATGCAAGC 1GCCACCTAGG"AA (CAR3.l) CAGG"CCAACTGG"GCAG"CCGGAGCCGA QVQLVQSGAEVKK.

Clone GAAACCAGGTGCCTCCGTTAAAG SVKVSCKVSGY". 20C5.l THD TGAG""GCAAAGTCTC"GGATACACTCTG SWiWVRQA?GKG.H CAR DNA. ACCGAGCTCTCTA"GCAC"GGG"CCGGCA GGbDPﬁDG."IYAQKF HXL GGCCCCCGGCAAGGGA"TGGAA"GGATGG QVTVTED"S"D"AY GCGGG"TCGATCC"GAGGACGGAGAGACr1 HISSIRS?DTAVYYC A"Cr 1CAAAAA""CCAGGGACGAGr1 ATESRGIGWPYFDYWG GACr CAC"AG"ACCGACA QG"LV"VSSGGGGSGG C""GCC" 1GGAAC""""""CCTC""C""GCGA GGSGGGGS DIQMTQSP r1CAGAAGA"ACCGCAG""G"AC"AC"GTGC SSJSASVGDRVTI"CR r1AC"GAA"C"AGGGGCA""GGA"GGCCCr1 ASQSISSYJNWYQQKP ACT"CGA""ACTGGGG"CAGGGAAC"CTG GKAPKJLISGASSJKS G"GACTG"C"CCAGCGG"GGAGG"GGCAG GV?SRFSGSGSG"DFr1 CGG"GG"GGCGGAAGCGGGGGGGGCGGCr1 .TISSIP??DFA"YYC C"GA"A""CAGA"GAC"CAA""C""CC""TCr1 QQSYS"PI"FGQG"R4 r1C"C"G"CCGC""CCG"GGGCG r1AGAGr1 EIKRAAAJNTKSVGr1 "AC""G"AGGGCGTCCCAG"CAA IIHVKGKiuC?SP.F? AG"TA""""""GAA""GG"A"CAGCAG ?SKPFWV4VVVGGV4 AAGCCCGGGAAAGCACC"AAGC"G"""GAr1 4V"VAFIIFWV CAGCGGGGC""C"AGCC"GAAGAG"GGGG R. DYMNMr1 r1ACCTTCACGGW'"CAGCGGAAGCGGAAGC _ ?" GGAACCGAT""CACCC"GACTA"CAGCAG CC"GCCACC"GAGGAC""TGCAAC"TACr1 AC"GCCAACAG"CATACAGCAC"C AC"TTCGGCCAGGGCACCCGGC"CGAAAr CAAGCGCGC"GC"GC"""GGACAAF . . EAY AG"CAAACGGCACCA"CA"ACA"Gr . RGKGHD CATC"G"GTCCC"CCCCGCF ISTATKDTYDA CCCCGGCCC""CCAAACCG"TC"GGGF I??R "GG"GG"GG"CGGAGGCG"AC"CGC"F r1A"AG""C"GC"GGTAAC"G"CGCCT"CAr1 CA"C""""GGGTGAGA"CCAAAAGAAGCC GCC"GC"CCA"AGCGA""ACA"GAA"ATG AC"CCACGCCGCCCTGGCCCCACAAGGAA ACAC"ACCAGCCT"ACGCACCACC"AGAG A"""CGCTGCC"A"CGGAGCAGGG"GAAG I'"""""""CCAGA"C""GCAGATGCACCAGCGTA r1CAGCAGGGCCAGAACCAACTGTA"AACG AGC"CAACC"GGGACGCAGGGAAGAGTAT GACGTTTTGGACAAGCGCAGAGGACGGGA CCC"GAGA"GGGTGGCAAACCAAGACGAA AAAACCCCCAGGAGGG"CTC"A"AATGAG C"GCAGAAGGATAAGA"GGC"GAAGCCTA I""1C"GAAA""AGGCA""GAAAGGAGAGCGGA GAAGGGGAAAAGGGCACGACGG"TTG"AC C"CAGCAC"GC"ACGAAGGA"AC I'"""A""GACGCTC"CCACA""GCAAGCCC""GC CACC""AGG (CAR3 . 2) C"CCCCGTAAC""GCTCTGCTGC"1 MA .?VTA .

Clone GCCG""GGCA""GCTCC"GCACGCCGCAC HAAQPQVQ 20C5.l CHD GCCCGCAGGTGCAGCTTG"GCAGAGCGGG ?GASVK CAR DNA. GCCGAGG"GAAGAAGCCCGGGGCCAGCGr1 "?LSW{ HXL CAAAG"G"CC"G"AAGG"CAGCGG"TACA .ﬁWMGGt CCCTCACCGAGC"GAGCA"GCAC"GGG"A YAQKFQG GC"CCCGGCAAAGG"CT"GAG"G 13""AYM'4".

"GGA"T"GATCCAGAAGA"GGAG AVYYCATESRGIGWPY AGACTA"C"ACGCCCAGAAG"TCCAGGGC FDYWGQG"LV"VSSGG [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by eadley ation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley CGGGTCACCG"AACAGAAGACACC"CAAC GGSGGGGSGGGGS 1GACACCGCT"ACATGGAGCTGAG""CAC MTQSPSSLSASVGDRV 1GCGGTCCGAGGACACGGCCGTGTA"TAT TI"CRASQSISSY4NW 1GTGCCACCGAGAGCCGCGGAATCGGA"G YQQKPGKAPKIIISGA GCC""AC""CGACTACTGGGGACAGGG"A VPSRFSGSGS CAC""GT"ACAGTATCA"CCGGGGG"GGC 1DEWITISS ?P?DF GGC"C"GG"GGGGGCGGCTCCGGAGGGGG _ 1FG T GGA"1 CAGA""A"1 CCAAA"1 GAC"1 CAAAG"1 C ' 2" CAAG""CCC"G"C"GCC"CAG"CGGAGAT H _ 1II AGAG"CACCA"AACC"GCAGGGCAAGTCA G"CCA"CTCC"CC"A"C"GAAC"GG"ACC AACAGAAACC"GGAAAGGCGCC"AAGC"C C"GA"CTCCGGAGCC"CATC"""GAAAﬁC CGG"G"CCCA"CTCGC""CAG"GGC"C"G G"ACAGATT""AC"""GACCA"T AGCAGCC"CCCACCGGAAGAC""TGCTAC ATA""AC"GCCAGCAG"CT"AC"CAACCC CAA"CACCTTCGGGCAAGGCACCAGAC"C GAAA"AAAAAGAGCAGCTGC"A"CGAGGr1 TA"G"ACCCACCGCCG"AC""GGA"AACG AAAAAAGCAATGGGACCATCA""CA"G"G AAGGGTAAGCACC"T"GCCC"AGCCCACr1 GT""CC"GGCCCGAG"AAACCC"T""GGG r1AC"TG"GG"CGTCGGCGGCG"GC"GGCC r1GC"AC""CAC"CC"GGT"ACCG"CGCATr1 CA"CA"C"T""GGGTGAGA"CCAAAAGAA GCCGCC"GC"CCA"AGCGA"TACA"GAAr1 ATGAC"CCACGCCGCCC"GGCCCCACAAG GAAACAC"ACCAGCCT"ACGCACCACC"A GAGA"""CGCTGCC"A"CGGAGCAGGG"G AAG""""CCAGA"C"GCAGATGCACCAGC GTA"CAGCAGGGCCAGAACCAACTGTA"A ACGAGC"CAACC"GGGACGCAGGGAAGAG TATGACGTTTTGGACAAGCGCAGAGGACG GGACCC"GAGA"GGGTGGCAAACCAAGAC GAAAAAACCCCCAGGAGGG"CTC"A"AAT GAGC"GCAGAAGGATAAGA"GGC"GAAGC CTA""C"GAAA"AGGCA"GAAAGGAGAGC GGAGAAGGGGAAAAGGGCACGACGG"TTG 1ACCAGGGAC"CAGCAC"GC"ACGAAGGA 1AC""A"GACGCTCTCCACA"GCAAGCCC 1GCCACC""AGG""AA (CAR3.2) CAGG"GCAGC""G"GCAGAGCGGGGCCGA QVQLVQSGAEVKK?GA Clone GG"GAAGAAGCCCGGGGCCAGCGTCAAAG SVKVSCKVSGY"."?L 20C5.l CHD r1G"CC"G"AAGGTCAGCGG"TACACCCTC SWiWVRQA?GKG.?WM CAR DNA C"GAGCA"GCAC"GGG"ACGGCA GGJ: DP ﬁDG *.""IYAQKF HXL GGC"CCCGGCAAAGG"CT"GAG"GGATGG RVTVTED"S"D"AY G"GGA"T""GATCCAGAAGA"GGAGAGACr1 HISSIRS?DTAVYYC A"C"ACGCCCAGAAG""CCAGGGCCGGGr1 ATESRGIGWPYFDYWG CACCG"AACAGAAGACACC"CAAC"GACA QG"LV"VSSGGGGSGG CCGCTF1ACATGGAGC"GAG"""CAC""GCGG GGSGGGGS SP TCCGAGGACACGGCCG"GTA"TAT"GTGC SSJSASVGDRVTI"CR CACCGAGAGCCGCGGAATCGGA""GGCC"""1 ASQSISSYJNWYQQKP ACW'"CGACTACTGGGGACAGGG""ACAC"""1 GKAPKJLISGASSJKS GT"ACAGTATCA"CCGGGGG"GGCGGC"C GV?SRFSGSGSG"DFr1 GGGGCGGCTCCGGAGGGGGTGGAr1 .TISSIPP?DFA"YYC CAGA"A"CCAAA"GAC"CAAAG"CCAAGr1 QQSYS"PI"FGQG"R4 r1CCC"G"CTGCC"CAG"CGGAGATAGAGr1 EIKQAAAIEVMYP?PY CACCATAACCTGCAGGGCAAGTCAGTCCA .DN?KSNG"IIHVKGK [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley r1CTCC"CCTA"C"GAAC"GG"ACCAACAG {JCPSPLF?G?SKPFW AAACC"GGAAAGGCGCC"AAGC"CCTGAT VJVVVGGVIACYSL AGCC"CATC"""GAAA"CCGG"G r1VAFIIFWVRSKRS "CTCGC""CAG"GGC"C"GGAAGC 4{SDYMNM"?RRPG_ GG"ACAGATT""AC"""GACCA"TAGCAG RKHYQPYA??RDFAAY CC"CCCACCGGAAGAC""""TGCTACA.T‘A"""1 QSRVKFSRSA AGCAG"CT"AC"CAACCCCAA"C QGQNQLYN7 ACCTTCGGGCAAGGCACCAGAC"CGAAAr1 EYDVLDKRRG AGCAGCTGC"A"CGAGG"TA""Gr1 GKPRRKVPQ.

ACCCACCGCCG"AC""GGA"AACGAAAAA QKDKMAﬁAYS GGGACCATCA""CAF ERRRGKGHDG TAAGCACC"T"GCCC"AGCC TATKDTYDA C"GGCCCGAG"AAACCC"TF ?R G"GG"CGTCGGCGGCG"GCF C"CAC"CC"GGT"ACCGF r1C"T""""GGGTGAGA"CCAAAAGAAGCCGC C"GC"CCA"AGCGA"TACA"GAA"ATGAC r1CCACGCCGCCC"GGCCCCACAAGGAAAC AC"ACCAGCCT"ACGCACCACC"AGAGAr1 I'"""CGCTGCC""A""CGGAGCAGGG"GAAGW'1 I""1CCAGA"C"GCAGATGCACCAGCGTA""C AGCAGGGCCAGAACCAACTGTA"AACGAG C"CAACC"GGGACGCAGGGAAGAGTATGA CGTTTTGGACAAGCGCAGAGGACGGGACC C"GAGA"GGGTGGCAAACCAAGACGAAAA CAGGAGGG"CTC"A"AATGAGCr1 GCAGAAGGATAAGA"GGC""GAAGCCTA"""1 C"GAAA"AGGCA"GAAAGGAGAGCGGAGA AGGGGAAAAGGGCACGACGG"TTG"ACCA GGGACTCAGCAC"GC""ACGAAGGA""AC"""1 A"GACGC"CTCCACA"GCAAGCCC"GCCA CCTAGG (CAR3.3) A"GGCAC"CCCCGTAAC"GCTCTGCTGCr1 MA.?VTA.

Clone GCCGT"GGCA"TGC"CC"GCACGCCGCAC HAAQPQVQ 20C5.l CD8 GCCCGCAGG"GCAG"TGG"GCAAAGCGGC ?GASVK CAR DNA. GCAGAAG""AAGAAACC"GGGGCGTCAGr1 "?LSW{WVRQA.

HXL TAAGG"G"C""GCAAAG"ATCTGGCTA"A :ﬁWMGGb CCCTCAC"GAGC"G"CCA"GCA"TGGG"A G GC"CC"GGAAAGGGGC"CGAA"G MT.

GATGGGAGGA"""GACCC"GAAGACGGAG AVYYCATESRGIGWPY AGACCATC"ACGCCCAGAAA""CCAGGGT FDYWGQG"LV"VSSGG AGAGTAACAGF 1GAGGACAC"AGCAC GGSGGGGSGGGGSDIQ "GACACAGCGF "GGAGC"GAGTTCF MTQSPSSJSASVG G"GAGGACACAGCCG""F TI"CRASQSISSY.

"GCGC"ACCGAG"CCAGAGG"A"F YQQKPGKAPK.

GCCA"AC"TCGACTAT"GGGG"CAGGGCA SSJKSGV?SRF CCC"GGT"ACAG"GAG""CAGGAGGCGGG G"DF".TISS.__H GGC""C""GGGGGGGGCGG"TCCGGAGGGGG A"YYCQQSYS"1 _ GGGC"CAGA"A"ACAGA"GACGCAGAGTC QG"R.?IK%AAA CATCAAG"C"C"CAGCCAGCG"GGGAGAr1 IMYFSiFV?VF__ CGCG"GAC"A""ACT"GCCGCGCCAGCCA "r. _ ?TPA.

GAG"A""AGC"CCTA"CF 1ACC SQP ' r 1GGCGCC"C 1C AGG"G"GCCAAGCAG r 1G GAAGF1GGCACTGACT"" ""AC" 1C TCCAGCC"GCCCCCAGAGGA"""TGCCAC ATA"1 I'"AC"1 G"1 CAGCAAAGCTAC"1 CTAC"1 C CAA"CAC"T"CGGCCAGGGCACAAGAT"G [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley 1AAGAGGGC"GCCGCAC"""CAAA EYDVLDKRRG 1CA"G"AT""CAGCCA""""GTGC GKPRRKVPQ.

