Bispecific chimeric antigen receptors and use thereof

Abstract

The present disclosure relates to bispecific chimeric antigen receptors (CARs) targeting both Claudin-18 splice variant 18.2 (CLDN18.2) and transforming growth factor-β (TGF-β). Also disclosed are chimeric antigen receptor (CAR) T cells expressing the bispecific CAR and methods of treating gastric cancer by administering the CAR T cells to a subject in need thereof.

Description

BISPECIFIC CHIMERIC ANTIGEN RECEPTORS AND USE THEREOF RELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 537943, filed September 12, 2023, the content of which is hereby incorporated by reference in its entirety into this disclosure. INCORPORATION BY REFERENCE OF AN ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The computer readable file, created on September 5, 2024, is named 67944__SequenceListing.xml and is 105,976 bytes in size. INCORPORATION BY REFERENCE

[0003] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein controls. BACKGROUND

[0004] Chimeric antigen receptors (CARs) are engineered molecules capable of redirecting the specificity of T cells to predetermined antigens, e.g., those predominantly expressed in cancer cells. The coding sequence for a CAR can be introduced into a T cell via a viral vector (e.g., a lentiviral or retroviral vector) to produce a chimeric antigen receptor (CAR) T cell. CAR T cells have been useful in the treatment of a variety of diseases, including but not limited to, cancers. Nevertheless, CAR T cell-based therapy can lead to undesirable and unsafe effects. Toxic effects such as cytokine release syndrome (CRS), and neurotoxicity are potential adverse effects of CAR T cell therapy and are potentially fatal. CRS is the most common acute toxicity associated with CART cells. CRS occurs when lymphocytes are highly activated and release excessive amounts of inflammatory cytokines, including interleukin 2, interleukin 6, and interferon-gamma.

[0005] Gastric cancer (GC) is the fifth most common cancer and the third most common cause of cancer death globally. Gastric cancer is diagnosed histologically after endoscopic biopsy and staged using CT (computerized tomography), endoscopic ultrasound, PET, and laparoscopy. It is a molecularly and phenotypically a highly heterogeneous disease. Currently, the main treatment for early gastric cancer is endoscopic resection. Recently, the potential of CAR T cell therapy in GC has been explored, yet the clinical outcome has been limited.

[0006] Claudin-18 (CLDN18) is a member of a large family of four-span transmembrane proteins, Claudins, which are the essential components of the mammalian tight junctions (TJs) in the epithelial cells. Claudin-18 has two splice variants, CLDN18.1 and CLDN18.2. While CLDN18.1 is specifically expressed in the lung tissue, CLDN18.2 expression in normal tissue is more restricted and is only detected in small patches of stomach mucosal. CLDN18.2 expression is elevated in many types of epithelial cancers including stomach, esophagus, pancreatic, and ovarian cancers. The expression of CLDN18.2 is not only detected in primary tumors, but also in metastatic sites.

[0007] Transforming growth factor beta 1 or TGF-β1 is a polypeptide member of the transforming growth factor beta superfamily of cytokines. It is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation, and apoptosis. The TGF-β signaling pathway governs key cellular processes under physiologic conditions and is deregulated in many pathologies, including cancer. In the early phase of tumorigenesis, TGF-β has tumor suppressive functions, primarily through cell cycle arrest and apoptosis. However, in the late stage of cancer, TGF-β acts as a driver of tumor progression and metastasis by increasing tumor cell invasiveness and migration and promoting chemo-resistance.

[0008] Thus, there is an urgent need in the field to develop safer and more effective CAR T cell therapies to treat GC by targeting CLDN18.2 and TGF-β1. SUMMARY OF THE INVENTION

[0009] Disclosed herein are bispecific chimeric antigen receptors (CAR) targeting CLDN18.2 and TGF-β. T cells expressing the bispecific CAR show (1) low cross-reactivity to CLDN18.1; (2) potent cytotoxicity toward CLDN18.2-expressing cells; and (3) sufficient responses to TGF-β. These bispecific CAR T cells may be useful in treating gastric cancer.

[0010] In one aspect, provided is a bispecific chimeric antigen receptor (CAR) comprising, in order:a) a first antigen-binding fragment that specifically binds to CLDN18.2; b) a second antigen-binding fragment that specifically binds to TGF-β; c) a spacer domain; d) a transmembrane domain; e) a costimulatory domain; and f) a signaling domain. Each of the first antigen-binding fragment and the second antigen-binding fragment of the bispecific CAR of the present disclosure may be a single-chain variable fragment (scFv), a Fab fragment, a single domain antibody, or a single domain antibody fragment.