"Tr ""CCGGCCAAACC"ACAACC QKDKMAﬁAYS.

ACTCCCGCCCCACGCCCACCTAC"CCCGC ERRRGKGHDG CCC"ACCA""GCC"CCCAGCC"C"G"CTC TATKDTYDA C"GAGGC""G"AGACC"GC"GCC ?R GGCGGAGCCG"GCACACTCGCGG"CF C"TCGCC"GCGACA"C"ATATCF C"C"GGCCGGCACC"GCGGCG""Cr CmCmCACmCGmAAr 1Ar "CACAGGAACAGAF r1GC"CCA"AGCGA""ACATGAA" CGCCCTGGCCCCACAAGGAAACA CTACCAGCCT""ACGCACCACC""AGAGA"""1 GCC""A""CGGAGCAGGG"GAAGW'"1 r1CCAGA"C"GCAGATGCACCAGCGTA""CA GCAGGGCCAGAACCAACTGTA"AACGAGC r1CAACC"GGGACGCAGGGAAGAGTATGAC GTTTTGGACAAGCGCAGAGGACGGGACCC r1GAGA"GGGTGGCAAACCAAGACGAAAAA ACCCCCAGGAGGG"CTC"A"AATGAGC"G CAGAAGGATAAGA"GGC"GAAGCCTAD"C r1GAAA"AGGCA"GAAAGGAGAGCGGAGAA GGGGAAAAGGGCACGACGG"TTG"ACCAG GGAC"1CAGCAC"1 GC""ACGAAGGA""AC"1"A CTCTCCACA"GCAAGCCC"GCCAC CTAGGF (CAR3.3) CAGG"GCAG"TGG"GCAAAGCGGCGCAGA QVQLVQSGAEVKK Clone AG""AAGAAACC"GGGGCGTCAG"TAAGG SVKVSCKVSGY". 20C5.l CD8 TGF ""GCAAAG"ATCTGGCTA"ACCCTC SWiWVRQA?GKG.?WM CAR DNA AC" 1G"CCA"GCN‘TGGG""AAGGCA GGJ: DP *.DG *."‘IYAQKF HXL GGCF 1GGAAAGGGGC"CGAA"GGATGG QVTVTED"S"D"AY GAGGAF‘"GACCC"GAAGACGGAGAGACC H.SS.RS?DTAVYYC ATC"ACGCCCAGAAA""CCAGGGTAGAGT ATESRGIGWPYFDYWG AACAGF CAC"AGCAC"GACA QG"LV"VSSGGGGSGG CAGCG" 1GGAGC"GAGTTC""C""GAGA GGSGGGGS DIQMTQSP AG"GAGGACACAGCCG"""ACTAC"GCGC SSJSASVGDRVTI"CR r1ACCGAG"CCAGAGG"A""GGC"GGCCAr1 ASQSISSYJNWYQQKP AC"TCGACTAT"GGGG"CAGGGCACCC"G GKAPKIIISGASSJKS G"GAG""CAGGAGGCGGGGGC"C GV?SRFSGSGSGr1 r1GGGGGGGGCGG"TCCGGAGGGGGGGGCr1 .TISSI???DFAF CAGA"A"ACAGA"GACGCAGAGTCCATCA QQSYS"?IF AG"C"C"CAGCCAGCG"GGGAGA"CGCGr1 EIKQAAA GAC"A""ACT"GCCGCGCCAGCCAGAG"A ?VF4_ _ I'"""AGC""CCTA"C"GAAW'"GG""ACCAGCAA _ __ _ _"IASQ_JS AAGCCCGGGAAGGCCCC"AAGC""CTG " .3 ?AAGGAV{T I""1C"GGCGCC"CC"C""""""GAAG""CAGG""G _ DIYIWA. _ r1GCCAAGCAG I'"""""AGCGGG""C""GGAAG"" ISIVIT GGCACTGACT"""ACAC""""AC""A""CTCCAG RS R .

CC"GCCCCCAGAGGA""""1"GCCACA""A"""1 _ ?G?"RK AC"G"CAGCAAAGCTAC"C""AC""CCAA"C _ _ _ RDFAAY RS AC"""CGGCCAGGGCACAAGA""GGAG " GGC"GCCGCAC"""CAAAT"CCA 1CAmeATmmCAGCCAmmmmeGCCmem "Tr 1"CCGGCCAAACC"ACAACCACTCC CGCCCCACGCCCACCTAC"CCCGCCCC"A CC"CCCAGCC"C"G"CTCT"AGA CC"GAGGC"TGTAGACC"GC"GCCGGCGG AGCCGTGCACACTCGCGG"C"GGAC"TCG [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley CC"GCGACA"CTATATC"GGGCCCCF GCCGGCACC"GCGGCG""C"CCT"CF AC"CGTAA"CACACTC"AT"GCAAF GGAACAGA"CCAAAAGAAGCCGCCF CAF1AGCGA"1I'"ACAT GAA""A"1 GAC" CCGCCCTGGCCCCACAAGGAAACA ACGCACCACC"AGAGA"F GCC""A""CGGAGCAGGG"1GAAGW'" A"C"GCAGATGCACCAGCGTA"CA GCCAGAACCAACTGTA"AACGAGCF C"GGGACGCAGGGAAGAGTATGACGTTTT GGACAAGCGCAGAGGACGGGACCC"GAGA GCAAACCAAGACGAAAAAACCCC 1CTC"A"AATGAGC"GCAGAA "GGC"GAAGCCTA"F 1AGGCA"GAAAGGAGAGCGGAGAA AAAGGGCACGACGG"TTGF CAGCAC"GC"ACGAAGGAF C"CTCCACA"GCAAGCCCF (CAR4.l) A"GGCAC"C MAIPVTA.

Clone GCCGT"GGCAF HAARPQVQ 20C5.2 THD GCCCGCAGGF LQ CAR DNA. "GG"G"AG"GCAGCCGGGCCGCAGF TFSSYGMiWV HXL "T"CC"G"GCGGC"TCAGGC"F GLEWVAVISY "CCAGC"A"GGAA"GCAC"GGGF YVDSVKGRFTISRDNS CCCCCGGCAAAGGACT"GAGF KNR.YLQWNSIRA?DT "GACGGA"CAG AVYYCARERYSGRDYW 1GGACAGCG"CAAGGGC GQG"LVTVSSGGGGSG "TCACCAF 1C"AGGGACAACAG"AA GGGSGGGGSEIVM"QS 1AGAC"C"ACC"CCAGA"GAA"AGCC S?G?RATISC 1CAGAGCTGAAGACACGGCCG"C"ACF RASQSVSSLJTWYQQK 1G"GCTCGGGAGCGGTATAG"GGC PGQAPRIIIFGAS"RA CTAC"GGGGGCAGGGCACAC"CG"F 1GI?ARFSGSGSG"GF r1GAG"AGCGGCGGAGGAGGGAGTGGGGGC 1LTISSIQSTDFAVYY GG"GGCTCCGG"GGAGGAGG"TC"GAGAr1 ?F"FGPG"K "G""A"GACCCAGAGTCC"GCGACCC"CF .DN?KSNG GCCCCGGGGAGCGCGCAAC""F 1IIHVKGK-IIC?SP.F "C""GCAGAGC"AGTCAG"CCG"G"CCF ?G?SKPFWV4VVVGGV r1C""C"GACATGG"ACCAGCAAAAG .ACYSLIV"VAFIIFW GGCAGGC"CCGCGCC""""GATC"""" RSKRS R .

GC""CAACAAGAGCCAC"GGGA"r D RPGDF1 ACG I'"""C""C" AGCGGF _D DFAAY GT"""1 CACCC" "AGCAGF1CF AGCGAGGAC" "ATACF1ACF GCAG"ACGAF ""CACF (3]\(I(I]X(3(3(3]\(Ir "(3(31Xr1r1r1r GCCGCCGC"CF "AACGAAAAGF r1GGCACCA"AA"CCACGTCAAAGGCAAGC ACC"G"GCCCr"CCCCGC"C""CCCCGGA CCCAG"AAACCA"T"TGGG"GC"GG""Gr1 1G"GGGGGGCG"GC"GGCC"GC"A"AGCC TCACmeAGCCTmCAmmAmmme 1GGGTCAGA"CCAAAAGAAGCCGCC"GCr1 CCAF1AGCGA"1I'"ACA"1 GAAF1AT GAC"1CCAC GCCGCCCTGGCCCCACAAGGAAACAC"AC CAGCCT"ACGCACCACC"AGAGA"""CGC TGCC"A"CGGAGCAGGG"GAAG"""F GATC"GCAGATGCACCAGCGTA"CA GGCCAGAACCAACTGTA"AACGAGCr [Annotation] Anne.Headley None set by eadley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley CCTGGGACGCAGGGAAGAGTATGACGTTT AGCGCAGAGGACGGGACCC"GAG 1GGGTGGCAAACCAAGACGAAAAAACCC 1CTC"A"AATGAGC"GCAGA "GGC"GAAGCCTA"F 1AGGCA"GAAAGGAGAGCGGAGAA AAAAGGGCACGACGG"TTGF TCAGCAC"1GCF1ACGAAGGAF1ACF GC"C"CCACA"GCAAGCCC"GCCACC"AG (CAR4.l) CAGG"CCAGT"GG"CGAAAG"GGCGG"GG QVQ.V?SGGGVVQ?GR Clone GCAGCCGGGCCGCAG""TGAGGC SLRJSCAASGFTFSSY 20C5.2 THD """CCF "TCAGGC"""AC""TT GMiWVRQA?GKGLEWV CAR DNA. "CCAGCF 1GCAC"GGG"GCGGCA AVISYDGSDKYYVDSV HXL CGGCAAAGGACT"GAG"GGG"GG KGRFTISRDNSKNQJY ""CTTA"GACGGA"CAGA"AAG LQWNSIRA?DTAVYYC 1GGACAGCG"CAAGGGCAGA"T GRDYWGQG"L 1C"AGGGACAACAG"AAAAA"A VTVSSGGGGSGGGGSG 1ACC"CCAGA"GAA"AGCC"CAGA GGGSEIVM"QSPATJS GCTGAAGACACGGCCG"C"AC"AT"G"GC VS?G?RAT.SCRASQS TCGGGAGCGGTATAG"GGCAGAGACTACr1 VSSLJTWYQQKPGQAP GGGGGCAGGGCACAC"CG""ACAG"GAGr1 RIIIFGAS"RA"GI?A AGCGGCGGAGGAGGGAGTGGGGGCGG"GG RFSGSGSG"GF"LTIS CTCCGG"GGAGGAGG"TC"GAGA""G""A S.QS?DFAVYYCQQYD r1GACCCAGAGTCC"GCGACCC"C"CAG"C TW?F"FGPG"KVDFKR AGCCCCGGGGAGCGCGCAAC"""G"C""G AAA.DN?KSNG"IIHV CAGAGC"AGTCAG"CCG"G"CC"C"C""C KGK{.C?SP.F?G?SK TGG"ACCAGCAAAAGCCCGGGCAG PFWVJVVVGGVJACYS GC"CCGCGCC""""GATC"""GGGGC""C 4V"VAFIIFWV AACAAGAGCCAC"GGGAF1F1CCCGCACG r" DYMNM"1 _ RP "C"Cr 1CCGGGAGCGG"AC"GGT""C _ __ 3F ACCC" 1AGCAG"C""CCAGAGCGA GGAC" "ATAC""AC""GCCAGCAG"1 ACGAF 1GGCCA""CAC"TT"GGACCA GGGACF 1GGA""""AAGCGCGCCGC CGC"CF 1AACGAAAAG"CAAA"GGCA CCA"AA"CCACGTCAAAGGCAAGCACC"G r1GCCC""CCCCGC"C""CCCCGGACCCAG "AAACCA"T"TGGG"GC"GG""G""G"GG GGGGCG"GC"GGCC"GC"A"AGCC"T""G GTCACmeAGCCTmCAmmAmmmTr CAGA"CCAAAAGAAGCCGCC"GCF 1A GCGA""ACA"GAA"ATGAC"CCACGCCGC CCTGGCCCCACAAGGAAACAC"ACCAGCC T"ACGCACCACC"AGAGA"""CGCTGCCr1 A"CGGAGCAGGG"GAAG""""CCAGA"Cr1 GCAGATGCACCAGCGTA"CAGCAGGGCCA GAACCAACTGTA"AACGAGC"CAACC"GG GACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCC"GAGA"GGG ACCAAGACGAAAAAACCCCCAGG AGGG"CTC"A"AATGAGC"G AAGA"GGC"GAAGCCTA""CF CA"GAAAGGAGAGCGGAGAA GGCACGACGG"TTG"ACCAGGGACTCAGC AC"GC"ACGAAGGAF ""A"GACGCTC" CCACA"GCAAGCCC"GCCACCTAGG ATGGCACTCCCCGTAACF "CTGCTGCF 217 MALPVTAL. ?LALLL 218 GCCGT"GGCATTGC"CC"GCACGCCGCAC HAARPQVQ ?SGGGV [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley 20C5.2 CHD GCCCGCAGGTGCAGCTCG"GGAG"C"GGC VQ?GRSLQLSCAASGF CAR DNA. GGCGGCG"GG"CCAGCCCGGCCGGTCCCT MiWVRQA?GK HXL GCGCC"G"CC"GCGCCGCCAGCGGG"T"A GLEWVAVISYDGSDKY CT"TT"CC"CC"ACGGCA"GCAC"GGG"G YVDSVKGRFTISRDNS CGCCAGGC"CCCGGCAAGGGCC"CGAG"G KNR.YLQWNSLRA7DT GG"CGCCG"GA"C"CA"ACGA"GGG"CAG AVYYCARERYSGRDYW ACAAA"AC"A"G"CGA"TCTG""AAAGGG GQG"LVTVSSGGGGSG CGG"T"ACCA"""CAAGAGA"AACTC"AA GGGSGGGGSEIVM"QS GAA"AGGC"G"A""TGCAGA"GAACAGCC PATISVS?G?RAT.SC "GAGGGC"GAAGA"ACCGCAGF 1Ar1 RASQSVSSLJTWYQQK 1GCGC"AGGGAGCGG"ATAG"GG PGQA?R..IFGAS"RA "TACTGGGGACAGGG"ACACF 1GACCG r1GI?ARFSGSGSG"GF 1GAGC"C"GGGGG"GGCGGAAGCGGGGGr1 r1.TISS.QS':‘.DFAVYY AGCGGCGGAGGGGG"AG"GAAAF CQQYDTW?F"FGPGTK "G"GA"GACCCAG"C"CCGGCTACAC"F V QAAAIEVMYPP? CAG"C"CCCC"GGGGAGAGAGC"ACACr Y ?KSVG"IIHVKG "CA"GCAGAGCGTCCCAG"CCG"CF K ?SP.F?G "C"CC"TACC"GG"AF GGVIACYSL GCCAGGC"CC"CGACr V"VAFIIFWV GCC"CCACAAGGGCGACCGGGAW' u-IS DYMNM"1 .3 CCGC"""C"CAGGT""C"GGGAGCGGAACTG YA? .3 GTT"CAC"""GACAA"CAG""CAC"GCAG RSRVKFSRSA TCAGAGG I'"""""CGCCG""GTAC""AC""GCCA QGQVQLYN7 GCAA"ACGACACA"GGCCA""CAC"TTCG .ﬁYDVLDKR GACCCGG"ACCAAAG"CGA"""CAAGAGA RRKVPQ.

GCCGCGGCCA"CGAGG""A"G"ACCCACC _ ﬁAYS ACCA"A"C"GGACAA"GAAAAAAGCAATG I RGKGHDG GAACCA"1 I'"A"1 C CA"1 G"1 GAAGGGTAAACAC C"CTGCCC"AGCCCAC""T"CCC"GGCCC A"CAAAGCCC""C"GGG"C""GG"GG"CG TGGGGGG"G"GC"GGCC"G""ACAGCC"T C"GGTGACGG""GCTT"CA""A"C"TC"G GGT"AGA"CCAAAAGAAGCCGCC"GC"CC A"AGCGA"TACA"GAA"ATGAC"CCACGC CGCCCTGGCCCCACAAGGAAACAC"ACCA GCC""ACGCACCACC"AGAGA"""CGCTG GGAGCAGGG"GAAG""""CCAGA r1C"GCAGATGCACCAGCGTA"CAGCAGGG CCAACTGTA"AACGAGC"CAACC r1GGGACGCAGGGAAGAGTATGACGTTTTG GACAAGCGCAGAGGACGGGACCC"GAGAr1 GGGTGGCAAACCAAGACGAAAAAACCCCC AGGAGGG"CTC"A"AATGAGC"GCAGAAG A"GGC"GAAGCCTA"r GAAAGGAGAGCGGAGAA AAGGGCACGACGG"TTGF AGCAC"GC"ACGAAGGA"ACF 1GACGC 1CCACA"GCAAGCCC"GCCACCTAGGT (CAR4.2) CAGGTGCAGCTCG"GGAGF 1GGCGGCGG QVQ.V?SGGGVVQ?GR Clone CG"GG"CCAGCCCGGCCGGTCCCTGCGCC SLRJSCAASGFTFSSY 20C5.2 CHD "GCGCCGCCAGCGGG"T"ACT"TT GMiWVRQA?GKGLEWV CAR DNA I'1CC"CC""ACGGCA""GCAC""GGG""GCGCCA AVI SY DGS DKYYVDSV HXL GGC"CCCGGCAAGGGCC"CGAG"GGG"CG KGRFTISRDNSKNRJY CCG"GA"C"CA"ACGA"GGG"CAGACAAA LQWNSIRA?DTAVYYC r1AC"A""G"CGA"TCTG""AAAGGGCGG"T ARERYSGRDYWGQG"L r1ACCA"""""CAAGAGA"AACTC""AAGAA"A VTVSSGGGGSGGGGSG GGC"G"A""TGCAGA"GAACAGCC"GAGG GGGSEIVM"QSPATJS GCTGAAGA"ACCGCAG"GTAC"AT"GCGC VS?G?RAT.SCRASQS [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley "AGGGAGCGG"ATAG"GGCCGCGAF VSSLJTWYQQKPGQA? GGGGACAGGG"ACAC"GG"GACCGF RIIIFGAS"RA"GIPA "CTGGGGG"GGCGGAAGCGGGGGF SG"GF"4TIS AAGCGGCGGAGGGGG"AG"GAAAF S.QS?DFAVYYCQQYD "GACCCAG"C"CCGGCTACAC""F TW?F"FGPGTKVDFKQ I'1CCCC""GGGGAGAGAGC""ACAC""G" AAAIEVMYPP ?Y .DN'T.

CAGAGCGTCCCAG"CCG"C"C""CF KSNG"IIHVKGK{4C? r1TACC"GG"A"CAGCAGAAGCCCGGCCAG SPJF?G?SKPFWV4VV GC"CC"CGACF ""CGGTGCC"C VGGVIACYSL.V"VAF CACAAGGGCGACCGGGA""CCAGCCCGCr1 IIFWV RuuiSD I'1C"CAGGT"C"GGGAGCGGAACTGGTT""C YMNM"1 _ ?" RKHY AC"""GACAA"CAG""CAC"GCAGTCAGA QPYA?_ GG I'"""""CGCCG"GTAC""AC""GCCAGCAA" KFSRSA ACGACACA"GGCCA""CAC"TTCGGACCC QLYN? GG"ACCAAAG"CGA"""CAAGAGAGCCGC LDKR GGCCA"CGAGG""A"G"ACCCACCACCAr1 RKVPQﬁG A" C"1 "1GAAAAAAGCAATGGAACC MA *.AYS A""A"CCA"G"GAAGGGTAAACACC"CTG GKGHDG CCC"AGCCCAC""T"CCC"GGCCCA"CAA DTYDA.

"C"GGG"C""GG"GG"CGTGGGG GG"G"GC"GGCC"G""ACAGCC"TC"GGT GACGG""GCTT"CA""A"C"TC"GGGT"A GA"CCAAAAGAAGCCGCC"GC"CCA"AGC GA"TACA"GAA"ATGAC"CCACGCCGCCC CACAAGGAAACAC""ACCAGCCW'1 ACGCACCACC"AGAGA"""CGCTGCC"Ar1 CGGAGCAGGG"GAAG""""CCAGA"C"GC AGATGCACCAGCGTA"CAGCAGGGCCAGA ACCAACTGTA"AACGAGC"CAACC"GGGA CGCAGGGAAGAGTATGACGTTTTGGACAA AGGACGGGACCC"GAGA"GGGTG GCAAACCAAGACGAAAAAACCCCCAGGAG GG"CTC"A"AATGAGC"GCAGAAGGATAA "GAAGCCTA""C"GAAA"AGGCA r1GAAAGGAGAGCGGAGAAGGGGAAAAGGG CACGACGG"TTG"ACCAGGGAC"CAGCAC 1ACGAAGGA"AC""A"GACGCTCTCC 1GCAAGCCC"GCCACC"AGG ATGGCAC"CCCCGTAAC"GC"C"GCTGCr1 MAIPVTA.

Clone GCCGT"GGCA"TGC"CC"GCACGCCGCAC HAARPQVQ 20C5.2 CD8 GCCCGCAGG"GCAG""GG""GAA"CAGGA VQ?GRSL% CAR DNA. GGGGG"G"GG"GCAACCCG 1CGGTCACr1 TFSSYGMiWV HXL GCGCC"CAG""GTGC"GC""CCGGG"T"A GLEWVAVISY GC"CA"ATGGGA"GCAC"GGG"A YVDSVKGRFTISRDNS CGGCAGGC"CCAGG"AAAGGC"TGGAA"G KNR.YLQWNSIRA?DT GG"GGCGG"GA"CAGCTA"GACGGC"C"G AVYYCARERYSGRDYW ACAAATAT"A"GTGGACTCCG"GAAAGGC GQG"LVTVSSGGGGSG AGA"TCACCA"CAG"CGAGACAAC"CAAA GGGSGGGGSEIVM"QS GAA"AGAC"C"AC""GCAGA"GAA"AGCC PATISVS?G?RAT.SC r1CCGGGCCGAAGA"ACTGCAG"C"A"TAT RASQSVSSLJTWYQQK r1GCGCCCGGGAGCGCTACAG"GGAAGAGA PGQA?R..IFGAS"RA CTA"TGGGGGCAAGGAAC"C""G"CACAG r1GI?ARFSGSGSG"GF r1C"CA"CTGGCGGCGGCGGCAGCGG"GGG r1.TISS.QS':‘.DFAVYY GGCGGATCTGGCGGGGGCGGCAGCGAAAr1 CQQYD"W?F"FGPG"K CG""A"GACTCAGAGTCC"GCCACACTGA VDFKQAAALSNSIMYF GCG""AGCCC"GG"GAGAGAGCAACACTr1 S ?VF4PAKPTT"? AGC"GCAGAGCTAGTCAGAGTGT""CCAG A. ?"PAP"IASQP4 GACA"GG"ACCAACAGAAGCCCG S ?ACRPAAGGAV{ GTCAAGCTCCACGACTGCTCATC""CGGr1 T DFACDIYIWAPJ [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GCA"CCACCCGCGCAACCGGGA"ACCCGC AGTCGV CCGG"TTTCCGGT"C"GGAAGF CNHRV GA"1 r" CACGC"1 CACCA"1 ' YMNMT _ GAC"""GCCG"GTAF QPYA?? GCAG"ACGA"ACC"GGCCC"F KFSRSA GCCCAGGTAC"AAAG"GGAC"F QLYN? GC"GCTGCAC"""CCAATAG"AF LDKR C""T"CACA""""GTGCCCG"GF RKVPQ.

C"GCGAAGCC"ACGACAACCCCAGCCCCr1 MAﬁAYS AGGCCGCCCACACCGGCCCCAAC"A""GC GKGHDG C"CCCAGCCA""G"CTC"GAGACCCGAAG DTYDA C""""GCAGACC"GC""GCTGGAGGCGCCG"""1 CGAGGA""GGA"""CGCA"G"GA CA"TTACA"C"GGGCCCC"""GGCCGGAA CC"GCGG"G"GC"GCTGC"G"CAC"CG"G ZXF"r"]X(I]X(Ir"r"r"]X(Ir"(3(I]\Z\(I(IZX(I(I(3]X]X]X(IZXC§ A"CCAAAAGAAGCCGCC"GC"CCA"AGCG A""ACATGAATA"GAC"CCACGCCGCCCT ACAAGGAAACACTACCAGCCT"A CGCACCACC"AGAGA"""CGCTGCC"AF GGAGCAGGG"1GAAG"1 r" r" r" CCAGA"1 C" GATGCACCAGCGTA"CAGCAGGGCCAGAA CCAACTGTA"AACGAGC"CAACC"GGGAC GCAGGGAAGAGTATGACGTTTTGGACAAG CGCAGAGGACGGGACCC"GAGA"GGGTGG CAAACCAAGACGAAAAAACCCCCAGGAGG G"CTC"A"AATGAGC"GCAGAAGGATAAG A"GGC"GAAGCCTA""C"GAAA"AGGCAr1 GAAAGGAGAGCGGAGAAGGGGAAAAGGGC ACGACGG"1T r" G""ACCAGGGACT CAGCAC"1 GC"ACGAAGGA"AC""A"GACGC"CTCCA "GCAAGCCCF 1AGG"AA (CAR4.3) CAGG"GCAG""GG""GAA"CAGGAGGGGG QVQ.V?SGGGVVQ?GR Clone r1G"GG"GCAACCCG 1CGGTCAC"GCGCC AASGFTFSSY 20C5.2 CD8 r1CAG"""GTGC"GC""CCGGG"T"ACT"TC GMiWVRQA?GKGLEWV CAR DNA. AGC"CA"ATGGGA"GCAC"GGG"ACGGCA AVISYDGSDKYYVDSV HXL GGC"CCAGG"AAAGGC"TGGAA"GGG"GG KGRFTISRDNSKNRJY CGG"GA"CAGCTA"GACGGC"C"GACAAA LQWNSIRA?DTAVYYC TAT"A"GTGGACTCCG"GAAAGGCAGA"T ARERYSGRDYWGQG"L CACCA"CAG"CGAGACAAC"CAAAGAA"A VTVSSGGGGSGGGGSG GAC"C"AC""GCAGA"GAA"AGCC"CCGG VM"QSPATJS GCCGAAGA"ACTGCAG"C"A"TAT"GCGC AT.SCRASQS CCGGGAGCGCTACAG"GGAAGAGACTA"T VSSLJTWYQQKPGQA? GGGGGCAAGGAAC"C""G"CACAG"C"CA RIIIFGAS"RA"GI?A r1CTGGCGGCGGCGGCAGCGG"GGGGGCGG RFSGSGSG"GF"4TIS CGGGGGCGGCAGCGAAA"CG""A S.QS?DFAVYYCQQYD ""GACTCAGAGTCC""GCCACACTGAGCG"""1 1W?F"FGPG"KVDFKR AGCCC"GG"GAGAGAGCAACACT"AGC"G AAAJSNSIMYFS{FV? CAGAGCTAGTCAGAGTG"""" 1"" VFJ ?AKPTT"1 _ _ .3 r1GACA"GG"ACCAACAGAAGCCCGGTCAA IASQP ? GC"CCACGAC"GCTCA"CF ACR?AAGGAV D CACCCGCGCAACCGGG FACDIYIWAPJAGTCG "T"CCGGT"C"GGAAGF "r VT..SLVITLYCVHRV ACGC"CACCA""TC""CF "GCAGTCF RSKRSRIIHSDYWNMT AGAC"""GCCG"GTA"F 1GCCAGCAGr1 PRR?GP"RKHYQ?YA? ACGA"ACC"GGCCC"""ACC"""GGCCCA PRDFAAYQSRVKFSRS GGTAC""AAAG""GGA"1 r" r"I'"AAACGAGC""GC ADA ?AYQQGQ‘\IQ T.