[0011] In another aspect, the first antigen-binding fragment of the bispecific CAR is an anti- CLDN18.2 scFv; and the second antigen-binding fragment of the bispecific CAR is an anti-TGF-β scFv. Optionally, the anti-CLDN18.2 scFv and the anti-TGF-β scFv may be separated by a bispecific linker selected from the group consisting of a Witlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 49), (G4S)1 (SEQ ID NO: 50), (G4S)2 (SEQ ID NO: 51), (G4S)3 (SEQ ID NO: 52), and (G4S)4 (SEQ ID NO: 53). The bispecific CAR may comprise a spacer domain comprising SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, or 63; a transmembrane domain comprising SEQ ID NO: 64 or SEQ ID NO: 65; a costimulatory domain comprising SEQ ID NO: 66; or a signaling domain comprising SEQ ID NO: 67. Optionally, the bispecific CAR may comprise SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

[0012] In another aspect, the anti-CLDN18.2 scFv may comprise a variable heavy (VH) region comprising SEQ ID NO: 68 (HCDR1), SEQ ID NO: 69 (HCDR2), and SEQ ID NO: 70 (HCDR3); and a variable light (VL) region comprising SEQ ID NO: 71 (LCDR1), SEQ ID NO: 72 (LCDR2), and SEQ ID NO: 73 (LCDR3).

[0013] Optionally, the VH region and VL region of the anti-CLDN18.2 scFv are separated by an scFv linker selected from the group consisting of a Witlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 49), (G4S)1 (SEQ ID NO: 50), (G4S)2 (SEQ ID NO: 51), (G4S)3 (SEQ ID NO: 52), and (G4S)4 (SEQ ID NO: 53). The VH region and VL region may have been humanized.

[0014] The VH region of the anti-CLDN18.2 scFv may comprise SEQ ID NO: 2, 6, 8, or 10. The VL region of the anti-CLDN18.2 scFv may comprise SEQ ID NO: 4, 12, 14, or 16.

[0015] The anti-CLDN18.2 scFv may have a VH-VL orientation, optionally wherein the anti-CLDN18.2 scFv may comprise SEQ ID NO: 22. Alternatively, the anti-CLDN18.2 scFv may have a VL-VHorientation, optionally wherein the anti-CLDN18.2 scFv comprises SEQ ID NO: 24.

[0016] In yet another aspect, the anti-TGF-β scFv of the bispecific CAR may comprise a variable heavy (VH) region comprising SEQ ID NO: 18 and a variable light (VL) region comprising SEQ ID NO: 20, and optionally wherein the VHregion and VLregion may be separated by a Witlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 49) or a GS18 linker (GSTSGGGSGGGSGGGGSS, SEQ ID NO: 54). The anti-TGF-β scFv may have a VH-VL orientation, optionally wherein the anti-TGF-β scFv may comprise SEQ ID NO: 26. Alternatively, the anti-TGF-β scFv may have a VL-VHorientation.

[0017] In another aspect, provided is a nucleic acid encoding the bispecific CAR, optionally, wherein the nucleic acid may comprise SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47. Also provided is a viral vector comprising the nucleic acid encoding the bispecific CAR, optionally wherein the viral vector may be a lentiviral vector.

[0018] In yet another aspect, provided is a method for making an engineered cell comprising transducing the nucleic acid encoding the bispecific CAR into a T cell.

[0019] In a further aspect, provided is a T cell comprising the nucleic acid encoding the bispecific CAR.

[0020] Further provided is a method of treating gastric cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a population of cells comprising the T cell comprising the nucleic acid encoding the bispecific CAR. The population of cells may be a population of autologous cells. The method of treating gastric cancer may further comprise administering another therapeutic agent, optionally wherein the another therapeutic agent may be nivolumab, pembrolizumab, ramucirumab, or trastuzumab. Alternatively, the method of treating gastric cancer may further comprise administering a PD-1 inhibitor or a PD-L1 inhibitor, optionally wherein the PD-1 inhibitor may be pembrolizumab, nivolumab, or cemiplimab, and optionally wherein the PD-L1 inhibitor may be atezolizumab, avelumab, or durvalumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 depicts a sequence alignment between the human CLDN18.1 (SEQ ID NO: 74) and the human CLDN18.2 (SEQ ID NO: 75). The alignment was obtained by running CLUSTALW with additional annotations of various structural elements.

[0022] FIGs. 2A, 2B, 2C, and 2D collectively depict the cytotoxicity potency of various single input CARs and bispecific CARs against gastric target cells as described in Example 2.

[0023] FIG. 3 depicts the potency of various bispecific CARs against human TGF-β1 as described in Example 2. DETAILED DESCRIPTION

[0024] 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 limitations of the measurement system. For example, "about" or "comprising essentially of" can mean within one or more than one standard deviation per the practice 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 acceptable error range for that particular value or composition.