TGCAC"""""CCAATAG"A""A"G"AC""Tr1 LNIGRRﬁﬁYDVL R CACA""""GTGCCCG"G""CC"GCC"GCG GRD?EMGGKPRRKNPQ [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley AAGCC"ACGACAACCCCAGCCCC"AGGCC ﬁGLYNﬁLQKDKMAEAY GCCCACACCGGCCCCAACTAT"GCC"CCC ﬁIGMKGﬁRRRGKGHD AGCCA"TG"CTC"GAGACCCGAAGC""GC TATKDTYDA AGACC"GC"GCTGGAGGCGCCG"""CACAC {MQA .P ?R CCGAGGA""GGA"""CGCA"G"GACACTT ACA"C"GGGCCCC"""GGCCGGAACC"GC GG"G"GC"GCTGC"G"CAC"CG"GA""AC ACr" r" I'"AC"1 GCAACCACCGAAACAGA"1CCA AAAGAAGCCGCC"GC"CCA"AGCGA""AC ATGAATA"GAC"CCACGCCGCCCTGGCCC CACAAGGAAACACTACCAGCCT"ACGCAC CACC"AGAGA"""CGCTGCC"A"CGGAGC AGGG"GAAG""""CCAGA"C"GCAGATGC ACCAGCGTA"CAGCAGGGCCAGAACCAAC TGTA"AACGAGC"CAACC"GGGACGCAGG GAAGAGTATGACGTTTTGGACAAGCGCAG AGGACGGGACCC"GAGA"GGGTGGCAAAC CAAGACGAAAAAACCCCCAGGAGGG"CTC r1A"AATGAGC"GCAGAAGGATAAGA"GGC r1GAAGCCTA""C"GAAA"AGGCA"GAAAG GAGAGCGGAGAAGGGGAAAAGGGCACGAC GG"TTG"ACCAGGGACTCAGCAC"GC"AC GAAGGA"AC""A"GACGCTC"CCACA"GC AAGCCC"GCCACC"AGG (CAR4.4) ATGGCAC"CCCCG"AAC"GC"CTGCTGCr1 MAL?VF .PLAL.

Clone GCCGTTGGCA"TGCTCC"GCACGCCGCAC HAARPI 1QSPA". 20C5.2 THD GCCCGGAGAW'"G"GA""GACCCAG""CCCCr1 SVS?G?RA".SCRASQ CAR DNA. GCTACCC"G"CCG"CAG"CCGGGCGAGAG TWYQQKPGQA LXH AGCCACC"TG"CA"GCCGGGCCAGCCAGr1 PRIIIFGAS"RA"GI? CCG"CAGCAG"C"CC"GAC"TGG"A"CAG ARFSGSGSG"GF"4TI CAAAAACCAGGGCAGGCACCGCGGC""T"1"1 SS.QS?DFAVYYCQQY GA""""TGGTGCAAGCACACGCGCCAC"G D"W?FTFGPGTKVDFK GCA""CCAGC"AGG""T"C"GGAAG"GGA RGGGGSGGGGSGGGGS r1C"GGGACAGGC"TCAC"C""GACAA""CAG QVQ.V?SGGGVVQ?GR r1AGCC"GCAGAG"GAGGAC"T"GCTGT"T SIRISCAASGFTFSSY AC"AC"G"CAACAG"ACGACACC"GGCCA GMiWVRQA?GKGLEWV I""1CACAT"CGGGCCCGGCACCAAGG""CGA AVI SY DGS DKYYVDSV C""CAAGAGGGGCGG"GGAGG"TCAGG"G KGRFTISRDVSKNRJY G"GGCGGGTCAGGCGGCGG"GGG"C"CAG .QWNS.RA?DTAVYYC Gm'"CAAC"GG"GGAA"CAGG"GGCGGCGr1 ARERYSGRDYWGQG"L "1G"CCAACCGGGGCGATCAC""""CGAC"""""1 VTVSSAAA.DN?KSNG CC"GTGC"GCCTCAGGC"T"ACT""""CA r1IIHVKGK-IIC?SP.F 1CC"A""GGGA"GCAC"GGG""CGGCAGGC ?G?SKPFWV4VVVGGV 1CCCGGAAAAGGACTCGAG"GGG""GCAG .ACYSL.VF TACGA""GGC""CAGACAAG""AT RSKRS R . 1G"GGACTCAG"CAAGGGGAGA"TCAC D RPG?r1 GAGACAACTCCAAAAACCGGC _D DFAAY 1A"CTCCAGA"GAACAGCC""AGAGCG GAAGATACCGCGG"A"ACTACF CGAGAGGTA""CCGGCAGAGACr GCACAC"GG"CACCGF GCCGCAGCGC"CGATAACGAAAAGAGCAA CGGAACCAT"A"CCACGT"AAGGGCAAGC ISTATKDTYD ACC"G"GCCCCAG"CCCC"C""CCCAGGA I??R CCAHcmAAAcccmmcmGGGmmcmGGmAGm AGGGG"CC""GCA"G""AC"CCC r1Tm'"GGTCACCG"CGCCT"CA""A""TTC r1GGGTGAGA"CCAAAAGAAGCCGCC"GCr1 CCA""AGCGA"1TACA"1 GAA""AT GAC"1CCAC [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley GCCGCCCTGGCCCCACAAGGAAACACTAC CAGCCT"ACGCACCACC"AGAGA"""CGC TGCC"A"CGGAGCAGGG"GAAG""""CCA CAGATGCACCAGCGTA"CAGCAG GGCCAGAACCAACTGTA"AACGAGC"CAA CC"GGGACGCAGGGAAGAGTATGACGTTT TGGACAAGCGCAGAGGACGGGACCC"GAG 1GGGTGGCAAACCAAGACGAAAAAACCC "AATGAGC"GCAGA "GGC"GAAGCCTA"F 1AGGCA"GAAAGGAGAGCGGAGAA GCACGACGG"TTGF TCAGCAC"GC"ACGAAGGAF GC"C"CCACA"GCAAGCCCF (CAR4.4) GAGA""G"GA"GACCCAG"CCCCF SPATLSVS?G Clone CC"G"CCG"CAGTCCGGGCGAGAGAGCCA ?RAT.SCRASQSVSSL 20C5.2 THD CC"TG"CA"GCCGGGCCAGCCAG"CCG"C JTWYQQKPGQAPR.

CAR DNA. AGCAG"C"CC"GAC"TGG"A"CAGCAAAA FGAS"RA"GIPARF LXH ACCAGGGCAGGCACCGCGGC""T"1""GA."""""1 SGSG"GF".TISS.QS r1TGGTGCAAGCACACGCGCCAC"GGC "" EDFAVYYCQQYD"W.

AGG""T"C"GGAAG"GGA"C"GG TFGPGTKVDFKRGGGG GACAGGC"TCAC"C"GACAA"CAG"AGCC SGGGGSGGGGSQVQJV r1GCAGAG"GAGGAC"T"GCTGT"TAC"AC ESGGGVVQ?GRS.RIS r1G"CAACAG"ACGACACC"GGCCA""CAC CAASGFTFSSYGMiWV AT"CGGGCCCGGCACCAAGG"CGAC""CA ?GKGLEWVAVISY AGAGGGGCGG"GGAGG"TCAGG"GG"GGC DKYYVDSVKGRFT GGGTCAGGCGGCGG"GGG"C"CAGG""CA DVSKNRIYIQWNS AC"GG"GGAA"CAGG"GGCGGCG""G"CC HDTAVYYCARERY AACCGGGGCGATCAC""CGAC"""CC"GT DYWGQG"LVTVSS TCAGGC"T"ACT""""CA"CC"A DN?KSNG"IIHV r1GGGA"GCAC"GGG""CGGCAGGC"CCCG .C?SP.F?G?SK GAAAAGGACTCGAG"GGG""GCAG"GA"C JVVVGGVJACYS r1C"TACGA"GGC"CAGACAAG"AT"AT"Gr1 _ 1VAFIIFWVRSKR GGACTCAG"CAAGGGGAGA"TCACAA§AA _RRP GCCGAGACAACTCCAAAAACCGGC"T"Ar1 _ __RDF CTCCAGA"GAACAGCC""AGAGCGGAAGA TACCGCGG"A"ACTAC"GTGCCCGCGAGA '. .G GGTA""CCGGCAGAGAC"AC"GGGGACAG GGCACAC"GG"CACCG"GAG"TCTGCCGC AGCGC"CGATAACGAAAAGAGCAACGGAA .

CCAT"A"CCACGT"AAGGGCAAGCACC"G ERRRGKGHDGJYQ r1GCCCCAG"CCCC"C""CCCAGGACCA"C _ TATKDTYDA4iMQ mAAACCCmmCmGGGmmCmGGmAGmAGmmG __?R ﬁccﬁmGCAﬁGﬁmAcﬁcccﬁTﬁﬁG GTCACCG"CGCCT"CA""A"""TCr GAGA"CCAAAAGAAGCCGCC"GCF 1A GCGA"TACA"GAA"ATGAC"CCACGCCGC CCTGGCCCCACAAGGAAACAC"ACCAGCC T"ACGCACCACC"AGAGA"""CGCTGCCr1 A"CGGAGCAGGG"GAAG""""CCAGA"Cr1 GCAGATGCACCAGCGTA"CAGCAGGGCCA GAACCAACTGTA"AACGAGC"CAACC"GG GACGCAGGGAAGAGTATGACGTTTTGGAC AAGCGCAGAGGACGGGACCC"GAGA"GGG TGGCAAACCAAGACGAAAAAACCCCCAGG AGGG"CTC"ATAATGAGC"GCAGAAGGAT AAGA"GGC"GAAGCCTAT"C"GAAA"AGG CATGAAAGGAGAGCGGAGAAGGGGAAAAG [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GGCACGACGGTTTG"ACCAGGGACTCAGC ACTGC"ACGAAGGAF 1TA"GACGCTCr1 GCAAGCCC"GCCACCTAGG (CAR4.5) ATGGCAC"CCCCGF 1GC"CTGC"GCr1 MAL?VF .PLAL.

Clone GCCGT"GGCA"TGCF 1GCACGCCGCAC HAARPI 1QSPA". 20C5.2 CHD GCCCGGAGATCG"CA"GACACAGAG"CCA SVS?G?RA".SCRASQ CAR DNA. GCTACCC"GAGCG"G"CCCC"GGAGAGAG SVSSLJTWYQQKPGQA LXH AGCCACCC"G"CC"G"AGGGCTAGTCAGA PRIIIFGAS"RA"GI? GTG"G"CCAGCC"CC"CACC"GG"A"CAA ARFSGSGSG"GF"4TI CAGAAGCC"GGTCAAGC"CCCCGGC"GCT .QS?DFAVYYCQQY TA"CT"CGGGGCCAGCACGCGAGCCACAG GTKVDFK GCA"CCCGGCCAGA""C"C"GGC"CF ACCGGGTTCAC"C"CACGAF A"CCC"GCAGTCAGAGGAT"TCGCF A"I'"AC"1 G"1 CAGCAGF1ACGAF1ACA" I""1CACCT"CGGCCCGGGCACAAAAG" I"W"CAAGCGCGGCGGCGGGGGTAG" GCGGGGGATCAGGAGGAGGGGGCF G"ACAGC"GG""GAGAGCGGCGGCGGGGr GG""CAGCCCGGGCGCAGCCTCAGGCTGA Gm'"GCGCAGCA"CAGGATTCACA"TCAGr1 1C""ATGGAA"GCA""GGG"CAGACAGGC GAAGGGCC""GAA"GGG"GGCAG 1CA""AGCTACGACGGAAGCGATAAG"AC 1A" G"1 GGACT CAG"1I'"AAAGGGAGATT""AC 1A"CAGCCGCGACAA"TCCAAAAACAGAT 1G"A"TTGCAGA"GAAC"CCC"CAGGGCG GAGGACAC"GC"G"A"A"TAC"GCGCACG AGAGAGATAC"CCGGCCGAGACTAF GAACA"1 r" GGF1AACr" G" GCCGCAGC"A""GAGG"CA"GF ACC""A"C"CGA"AA"GAGAAGAGr1 GGAC""A""AA"1 r" CACGr1AAAGGGCAAACAC C"G"GCCC""CCCCGC"GT""CCAGG"CC AAG"AAGCCG"TCTGGG"CC"GG""G"GG r1GGGAGGGG"GC"GGCC"GC"A"""C"C"G mr 1ACCGmGGCCTmmAmCAmr 1cmG GGTGAGA"CCAAAAGAAGCCGCC"GC"CC A"AGCGA"TACA"GAA"ATGAC"CCACGC CGCCC"GGCCCCACAAGGAAACAC"ACCA GCCT"ACGCACCACC"AGAGA"""CGCTG CC"A"CGGAGCAGGG"GAAG""""CCAGA r1C"GCAGATGCACCAGCGTA"CAGCAGGG CCAGAACCAACTGTA"AACGAGC"CAACC CGCAGGGAAGAGTATGACGTTTTG GACAAGCGCAGAGGACGGGACCC"GAGAr1 GGGTGGCAAACCAAGACGAAAAAACCCCC AGGAGGG"CTC"A"AATGAGC"GCAGAAG GATAAGA"GGC"GAAGCCTA"F AGGCA"GAAAGGAGAGCGGAGAA AAGGGCACGACGG"TTGr AGCAC"GC"ACGAAGGA"ACF 1GACGC TC"CCACA"GCAAGCCC"GCCACCTAGGT (CAR4.5) GAGATCG"CA"GACACAGAG"CCAGCTAC EIVM"QSPATLSVSPG Clone CC"GAGCG"G"CCCC"GGAGAGAGAGCCA CRASQSVSSL 20C5.2 CHD CCC"GTCC"G"AGGGCTAGTCAGAGTGTG KPGQAPRJLI CAR DNA. TCCAGCC"CC"CACC"GGTA"CAACAGAA A"GIPARFSG GCC"GGTCAAGCTCCCCGGC"GCTTATCT SGSG"GF"LTISSJQS [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley r1CGGGGCCAGCACGCGAGCCACAGGCATC YCQQYD"W?F CCGGCCAGA"TC"C"GGC"C"GGCAG"GG TFGPGTKVDFKRGGGG CACCGGGTTCAC"C"CACGA"CTCA"CCC GGGGSQVQJV r1GCAGTCAGAGGAT"TCGC"GTGTA""AC ESGGGVVQ?GRS.RIS r1G"CAGCAG"ACGA"ACA"GGCCC""CAC CAASGFTFSSYGM CT"CGGCCCGGGCACAAAAG"AGA""F QQA?GKGLEWVAVISY AGCGCGGCGGCGGGGGTAG"GGGGGC DGS DSVKGRFT GGATCAGGAGGAGGGGGC"CCCAAGr1 NS GC"GG""GAGAGCGGCGGCGGGG"GGF '. YYCARERY AGCCCGGGCGCAGCCTCAGGCTGAG"F _ 1VSS GCAGCA"CAGGATTCACA"TCAG""CF EVMY???Y.DN? TGGAA"GCA""GGG"CAGACAGGC"CCCG 1IIHVKGK-LJC? GGAAGGGCC""GAA"GGG"GGCAG"C "" _ _ ?FWV4VV AGCTACGACGGAAGCGATAAG""AC"A"1 G"1 . 1VAF GGACTCAG"1I'"AAAGGGAGATTI'"AC"A"CA GCCGCGACAA"TCCAAAAACAGAT"G"Ar1 TTGCAGA"GAAC"CCC"CAGGGCGGAGGA CAC"GC"G"A"A"TAC"GCGCACGAGAGA GATAC"CCGGCCGAGACTA"TGGGGCCAA GGAACA""GG"AAC"G"GAGCTCCGCCGC AGC"A""GAGG"CA"G"ACCCCCCACC"F A"C"CGA"AA"GAGAAGAG"AATGGGACF A""AA"1 r" CACGr1AAAGGGCAAACACC"1G" CCC""CCCCGC"GT""CCAGG"CCAAGF AGCCG"TCTGGG"CC"GG""G"GGr GGGGmGCmGGCCmGCmAmmCmCmGr mACCGmGGCCTmmAmCAmmmmCr GA"CCAAAAGAAGCCGCC"GCF GA"TACA"GAA"ATGAC"CCACGCCGCCC r1GGCCCCACAAGGAAACAC"ACCAGCCTr1 CACC"AGAGA"""CGCTGCC"Ar1 CGGAGCAGGG"GAAG""""CCAGA"C"GC AGATGCACCAGCGTA"CAGCAGGGCCAGA ACCAACTGTA"AACGAGC"CAACC"GGGA CGCAGGGAAGAGTATGACGTTTTGGACAA AGGACGGGACCC"GAGA"GGGTG GCAAACCAAGACGAAAAAACCCCCAGGAG GG"CTC"A"AATGAGC"GCAGAAGGATAA GA"GGC"GAAGCCTA""C"GAAA"AGGCA r1GAAAGGAGAGCGGAGAAGGGGAAAAGGG CACGACGG"TTG"ACCAGGGAC"CAGCAC 1ACGAAGGA"AC""A"GACGCTCTCC 1GCAAGCCC"GCCACC"AGG (CAR4.6) ATGGCAC"CCCCG"AAC"GCTC"GCTGCT MAL?VF .PLAL.

Clone GCCGTTGGCA"TGCTCC"GCACGCCGCAC HAARPI 1QSPA". 20C5.2 CD8 GCCCGGAAATAG"GA"GAC"CAG"CCCCG SVS?G?RA".SCRASQ CAR DNA. GCCACCC"CAGCG"G"CCCCCGGGGAGCG SVSSLJTWYQQKPGQA LXH AGCGACCC"G"CA"GCAGGGCTTCCCAGA PRIIIFGAS"RA"GI? GC"CCC"GC"CAC"TGG"AF ARFSGSGSG"GF"4TI CAAAAGCCGGGGCAGGC"CCCCGCCr SS.QS?DFAVYYCQQY CATC""CGGGGCA"CAACTAGGGCCACCG D"W?FTFGPGTKVDFK GCA""CC"GCAAG """"CCGGG"C"GGC RGGGGSGGGGSGGGGS ACCGGC""CACCC""ACCAT"AG QVQ.V?SGGGVVQ?GR C"C"C"GCAGTC"GAGGAC"F SIRISCAASGFTFSSY AC"A""G"CAGCAG"A"GA"ACF GMiWVRQA?GKGLEWV T""ACC""CGG"CCCGGAACF AVISYDGSDKYYVDSV C""CAAGCGCGGGGGGGGTGGA"CTGGAG KGRFTISRDNSKNQJY G"GG"GGCTCCGGGGGCGG"GGAAGCCAG LQWNSIRA?DTAVYYC "F 1GAGAGCGGCGGCGGAGT ARERYSGRDYWGQG"L [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GG"GCAGCCCGGGAGGF 1TGCGGC"GA JSNSIMYF GC""G"GCAGCCTCCGG""" 1AC""""TT""CT _ _ T_ AGC"ATGGAA"GCA"TG 1AAGACAGGC _ __ _ ?TIASQ?. 1CCCGGAAAAGGCC"CG 1GGG"GGCGG .3 ?AAGGAV "CA""AGCTA"GA"GGAF 1GA"AAA""AC _ DIYIWA__ 1A"G"GGACTCAG""AAGGGGCGC"TCAC ISIVIT CAAGAGACAAr1AGCAAAAAr1AGAC RS? . .HS r 1ACCTGCAGA"GAATAG"C"GCGCGCC _QQ?G?"RK{Y GAGGACACTGCCG"G"ACTAC"GCGCCCG _ __RDFAAYRSRV CGAGAGA"ACAGCGGACGGGA""AC"GGG GCCAGGG"ACCC"CG"AACGG"G"CC"CC GC"GCCGCCC""AGCAACAGCA""A"GTA CmmmTCTCAmmmCGmGCCAGTCmmTCmCC CAGCAAAGCCCACCAC"ACCCCGGCCCCC AGGCCGCCTAC"CC"GCCCCCAC"A"CGC G"C"CAGCC"C"C"CC""GCGGCCCGAGG CC"GCCGGCCAGCCGCAGGGGGCGCCG"A CA"ACTCGGGG"""GGA""TCGC""GCGA CA"A"ATAT"TGGGCCCCCC"CGCCGGCA CA"G"GGAG"GC"GCTCC""GAGTC""CG"""1 A"AACCCTC"A""GCAACCA"AGAAACAG A"CCAAAAGAAGCCGCC"GC"CCA"AGCG A""ACATGAATA"GAC"CCACGCCGCCCr1 GGCCCCACAAGGAAACACTACCAGCCT"A CGCACCACC"AGAGA"""CGCTGCC"Ar GGG"GAAG""""CCAGA"C" CCAGCGTA"CAGCAGGGCCAGAA CCAACTGTA"AACGAGC"CAACC"GGGAC GCAGGGAAGAGTATGACGTTTTGGACAAG CGCAGAGGACGGGACCC"GAGA"GGGTGG CAAACCAAGACGAAAAAACCCCCAGGAGG G"CTC"A"AATGAGC"GCAGAAGGATAAG A"GGC"GAAGCCTA""C"GAAA"AGGCAr1 AGAGCGGAGAAGGGGAAAAGGGC G"TTG"ACCAGGGACTCAGCACr1 GC"ACGAAGGA"AC""A"GACGCTCTCCA CA"GCAAGCCC"GCCACC"AGGTAA (CAR4.6) GAAATAG"GA"GAC"CAG"CCCCGGCCAC EIVM"QSPATLSVS?G Clone CC"CAGCG"G"CCCCCGGGGAGCGAGCGA CRASQSVSSL 20C5.2 CD8 CCC"GTCA"GCAGGGCTTCCCAGAGTG"C JTWYQQKPGQAPR.

CAR DNA. AGC"CCC"GC"CAC"TGG"A"CAGCAAAA A"GIPARF LXH GCCGGGGCAGGC"CCCCGCC"CCTCA.TCr1 SGSG"GF".TISS.QS "1CGGGGCA"CAACTAGGGCCACCGGCAW'1 EDFAVYYCQQYD"W.

CC"GCAAG """"CCGGG"C"GGCAGCGG TFGPGTKVDFKRGGGG CACCGGC""CACCC""ACCA""AGC"C"C GGGGSQVQJV r1GCAGTC"GAGGAC""CGCCG"TTAC"Ar1 ESGGGVVQ?GRS.RIS r1G"CAGCAG"A""GA"AC""GGCCCT""AC CAASGFTFSSYGM C""CGG"CCCGGAAC"AAGG"GGAC""CA ?GKGLEWVAVISY AGCGCGGGGGGGGTGGA"CTGGAGG"GGr1 DKYYVDSVKGRFT GGCTCCGGGGGCGG"GGAAGCCAGG"CCA DNSKNRJYLQWNS G""GG""GAGAGCGGCGGCGGAG"GG"GC ?DTAVYYCARERY GGAGG"CC"TGCGGC"GAGC"Gr1 DYWGQG"4V"VSS GCAGCC"CCGG""T"AC""TT"C"AGC"A JSNSIWYFSHFV? TGGAA"GCA"TGGG"AAGACAGGC""CCCG _ "1 _ RP .3 GAAAAGGCC"CGAG"GGG""GGCGG""CA"""1 ?TIASQP RP? AGCTA"GA"GGA"C"GA"AAA"AC"AT"Gr1 ?AAGGAV{TRGLD GGACTCAG""AAGGGGCGC"TCACAA"Cr1 DIYIWAPJAGTCG CAAGAGACAATAGCAAAAA"AGAC"G"AC .SLVITLYCViRV CTGCAGATGAATAG"CTGCGCGCCGAGGA RSRLLHSDYWVMT [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley CACTGCCGTG"ACTAC"GCGCCCGCGAGA _ RPG?TRKHYQPYA? GA"ACAGCGGACGGGA""AC"GGGGCCAG _ DFAAYRSRVKFSRS GG"ACCCTCG"AACGG"G"CC"CCGC"GC APAYQQGQNQLYNE CGCCC""AGCAACAGCA""A"GTAC"""T . YDVLDKRR CTCA"""CG"GCCAGTC""TC"CCCAGCA DPEWGGKPRRKVPQ AAGCCCACCAC"ACCCCGGCCCCCAGGCC ﬂ .YVﬁiQKDKMAEAY "CC"GCCCCCAC"A"CGCG"C"C ﬁIGWKGﬁRRRGKGHD AGCC"C"C"CC""GCGGCCCGAGGCC"GC .YQGISTATKDTYDA CGGCCAGCCGCAGGGGGCGCCG"ACA§AC .{MQA.??R TCGGGG"""GGA""TCGC""GCGACA"Ar1 ATAT"TGGGCCCCCC"CGCCGGCACA""Gr1 GGAG"GC"GCTCCr1 AGTC"CG""A"AAC CCTC"A""GCAACCA"AGAAACAGA"CCA AAAGAAGCCGCC"GC"CCA"AGCGA""AC ATGAATA"GAC"CCACGCCGCCC"GGCCC CACAAGGAAACACTACCAGCCT"ACGCAC CACC"AGAGA"""CGCTGCC"A"CGGAGC AGGG"GAAG""""CCAGA"C"GCAGATGC ACCAGCGTA"CAGCAGGGCCAGAACCAAC TGTA"AACGAGC"CAACC"GGGACGCAGG TATGACGTTTTGGACAAGCGCAG AGGACGGGACCC"GAGA"GGGTGGCAAAC CAAGACGAAAAAACCCCCAGGAGGG"CTC r1PFAATGAGC"GCAGAAGGATAAGA"GGC " AAGCCTA""C"GAAA"AGGCA"GAAAG GGAGAAGGGGAAAAGGGCACGAC GG"TTG"ACCAGGGACTCAGCAC"GC"AC GAAGGA"AC""A"GACGCTCTCCACA"GC AAGCCC"GCCACCTAGG In some embodiments, the polynucleotide of the t invention encodes a CAR, wherein the CAR comprises an amino acid sequence at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 178, 180, 190, 192, 202, 204, 214, 216, 226, and 228. In certain embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 178, 180, 190, 192, 202, 204, 214, 216, 226, and 228.

In some embodiments, the polynucleotide ofthe present invention comprises an nucleotide ce at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 133,135,137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,177, 179, a 191, 201, 203, 213, 215, 225, and 227. In certain embodiments, the polynucleotide [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley ses a nucleotide sequence selected from the group consisting of SEQ ID NOs: 133, 135, 137,139,141,143,145,147,149,151,153,155,157,159,161,163,165,167,177,179,189, 191, 201, 203, 213, 215, 225, and 227.

II. Vectors, Cells, and Pharmaceutical Compositions In certain aspects, provided herein are vectors comprising a polynucleotide of the present invention. In some embodiments, the present invention is directed to a vector or a set of vectors sing a polynucleotide encoding a CAR or a TCR comprising the truncated hinge domain ("THD") domain, as described above.

Any vector known in the art can be suitable for the present invention. In some embodiments, the vector is a viral . In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, a RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector (AAV), a lentiviral vector, or any combination thereof. id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221" id="p-221"

[0221] In an ment, a vector that can be employed in the t of the present invention is pGAR and has the coding ce: CTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCA CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCT TCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCC TTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGA CGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACT CAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCT ATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAAT ATTAACGCTTACAATTTGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG CGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTG CAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAAC GACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGACCCGGGGATGGCGCG CCAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTT ACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTG ACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTG TATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA CGTATTAGTCATCGCTATTACCATGCTGATGCGGTTTTGGCAGTACATCAATGGG CGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCA ATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATAT AAGﬁEAGCTGGTTTAGTGAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGCCT [Annotation] eadley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley GGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCC GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGA TCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTGGCGCCCGAACAG GGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGACTCGGCT TGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAA TGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCGTCAGTA TTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAAGGCCAGGGG GAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCIMEKA CGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATAC TGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTAT ATAATACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACA CCAAGGAAGCTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACC GCACAGCAAGCCGCCGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAA TTGGAGAAGTGAATTATATAAATATAAAGTAGTAAAAATTGAACCATTAGGAGT AGCACCCACCAAGGCAAAGAGAAGAGTGGTGCAGAGAGAAAAAAGAGCAGTGG GAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGC AGCGTCAATGACGCTGACGGTACAGGCCAGACAATTATTGTCTGGTATAGTGCA GCAGCAGAACAATTTGCTGAGGGCTATTGAGGCGCAACAGCATCTGTTGCAACT CACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTGGAAAGATA CCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGGAAAACTCATTTGC ACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGGAACAGATTT GGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACAAGCT TAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAG AATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAA TTGGCTGTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTA AGAATAGTTTTTGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCAC CATTATCGTTTCAGACCCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAG GAATAGAAGAAGAAGGTGGAGAGAGAGACAGAGACAGATCCATTCGATTAGTG AACGGATCTCGACGGTATCGGTTAACTTTTAAAAGAAAAGGGGGGATTGGGGGG TACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAACAGACATACAAACTAA ACAAAAACAAATTACAAAATTCAAAATTTTATCGCGATCGCGGAATGA AAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGC ATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTAGGAACAGAG AGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCG GCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCT AGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTG CCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCT CCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTTCGA AGTAGATCTTTGTCGATCCTACCATCCACTCGACACACCCGCCAGCGGCCGCﬂﬁ: 40 CAAGCTTCCGAGCTCTCGAATTAATTCACGGTACCCACCATGGCCTAGGGAGACT AGTCGAATCGATATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTAT TCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGT ATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGT TGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTG 45 CACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAG CTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGC CGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCC CCTﬁTTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGTG T TTGTCGGGGAAGCTGACGTCCTTTTCATGGCTGCTCGCCTGTGTTGCCA [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley GGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCC TTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGGTTAATTA AAGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTT AAAAGAAAAGGGGGGACTGGAAGGGCGAATTCACTCCCAACGAAGACAAGATC TGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTC TCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGC TAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAG ACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGGCATGCCAGACATGATAAGATA CATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATT TGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACA AGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGG GAGGTTTTTTGGCGCGCCATCGTCGAGGTTCCCTTTAGTGAGGGTTAATTGCGAG CTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAA TTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAAT GAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGG AAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCC TGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCG GTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT GTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGC GCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCA AACAAACCACCGCTGGTAGCGGTGGTTFNﬂTGTTTGCAAGCAGCAGATTACGCG CAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTA TATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTNT CTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG GCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTG 40 TTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGT GTTATCACTCATGGTTATGGCAGCAﬂTGCATAATTCTCTTACTGTCATGCCATCCG TAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG 45 GCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCA CATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCAC AGG‘EIKKAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAACCAA,TGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAAC [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCG CGCACATTTCCCCGAAAAGTGCCAC (SEQ ID NO: 252).

The pGAR vector map is set forth in . Suitable additional exemplary vectors include e.g., pBABE-puro, pBABE-neo largeTcDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV- loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES Luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc. id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223" id="p-223"

[0223] In other aspects, provided herein are cells comprising a cleotide or a vector of the present invention. In some embodiments, the present invention is directed to cells, e.g., in vitro cells, comprising a polynucleotide ng a CAR or a TCR comprising a TCD described . In other embodiments, the present invention is directed to cells, e.g., in vitro cells, comprising a polypeptide encoded by a CAR or a TCR comprising a TCD described herein.

Any cell may be used as a host cell for the polynucleotides, the vectors, or the polypeptides of the present invention. In some embodiments, the cell can be a prokaryotic cell, fungal cell, yeast cell, or higher eukaryotic cells such as a m ammalian cell. Suitable prokaryotic cells include, without limitation, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobactehaceae such asEscherichia, e.g., E. coli; bacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; Serratia, e.g., [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley Serratl'a marcescans, and Shigella, Bacillz' such as B. sublilz's and B. licheniformz’s, monas such as P. aeruginosa, and Slreptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some ments, the immune cell is ed from the group consisting of a T cell, a B cell, a tumor inﬁltrating lymphocyte (TIL), a TCR expressing cell, a natural killer (NK) cell, a dendritic cell, a granulocyte, an innate lymphoid cell, a megakaryocyte, a monocyte, a macrophage, a platelet, a thymocyte, and a myeloid cell. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is an NK cell. In certain embodiments, the T cell is a tumor- rating lymphocyte (TIL), autologous T cell, engineered autologous T cell (eACTTM), an neic T cell, a heterologous T cell, or any combination thereof.

The cell of the present invention may be ed through any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained from, e.g., peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural on, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood ted from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM tion and/or apheresis. In certain embodiments, the cells collected by apheresis are washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent sing. In some embodiments, the cells are washed with PBS. As will be appreciated, a washing step can be used, such as by using a semiautomated ﬂowthrough centrifuge, e.g., the CobeTM 2991 cell processor, the Baxter CytoMateTM, or the like. In some embodiments, the washed cells are resuspended in one or more biocompatible s, or other saline on with or without buffer. In certain embodiments, the undesired components of the apheresis sample are removed. Additional s of isolating T cells for a T cell therapy are disclosed in US. Patent Publication No. 2013/0287748, which is herein incorporated by references in its entirety.

In certain ments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, e.g., by using centrifugation through a PERCOLLTM gradient. In some embodiments, a speciﬁc subpopulation of T cells, such as CD4+, CD8+, CD281 CD45RA+, and CD45RO+ T cells is further isolated by positive or ve selection techniques known in the art. For example, enrichment of a T cell population by negative selectDcan be accomplished with a combination of antibodies directed to surface markers [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley unique to the negatively selected cells. In some embodiments, cell sorting and/or ion via negative magnetic immunoadherence or ﬂow cytometry that uses a cocktail of monoclonal antibodies directed to cell e markers present on the cells negatively selected can be used.

For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD8, CD1 lb, CD14, CD16, CD20, and HLA-DR. In certain embodiments, ﬂow cytometry and cell sorting are used to isolate cell populations of interest for use in the present invention.

In some embodiments, PBMCs are used directly for c modiﬁcation with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolating the PBMCs, T cytes are further ed, and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and/or expansion.

In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface ns that are associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells es CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and are negative for granzyme B. In some embodiments, l memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, or T cells are negative for CCR7, CD28, CD62L, and CD127 and ve for granzyme B and perforin. In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell tions that have cell surface antigens.

In some embodiments, the immune cells, e.g., T cells, are genetically modified following isolation using known methods, or the immune cells are activated and expanded (or differentiated in the case of progenitors) in vitro prior to being genetically modiﬁed. In another embodiment, the immune cells, e.g., T cells, are genetically modified with the chimeric antigen receptors described herein (e. g., uced with a viral vector comprising one or more nucleotide sequences ng a CAR) and then are activated and/or expanded in vitro.

Methods for activating and expanding T cells are known in the art and are described, e. g., in US. Patent Nos. 6,905,874, 6,867,041, and 514, and PCT Publication No. WO 2012/079000, the contents of which are hereby incorporated by nce in their entirety.

Generally, such methods include contacting PBMC or isolated T cells with a stimulatory agent and chulatory agent, such as anti-CD3 and anti-CD28 antibodies, generally attached to a [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley bead or other surface, in a culture medium with appropriate cytokines, such as IL-2. Anti-CD3 and D28 antibodies attached to the same bead serve as a "surrogate" antigen presenting cell (APC). One example is The Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. In other embodiments, the T cells are activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in US. Patent Nos. 6,040,177 and 5,827,642 and PCT Publication No. WO 2012/ 1295 14, the contents of which are hereby orated by reference in their entirety.

In certain embodiments, the T cells are obtained from a donor subject. In some embodiments, the donor t is human patient afﬂicted with a cancer or a tumor. In other embodiments, the donor subject is a human patient not afﬂicted with a cancer or a tumor.

Other aspects of the present invention are directed to compositions comprising a polynucleotide described herein, a vector described herein, a polypeptide described herein, or an in Vitro cell bed herein. In some embodiments, the composition comprises a ceutically acceptable r, diluent, lizer, emulsiﬁer, preservative and/or adjuvant. In some embodiments, the composition comprises an excipient. In one embodiment, the composition comprises a polynucleotide encoding a CAR or a TCR comprising a truncated hinge domain ("THD") described . In another embodiment, the composition ses a CAR or a TCR comprising a TCD encoded by a polynucleotide of the present invention. In another embodiment, the composition comprises a T cell comprising a CAR or a TCR comprising a TCD described herein.

In other embodiments, the composition is selected for eral delivery, for inhalation, or for delivery through the digestive tract, such as orally. The preparation of such pharmaceutically able compositions is within the ability of one skilled in the art. In certain embodiments, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8. In certain embodiments, when parenteral administration is contemplated, the ition is in the form of a pyrogen-free, parenterally acceptable s solution comprising a composition described herein, with or without additional therapeutic agents, in a pharmaceutically acceptable vehicle. In certain embodiments, the e for parenteral injection is sterile distilled water in which composition described herein, with or without at least one additional therapeutic agent, is formulated as a sterile, isotonic solution, properly preserved. In n nts, the preparation involves the ation ofthe d molecule with polymeric [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that provide for the lled or sustained release of the product, which are then be delivered via a depot injection. In n embodiments, implantable drug delivery devices are used to introduce the desired molecule.

III. Methods ofthe Invention Another aspect of the invention is directed to a method of making a cell expressing a CAR or a TCR comprising transducing a cell with a polynucleotide disclosed herein under suitable conditions. In some embodiments, the method comprises transducing a cell with a polynucleotide encoding a CAR or a TCR, as disclosed herein. In some embodiments, the method comprises transducing a cell with a vector comprising the polynucleotide ng a CAR or a TCR.

Another aspect of the t invention is directed to a method of inducing an immunity against a tumor comprising administering to a subject an effective amount of a cell comprising a polynucleotide described herein, a vector described herein, or a CAR or a TCR described herein. In one embodiment, the method comprises administering to a subject an effective amount of a cell comprising a cleotide ng a CAR or a TCR disclosed herein. In another embodiment, the method comprises administering to a t an effective amount of a cell comprising a vector comprising a polynucleotide ng a CAR or a TCR disclosed herein. In another embodiment, the method comprises administering to a subject an effective amount of a cell comprising a CAR or a TCR encoded by a polynucleotide disclosed herein.

Another aspect of the present invention is directed to a method of inducing an immune response in a subject sing administering an effective amount of the engineered immune cells of the t application. In some embodiments, the immune response is a T cell-mediated immune response. In some embodiments, the T cell-mediated immune response is directed against one or more target cells. In some embodiments, the engineered immune cell comprises a CAR or a TCR, wherein the CAR or the TCR comprises a THD described in the present disclosure. In some embodiments, the target cell is a tumor cell.

Another aspect of the present ion is directed to a method for treating or preventing a malignancy, said method comprising administering to a t in need thereof an effective amount of at least one immune cell, n the immune cell comprises at least one CAR OCR, and wherein the CAR or the TCR comprises a THD described herein.

[Annotation] eadley None set by eadley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley ed set by Anne.Headley Another aspect of the present invention is directed to a method of ng a cancer in a subject in need thereof comprising administering to the subject a cleotide, a vector, a CAR or a TCR, a cell, or a composition disclosed herein. In one embodiment, the method comprises administering a polynucleotide encoding a CAR or a TCR. In another embodiment, the method comprises administering a vector comprising a polynucleotide encoding a CAR or a TCR. In another embodiment, the method ses administering a CAR or a TCR encoded by a polynucleotide disclosed herein. In another embodiment, the method comprises administering a cell comprising the polynucleotide, or a vector comprising the polynucleotide, encoding a CAR or a TCR. id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238" id="p-238"

[0238] In some embodiments, the methods of treating a cancer in a subject in need thereof comprise a T cell y. In one embodiment, the T cell therapy of the present invention is engineered Autologous Cell Therapy (eACTTM). According to this embodiment, the method can include collecting blood cells from the patient. The isolated blood cells (e.g., T cells) can then be engineered to express a CAR or a TCR ofthe present ion. In a particular embodiment, the CAR T cells or the TCR T cells are administered to the patient. In some embodiments, the CAR T cells or the TCR T cells treat a tumor or a cancer in the patient. In one embodiment the CAR T cells or the TCR T cells reduce the size of a tumor or a .

In some ments, the donor T cells for use in the T cell therapy are obtained from the patient (e.g., for an autologous T cell therapy). In other embodiments, the donor T cells for use in the T cell therapy are obtained from a subject that is not the patient.

The T cells can be administered at a therapeutically effective amount. For example, a therapeutically effective amount of the T cells can be at least about 104 cells, at least about 105 cells, at least about 106 cells, at least about 107 cells, at least about 108 cells, at least about 109, or at least about 1010. In another embodiment, the therapeutically ive amount of the T cells is about 104 cells, about 105 cells, about 106 cells, about 107 cells, or about 108 cells. In one particular embodiment, the eutically effective amount of the CAR T cells or the TCR T cells is about 2 X 106 cells/kg, about 3 X 106 cells/kg, about 4 X 106 cells/kg, about 5 X 106 cells/kg, about 6 X 106 cells/kg, about 7 X 106 cells/kg, about 8 X 106 cells/kg, about 9 X 106 kg, about 1 X 107 cells/kg, about 2 X 107 cells/kg, about 3 X 107 cells/kg, about 4 X 107 cells/kg, about 5 X 107 cells/kg, about 6 X 107 cells/kg, about 7 X 107 kg, about 8 X 107 cells/kg, or about 9 X 107 cells/kg.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley IV. Cancer Treatment The methods of the invention can be used to treat a cancer in a t, reduce the size of a tumor, kill tumor cells, prevent tumor cell proliferation, prevent growth of a tumor, eliminate a tumor from a patient, prevent relapse of a tumor, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In certain embodiments, the methods induce a complete se. In other embodiments, the methods induce a partial response.

Cancers that may be treated include tumors that are not vascularized, not yet substantially vascularized, or vascularized. The cancer may also include solid or non-solid tumors. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is of the white blood cells. In other embodiments, the cancer is of the plasma cells. In some embodiments, the cancer is leukemia, lymphoma, or myeloma. In certain embodiments, the cancer is acute lymphoblastic ia (ALL) (including non T cell ALL), acute lymphoid leukemia (ALL), and hemophagocytic lymphohistocytosis (HLH)), B cell prolymphocytic leukemia, B-cell acute lymphoid leukemia ("BALL"), c plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia (CIVIL), c or acute granulomatous disease, chronic or acute leukemia, diffuse large B cell lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, follicular lymphoma (FL), hairy cell leukemia, hemophagocytic me (Macrophage ting Syndrome (MAS), Hodgkin's Disease, large cell granuloma, leukocyte adhesion deﬁciency, ant proliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, monoclonal gammapathy of undetermined signiﬁcance (MGUS), multiple myeloma, myelodysplasia and ysplastic syndrome (MDS), d es including but not limited to acute myeloid leukemia (AML), non-Hodgkin's ma (NHL), plasma cell proliferative disorders (e. g., asymptomatic myeloma (smoldering multiple myeloma or indolent myeloma), blastic lymphoma, plasmacytoid dendritic cell neoplasm, cytomas (e. g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple cytoma), POEMS syndrome (Crow-Fukase syndrome, Takatsuki disease, PEP syndrome), primary mediastinal large B cell lymphoma (PMBC), small cell- or a large cell- follicular lymphoma, splenic al zone ma (SMZL), systemic amyloid light chain amyloidosis, T-cell acute id leukemia ("TALL"), T-cell lymphoma, transformed follicular lymphoma, Waldenstrom macroglobulinemia, or a combination thereof.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] eadley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley In one embodiment, the cancer is a myeloma. In one particular embodiment, the cancer is multiple myeloma. In another embodiment, the cancer is a leukemia. In one ment, the cancer is acute myeloid leukemia.

In some embodiments, the methods further comprise administering a chemotherapeutic. In n embodiments, the chemotherapeutic selected is a lymphodepleting (preconditioning) herapeutic. Beneﬁcial preconditioning treatment regimens, along with correlative beneﬁcial kers are described in U. S. Provisional Patent Applications 62/262,143 and 62/167,750 which are hereby incorporated by reference in their entirety herein. These describe, e.g., methods of conditioning a patient in need of a T cell therapy comprising administering to the t speciﬁed beneﬁcial doses of cyclophosphamide (between 200 mg/mZ/day and 2000 mg/mZ/day) and speciﬁed doses of ﬁudarabine (between 20 mg/mZ/day and 900 mg/mZ/day). One such dose regimen involves treating a patient comprising administering daily to the patient about 500 mg/mZ/day of cyclophosphamide and about 60 mg/mZ/day of ﬁudarabine for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.

In other embodiments, the antigen binding le, transduced (or otherwise engineered) cells (such as CARs or TCRs), and the chemotherapeutic agent are administered each in an amount effective to treat the disease or condition in the subject.

In certain embodiments, compositions comprising CAR- and/or TCR- expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXANTM), alkyl sulfonates such as an, improsulfan and piposulfan, ines such as benzodopa, uone, meturedopa, and uredopa, ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, ylenethiophosphaoramide and hylolomelamine resume, nitrogen mustards such as mbucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosureas such as carmustine, chlorozotocin, stine, lomustine, nimustine, ranimustine, antibiotics such as aclacinomysins, actinomycin, mycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, canninomycin, carzinophilin, chromomycins, omycin, daunorubicin, detorubicin, 6-diazooxo-L-norleucine, doxornzin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley acid; nogalamycin; olivomycins; peplomycin; potﬁromycin; puromycin; quelamycin; rodorubicin; streptonigrin; streptozocin; tubercidin; ubenimeX; zinostatin; zorubicin; anti- metabolites such as methotrexate and 5-ﬂuorouracil (5-FU); folic acid ues such as denopterin; methotrexate; pteropterin; trimetrexate; purine analogs such as ﬂudarabine; 6- mercaptopurine; thiamiprine; thioguanine; pyrimidine analogs such as ancitabine; azacitidine; 6-azaun'dine, cannofur; cytarabine; yuridine; doxiﬂuridine; enocitabine; ﬂoxuridine; 5- FU; androgens such as calusterone; dromostanolone propionate; epitiostanol; ostane; testolactone; anti-adrenals such as aminoglutethimide; mitotane; trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; evulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizoﬁran; ermanium; tenuazonic acid; triaziquone; 2; trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; ctol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids; e.g. paclitaxel (TAXOLTM; Bristol-Myers Squibb) and xel ERE®; Rhone-Poulenc Rorer); chlorambucil; gemcitabine; guanine; mercaptopurine; methotrexate; platinum analogs such as tin and carboplatin; vinblastine; platinum; etoposide (VP-l6); ifosfamide; cin C; mitoxantrone; vincristine; lbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-ll; topoisomerase inhibitor RFSZOOO; diﬂuoromethylomithine (DMFO); retinoic acid derivatives such as TargretinTM (bexarotene); PanretinTM; (alitretinoin); ONTAKTM (denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts; acids or derivatives of any of the above. In some ments; itions sing CAR- and/or TCR-expressing immune effector cells sed herein may be stered in conjunction with an anti-hormonal agent that acts to regulate or t hormone action on tumors such as anti-estrogens including for example tamoxifen; raloxifene; aromatase inhibiting 4(5)-imidazoles; 4-hydroxytamoxifen; trioxifene; keoxifene; LYll7018; onapristone; and toremifene (Fareston); and anti-androgens such as ﬂutamide; nilutamide; bicalutamide; leuprolide; and goserelin; and pharmaceutically acceptable salts; acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate; including; but not limited to CHOP; i.e.; [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley hosphamide (Cytoxan®), Doxorubicin (hydroxydoxorubicin), Vincristine (Oncovin®), and sone.

In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered from 1 to 4 weeks or from 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after the administration of the engineered cell or nucleic acid. In some embodiments, the herapeutic agent is administered at least 1 month before administering the cell or nucleic acid. In some embodiments, the methods further comprise administering two or more herapeutic agents.

A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-l inhibitors such as mab (OPDIVO®), pembrolizumab (KEYTRUDA®), pembrolizumab, pidilizumab (CureTech), and atezolizumab (Roche). id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249" id="p-249"

[0249] Additional therapeutic agents suitable for use in ation with the invention include, but are not limited to, ibrutinib (iMBRUVECA®), ofatumumab (ARZERR/W), rituximab (RITUXAN®), zumab IN®), trastuzumab (HERCEPTIN®), trastuzumab emtansine LA®), imatinib (GLEEVEC®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuXimab, alemtuzumab, gemtuzumab, erlotinib, gef1tinib, vandetanib, afatinib, lapatinib, neratinib, aXitinib, masitinib, pazopanib, nib, sorafenib, toceranib, lestaurtinib, aXitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, ntinib, imatinib, nib, nilotinib, ponatinib, radotinib, nib, lestaurtinib, tinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, inib, aﬂibercept,adipotide, denileukin diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, hedgehog inhibitors such as sonidegib and Vismodegib, CDK inhibitors such as CDK inhibitor (palbociclib).

In additional embodiments, the composition comprising CAR- and/or TCR- containing immune are administered with an anti-inﬂammatory agent. Anti-inﬂammatory agents or drugs can include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, prednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-iIDnmatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley sulfasalazine, leﬁunomide, anti-"INF medications, cyclophosphamide and mycophenolate. Exemplary NSAle include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, one, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include one, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modiﬁers include molecules directed against cell surface markers (e. g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF nists, (e. g., etanercept (ENBREL®), adalimumab A®) and inﬁiximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modiﬁers include monoclonal antibodies as well as inant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leﬂunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranoﬁn) and intramuscular), and minocycline.

In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. "Cytokine" as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are kines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N—methionyl human growth hormone, and bovine growth hormone, parathyroid e, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (F SH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH), hepatic growth factor (HGF), ﬁbroblast growth factor (FGF), prolactin, placental lactogen, mullerian-inhibiting nce, mouse gonadotropin-associated peptide, n, n, vascular elial growth factor, integrin, thrombopoietin (TPO), nerve growth factors (NGFs) such as NGF-beta, platelet- growth factor, transforming growth s (TGFs) such as TGF-alpha and TGF-beta, insulin- like growth factor-1 and -II, erythropoietin (EPO), osteoinductive factors, interferons such as interferon-alpha, beta, and -gamma, colony stimulating factors (CSFs) such as macrophage- CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (ILs) such as IL-1, lL-lalpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, lL-lO, IL-11, IL-12, IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta, and other ptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active s of the native ce cytokines.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley All publications, patents, and patent applications ned in this speciﬁcation are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was cally and individually indicated to be incorporated by reference. r, the citation of a reference herein should not be construed as an acknowledgement that such reference is prior art to the present invention. To the extent that any of the deﬁnitions or terms provided in the references incorporated by reference differ from the terms and discussion provided herein, the present terms and deﬁnitions control.

The present invention is further illustrated by the ing examples which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference.

EXAMPLES EXAMPLE 1 Plasmids ng a T7 promoter, CAR construct and a beta globin stabilizing sequence were ized by overnight ion of 10 ug DNA with EcoRI and BamHI (NEB).

DNA was then digested for 2 hours at 50°C with nase K o Fisher, 600 U/ml) puriﬁed with phenol/chloroform and precipitated by adding sodium acetate and two volumes of ethanol. Pellets were then dried, resuspended in RNAse/DNAse-free water and quantiﬁed using NanoDrop. One ug of the linear DNA was then used for in vitro transcription using the mMESSAGE mMACHINE T7 Ultra (Thermo Fisher) following the manufacturer’s instructions. RNA was further puriﬁed using the MEGAClear Kit (Thermo Fisher) following the cturer’s instructions and quantiﬁed using NanoDrop. mRNA integrity was assessed using mobility on an agarose gel. PBMCs were isolated from healthy donor leukopaks (Hemacare) using ﬁcoll-paque y fugation per manufacturer’s instructions. PBMCs were stimulated using OKT3 (50 ng/ml, Miltenyi Biotec) in R10 medium + 1L-2 (300 IU/ml, Proleukin®, Prometheus® Therapeutics and Diagnostics). Seven days timulation, T cells were washed twice in Opti-MEM medium (Thermo Fisher Scientiﬁc) and resuspended at a ﬁnal concentration of 2.5x107 cells/ml in Opti-MEM medium. Ten ug of mRNA was used per electroporation. oporation of cells was performed using a Gemini X2 system (Harvard Apparatus BTX) to deliver a single 400 V pulse for 0.5 ms in 2 mm cuvettes (Harvard Apparatus BTX). Cells were immediately transferred to R10 + IL-2 medium and allowed to recovﬁr 6 hours. To examine CAR sion, T cells were stained with FLT-=3 -HIS (Sino ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley Biological Inc.) or biotinylated Protein L (Thermo Scientiﬁc) in stain buffer (BD Phanningen) for 30 minutes at 4°C. Cells were then washed and d with anti-HIS-PE (Miltenyi Biotec) or PE Streptavidin (BD Pharmingen) in stain buffer for 30 minutes at 4°C. Cells were then washed and resuspended in stain buffer with propidium iodide (BD Pharmingen) prior to data acquisition. Expression of FLT3 CARs in electroporated T cells is shown in T cells were electroporated with ds encoding an LT3 CAR comprising a 10E3, 2E7, 8B5, 4E9, or llFll anti-FLT3 binding molecule and a hinge region selected from the full length hinge domain (a complete hinge domain or "CHD") or a truncated hinge domain ("THD"). The electroporated anti-FLT3 CAR T cells were then co-cultured with Namalwa (FLT3 negative), EoLl (FLT3 positive), HL6O (FLT3 positive), or MV4,11 (FLT3 positive) target cells at a 1:1 E:T ratio in R10 medium. Sixteen hours post-co-culture, supematants from Namalwa (FIGs. 4A-4F), EoLl (FIGs. 4G-4L), HL6O (FIGs. 4M-4R, and MV4,11 ) were analyzed by Luminex (EMD Millipore) for production of IFNy (FIGs. 4A, 4B, 4G, 4H, 4M, 4N, 4s, and 4T), IL-2 (FIGs. 4C, 4D, 41, 4J, 4o, 4P, 4U, and 4V), and TNFu (FIGs. 4E, 4F, 4K, 4L, 4Q, 4R, 4w, and 4X).

Target cell viability was assessed by ﬂow cytometric analysis of propidium iodide (PI) uptake by CD3-negative cells. The electroporated anti-FLT3 CAR T cells were co- cultured with Namalwa (FIGs. 5A-5B), EoLl (FIGs. 5C-5D), HL6O (FIGs. 5E-5F, and MV4,11 (5G—5H) target cells at 16 hours post-co-culture.

EXAMPLE 2 A third generation lentiviral transfer vector containing the different CAR constructs was used along with the ViraPower Lentiviral Packaging Mix (Life Technologies) to generate the lentiviral supematants. Brieﬂy, a ection mix was generated by mixing 15 ug of DNA and 22.5 ul of polyethileneimine ciences, 1 mg/ml) in 600 pl of OptiMEM medium. The mix was ted for 5 minutes at room temperature. Simultaneously, 293T cells (ATCC) were trypsinized, counted and a total of 10x106 total cells were plated in a T75 ﬂask along the transfection mix. Three days after the transfection, supematants were collected and ﬁltered through a 0.45 pm ﬁlter and stored at -80°C until used. PBMCs were isolated from healthy donor aks (Hemacare) using ﬁcoll-paque density fugation per manufacturer’s instructions. PBMCs were stimulated using OKT3 (50 ng/ml, Miltenyi ) in R10 medium + 1L-2 (300 IU/ml, PROLEUKIN®, PROMETHEUS® Therapeutics and Diagnn'cs). Forty eight hours post-stimulation, cells were transduced using lentivirus at an [Annotation] Anne.Headley None set by Anne.Headley ation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley MOI = 10. Cells were ined at 0.5-2.0 X 106 cells/ml prior to use in activity assays. To examine CAR expression, T cells were stained with FLTHIS (Sino Biological Inc.) or biotinylated Protein L (Thermo Scientiﬁc) in stain buffer (BD Pharmingen) for 30 minutes at 4°C. Cells were then washed and stained with IS-PE (Miltenyi ) or PE Streptavidin (BD Pharmingen) in stain buffer for 30 minutes at 4°C. Cells were then washed and resuspended in stain buffer with propidium iodide (BD Pharmingen) prior to data acquisition.

Expression of FLT3 CARs in T cells from two healthy donors is shown in -6B.

T cells from two y donors were transduced with lentiviral vectors encoding anti-FLT3 CAR T cells comprising a lOE3, 8B5, or llFll g molecule and a hinge region ed from the complete hinge domain ("CHD"), a ted hinge domain ("THD"), and the CD8 hinge region. Transduced T cells were co-cultured with target cells at a 1:1 E:T ratio in R10 medium. Sixteen hours post-co-culture, supematants were analyzed by Luminex (EMD Millipore) for production of IFNy (FIGs. 7A-7B), TNFOL (FIGs. 7C-7D), and IL-2 (FIGs. 7E-7F). id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259" id="p-259"

[0259] Target cell viability was assessed by ﬂow cytometric analysis of propidium iodide (PI) uptake by CD3-negative cells. e tic activity of lentivirus-transduced CAR T cells (from two healthy donors) co-cultured with Namalwa (), EoLl (), MV4, 11 (), and HL6O () target cells was measured.

To assess CAR T cell proliferation in response to FLT3 expressing target cells, T cells were labeled with CFSE prior to co-culture with target cells at a 1:1 E:T ratio in R10 medium. Five days later, T cell proliferation was assessed by ﬂow cytometric analysis of CFSE dilution. Proliferation of FLT3 CAR T cells is shown in FIGs. 9A-9B.

EXAMPLE 3 To examine in vivo anti-leukemic activity, FLT3 CAR T cells were generated for use in a xenogeneic model of human AML. CAR expression of the various effector lines used in the xenogeneic model of human AML are shown in FIGs. lOA-lOD. Luciferase-labeled MV4,ll cells (2 x 106 cells/animal) were injected intravenously into 5 to 6 week-old female NSG mice. After 6 days, 6 x 106 T cells (~50% CAR+) in 200 pl PBS were injected intravenously, and the tumor burden of the s was measured weekly using bioluminescence imaging (FIGs. lOE-lOG). Survival is was performed by injection of controls (mock) or lOE3-CHD (H), 10E3-THD (1), or 8B5-THD (]) expreﬁ CAR T cells.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] eadley ionNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley EXAMPLE 4 T cells were electroporated with plasmids encoding the anti-CLL-l CAR constructs 24C8_HL-CHD CAR (comprising a complete hinge domain of the costimulatory protein) and 24C8_HL-THD CAR (comprising a truncated hinge domain of the ulatory protein). Anti-CLL-l expression by electroporated T cells is shown in FIGs. llA-llD. The anti-CLL-l CAR T cells were then cultured with the target Namalwa (ATCC, CLL-l negative), U937 (ATCC, CLL-l positive), HL-6O (ATCC, CLL-l positive), EoL-l (Sigma, CLL-l positive), KGla (ATCC, CLL-l positive) and MV4,ll (ATCC, CLL-l positive) cells at a 1:1 E:T ratio in R10 media 6 hours after mRNA electroporation. Sixteen hours post-co-culture, supematants were analyzed by Luminex (EMD Millipore), according to the cturer's instructions, for tion of IL-2 (A), IFNy (B), and TNFu (C).

Target cell viability was assessed by ﬂow cytometric analysis of propidium iodide (PI) uptake. The electroporated anti-CLL-l CAR T cells were co-cultured with Namalwa (A), MV4,11 (B), EoL-l (C), HL-60 (D), or U937 (E) target cells for 16 hours. As expected, Namalwa cells co-cultured with the anti- CLL-l CAR T cells showed little change in target cell viability, relative to controls (A).

However, increased cytolytic activity was observed in MV,4ll cells co-cultured with 24C8_HL-CHD and 24C8_HL-THD T cells, relative to controls, with a r target cell cytolytic activity ed in the 24C8_HL-THD T cell co-culture (B). In addition, increased cytolytic activity was observed in EoL-l cells co-cultured with 24C8_HL-CHD and 24C8_HL-THD T cells, relative to controls (C). Increased cytolytic activity was observed in HL-60 cells co-cultured with L-CHD and 24C8_HL-THD T cells, relative to controls (D). sed cytolytic ty was observed in U937 cells co-cultured with 24C8_HL-CHD and 24C8_HL-THD T cells, relative to ls, with a greater target cell cytolytic activity observed in the 24C8_HL-THD T cell ture (E).

EXAMPLE 5 T cells transduced with lentiviral vectors comprising an anti-CLL-l CAR construct with a truncated hinge domain ("THD") of the costimulatory protein, lOE3_THD or 24Cl_LH_THD, were co-cultured with Namalwa, U937, HL-60, EoL-l, KGla and MV4,ll target cells at a 1:1 E:T ratio in RlO media 12 days after T cell stimulation. Sixteen hours post- co-culture, supernatants were ed by Luminex (EMD Millipore), ing to the manuUJrer's instructions, for production of the cytokines IFNy (A), IL-2 (B), [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley ation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley ation] Anne.Headley Unmarked set by Anne.Headley and TNFOL (C) in co-cultures of effector HD CAR T cells and 24C1_LH_THD CAR T cells with target Namalwa, HL-60, or MVA, 11 cells, as indicated.

Target cell viability was assessed by ﬂow cytometric analysis of propidium iodide (PI) uptake. Transduced effector 24C1_LH_THD CAR T cells were co-cultured with Namalwa, U937, HL-60, EoL-l, KGla, or MV4,ll target cells for 16 hours or 40 hours. Co- culture ofNamalwa target cells with transduced C _LH_THD CAR T cells had no effect on the percent of viable Namalwa target cells at 16 hours and 40 hours, as compared to mock controls (A). However, Cl_24Cl_LH_THD CAR T cells tured with either MV4,ll (B) or HL-60 (C) target cells resulted in a lower percent of viable target cells at both 16 hours and 40 hours, as compared to mock controls.

EXAMPLE 6 CAR T cells transduced with anti-BCMA CAR ucts sing a truncated hinge domain ("THD") of the costimulatory protein were cultured with target cells at a 1:1 effector cell to target cell (EzT) ratio in RlO media 12 days after T cell stimulation. Cell lines tested included EoL-l (Sigma, BCMA negative), NCI—H929 (Molecular Imaging, BCMA positive), and MMlS (Molecular Imaging, BCMA positive). Sixteen hours post-co-culture, supematants were analyzed by LumineX (EMD Millipore), according to the manufacturer's instructions, for production of the cytokines IFNy (FIGs. l6A-l6B), TNFu (FIGs. l6C-l6D), and IL-2 (FIGs. 16E-16F). IFNy (FIGs. 16A-16B), TNFu (FIGs. 16C-16D), and IL-2 (FIGs. l6E-l6F) were observed in the supernatant ofNCI—H929 and MMl S target cell co-cultures for each CMA CAR T cell tested in both donors (FIGs. l6A-l6B), however, IFNy (FIGs. l6A-l6B), TNFu (FIGs. l6C-l6D), and IL-2 (FIGs. l6E-l6F) were only observed in the atant of EoL-l target cell co-cultures above background for the IR negative control T cells (A). id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267" id="p-267"

[0267] Target cell viability was assessed by ﬂow cytometric analysis of propidium iodide (PI) uptake of CD3 negative cells. The anti-BCMA CAR T cells were co-cultured with EoLl (FIGs. l7A-l7B), NCI-H929 (FIGs. l7C-l7D), or MMlS (FIGs. l7E-l7F) target cells for 16 hours, 40 hours, 64 hours, 88 hours, or 112 hours. Little cytolytic activity was observed in the EoL-l co-cultures at any time period for the anti-BCMA CAR T cells (FIG. l7A-l7B).

However, co-culture of the anti-BCMA CAR T cells and the NCI—H929 or MMl S target cells resulted in a decrease in the percentage of viable target cells at each time point measured for each (ﬁe anti-BCMA CAR T cells.

[Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] Anne.Headley Unmarked set by Anne.Headley ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by eadley ation] Anne.Headley Unmarked set by Anne.Headley To examine proliferation, anti-BCMA CAR T cells were labeled with carboxyﬂuorescein succinimidyl ester (CFSE) prior to co-culture with EoL-l, NCI—H929, or MMlS target cells at a 1:1 E:T ratio in R10 media. Five days later, T cell eration was ed by ﬂow cytometric analysis of CFSE dilution (FIGs. 18A-18B).

EXAMPLE 7 Enhanced stability is a desired property of proteins. This is often ed by determining the melting temperature of a protein under various conditions. Proteins with a higher melting temperature are generally stable for longer times. When a CAR is more thermostable, it may be functionally active for longer periods of time on the surface of a cell. id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270" id="p-270"

[0270] Thermal stability of the CAR extracellular domain (ECD) with the longer hinge domain, i.e., the complete hinge domain ("CHD") and the thermal stability of the CAR ECD with a truncated hinge domain ("THD") was measured using a Bio-Rad C1000 thermal cycler, CFx96 Real-Time system. Unfolding of the proteins was monitored using the ﬂuorescent dye SYPRO Orange (Invitrogen) which binds to hydrophobic amino acids that become solvent exposed as the protein unfolds. A temperature gradient was set up from 25 0C to 95 0C with l 0C / 1 minute increments. Each sample contained 10 uM recombinant CAR ECD protein and 5X SYPRO Orange (Molecular ProbesTM SYPROTM Orange Protein Gel Stain (5,000X Concentrate in DMSO)). The assay was performed in PBS with or without 50 mM NaCl.

As shown in A and B, a CAR’s ECD which has a THD shows enhanced stability compared to a CAR’s ECD which has a CHD, e.g., including the PPY (SEQ ID NO: 250) motif. These method described in this example is a useful method for testing stability of mRNA encoding a CAR and the CAR itself, because once a T cell has been transduced with the mRNA encoding a CAR, the uced T cell will express the CAR and the stability of an individual mRNA or protein cannot be y assessed.

Claims

1.Claims 1. A chimeric antigen receptor (CAR) or a T cell receptor (TCR), which comprises (i) an antigen binding domain, (ii) a transmembrane domain, (iii) an intracellular costimulatory domain, and (iv) an activating domain, wherein the antigen g domain and the 5 transmembrane domain are ted through a truncated hinge domain that is r than 39 amino acid es in length and comprises a sequence that is at least 80% identical to amino acids 123 to 152 of SEQ ID NO: 1.

2. The CAR or TCR of claim 1 wherein the truncated hinge domain is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical 10 to amino acids 123 to 152 of SEQ ID NO: 1.

3. The CAR or TCR of claim 1 or 2, wherein the transmembrane domain is a transmembrane domain of 4-1BB/CD137, an alpha chain of a T cell receptor, a beta chain of a T cell receptor, CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD19, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a zeta chain of a T 15 cell receptor, or any combination thereof.

4. The CAR or TCR of claim 1 or 2, wherein the transmembrane domain comprises SEQ ID NO: 5.

5. The CAR or TCR of claim 1 or 2, wherein the intracellular domain comprises a signaling region of 4-1BB/CD137, activating NK cell receptors, B7-H3, BAFFR, BLAME 20 (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 ), CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8alpha, CD8beta, CD96 (Tactile), CDl la, CDl lb, CDl lc, CDl ld, CDS, CEACAM1, CRT AM, cytokine ors, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM 25 R), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, Immunoglobulin-like proteins, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGBl, KIRDS2, LAT, LFA-1, LFA-1, a ligand that specifically binds with CD83, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), 30 lymphocyte function-associated antigen-1 (LFA-1 (CDl la/CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG/Cbp, programmed 1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocytic activation molecules (SLAM proteins), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, a Toll 5 ligand receptor, TRANCE/RANKL, VLA1, or VLA-6, or a ation f.

6. The CAR or TCR of claim 1 or 2, wherein the intracellular domain comprises a 4- 1BB/CD137 signaling region.

7. The CAR or TCR of claim 1 or 2, wherein the intracellular domain comprises SEQ ID NO: 7. 10

8. The CAR or TCR of claim 1 or 2, wherein the antigen g domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises 3 complementarity ining regions (CDRs) and the VL comprises 3 CDRs.

9. The CAR or TCR of claim 1 or 2, wherein the antigen binding domain specifically binds an antigen selected from the group consisting of 5T4, alphafetoprotein, B cell 15 maturation antigen (BCMA), CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30 , CD33, CD56, CD123, CD138, c-Met, CSPG4, C-type lectin-like molecule 1 (CLL-1), EGFRvIII, lial tumor antigen, ERBB2, FLT3, folate binding protein, GD2, GD3, HER1-HER2 in combination, ER3 in combination, HER2/Neu, HERV-K, HIV-1 envelope glycoprotein gp41, HIV-1 envelope glycoprotein gpl20, IL- 20 llRalpha, kappa chain, lambda chain, melanoma-associated antigen, elin, MUC-1, mutated p53, d ras, prostate-specific antigen, ROR1, or VEGFR2, or a combination thereof.

10. The CAR or TCR of claim 1 or 2, wherein the activation domain comprises SEQ ID NO: 251. 25

11. A polynucleotide encoding the CAR or TCR of any one of claims 1 to 10. [Annotation] eadley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley ionNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley — gSEQ ID NO: 1! MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLH KGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFC KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLV V VGGVLACYSL Hinge Domain Transmembrane §YIYAEElFWYB§KB§BLLE§DXMHMTRBBR§BIBKH¥QB¥AERBDEAAXB§ Signaling Domain [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by r [Annotation] wilksar Unmarked set by wilksar ation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by Anne.Headley — Anti-CLL—l Binding Molecules FRI FR2 CDR2 24Cl VH QVQLQESGPCLURPSLTLQLTCTES SCSIbS--1’WQWIPQPPG'GLARIGVI’ISGS- 24GB VH 20C5.1_VE .VLVWSLALVLKJ’ 20Gb.“[\ vu DV;LVEUJB“\J ~LRLS(An er““ VMINVPUA‘GVGLUVPVL“ YYYY :YY : :Y. :: ::Y .YY ::: Y:YY YYYYYYY:. . ... . FR3 FR4 24C1HVH NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCVS VYCGGDCYSGFDZHGQGTL 24GB VH HYNPSLKSPVTISIDTSKNLFSLPLSSVTAADTAVYYCAS VYCGGDCYSGFDZHGQGTL '2005.1 VE YRUFFgPRVTVPjDTQPJKYMEThQTPQTDTPVYVFPP.Q\CIC-—WDYFDYNGQGTZ 20C5. "' VE FPPFTISQDWQRNPLYLQVWQLP.ADTrViVFPP ----- P.D‘HGQGTL Y . Y Y.. Y Y . ~ Y. . YYYYYYY er~b4r~b~lry~b .u.. . .u u u. u . 24c1_vn VTVSE 2409 vn VTVLd 200571 VH VTVQS 2005.2 VH VTVQS w+NY+ ; —---m [Annotation] wilksar None set by wilksar [Annotation] wilksar MigrationNone set by wilksar [Annotation] wilksar Unmarked set by wilksar [Annotation] Anne.Headley None set by Anne.Headley [Annotation] Anne.Headley MigrationNone set by Anne.Headley [Annotation] Anne.Headley Unmarked set by eadley [Annotation] Anne.Headley None set by eadley [Annotation] Anne.Headley MigrationNone set by eadley [Annotation] eadley Unmarked set by Anne.Headley —Anti—CLL-1 Binding Molecules 2 I31 CL VL DLIQL TQ SPS SLSASVGDFVS FT! ‘