[0025] The term "Administering" as used herein refers to the physical introduction of an agent to a subject, such as a modified T cell disclosed herein, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivoelectroporation. In some embodiments, 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 example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0026] The terms, "activated" and "activation" refer to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In one embodiment, activation may also be associated with induced cytokine production, and detectable effector functions. The term "activated T cells" refers to, among other things, T cells that are proliferating. Signals generated through the TCR alone may be insufficient for full activation of the T cell and one or more secondary or costimulatory signals may also be required. Thus, T cell activation comprises a primary stimulation signal through the TCR / CD3 complex and one or more secondary costimulatory signals. Costimulation may be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation through the TCR / CD3 complex.

[0027] The term "allogeneic" refers to any material derived from one individual which is then introduced to another individual of the same species, e.g., allogeneic T cell transplantation.

[0028] The term "antibody" (Ab) includes, without limitation, a glycoprotein immunoglobulin which binds specifically 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 constant region 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 VHand VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) ofthe classical complement system. In general, human antibodies are approximately 150 kD tetrameric agents composed of two identical heavy (H) chain polypeptides (about 50 kD each) and two identical light (L) chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a "Y-shaped" structure. The heavy and light chains are linked or connected to one another by a single disulfide bond; two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and the tetramer is formed. Naturally-produced antibodies are also glycosylated, e.g., on the CH2 domain.

[0029] The term "human antibody" is intended to comprise antibodies having variable and constant domain sequences generated, assembled, or derived from human immunoglobulin sequences, or sequences indistinguishable therefrom. In some embodiments, antibodies (or antibody components) may be considered to be "human" even though their amino acid sequences comprise residues or elements not encoded by human germline immunoglobulin sequences (e.g., variations introduced by in vitro random or site-specific mutagenesis or introduced by in vivo somatic mutation). The term "humanized" is intended to comprise antibodies having a variable domain with a sequence derived from a variable domain of a non-human species (e.g., a mouse), modified to be more similar to a human germline encoded sequence. In some embodiments, a "humanized" antibody comprises one or more framework domains having substantially the amino acid sequence of a human framework domain, and one or more complementary determining regions having substantially the amino acid sequence as that of a non-human antibody. In some embodiments, a humanized antibody comprises at least a portion of an immunoglobulin constant region (Fc), generally that of a human immunoglobulin constant domain. In some embodiments, a humanized antibody may comprise a CH1, hinge, CH2, CH3, and, optionally, a CH4 region of a human heavy chain constant domain.

[0030] Antibodies can include, e.g., monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, 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, intrabodies, 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')2fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti- Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as "antibody mimetics"), and antigen binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody populations. Antibodies may also comprise, for example, Fab' fragments, Fd' fragments, Fd fragments, isolated CDRs, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, single chain or Tandem diabodies, AnticalinsTM, and the like.

[0031] 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 include but are not limited to human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally 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 portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab.

[0032] An "antigen binding molecule," "antigen binding portion," "antigen binding fragment," or "antibody fragment" refers to any molecule that comprises the antigen 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 include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising peptide binding domains) are another example of suitable antigen binding molecules. In some embodiments, the antigen binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen binding molecule binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen. In certain embodiments an antigen binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0033] Amino acid sequences that specifically bind to desired antigens are known in the art or may be prepared using methods known in the art. Examples include immunoglobulins, variable regions of immunoglobulins (e.g., variable fragment ("Fv") or bivalent variable fragment ("Fab")), single chain antibodies, etc. In certain embodiments, the antigen binding molecule is an antibody fragment that specifically binds to the antigen, including one or more of the complementarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv).

[0034] In some instances, a CDR can be substantially identical to one found in a reference antibody (e.g., an antibody of the present disclosure) and / or the sequence of a CDR provided in the present disclosure. In some embodiments, a CDR is substantially identical to a reference CDR (e.g., a CDR provided in the present disclosure) in that it is either identical in sequence or contains between 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions as compared with the reference CDR. In some embodiments a CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In some embodiments a CDR is substantially identical to a reference CDR in that it shows at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments a CDR is substantially identical to a reference CDR in that one amino acid within the CDR is deleted, added, or substituted as compared with the reference CDR while the CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments a CDR is substantially identical to a reference CDR in that 2, 3, 4, or 5 (e.g., 2- 5) amino acids within the CDR are deleted, added, or substituted as compared with the reference CDR while the CDR has an amino acid sequence that is otherwise identical to the reference CDR. In various embodiments, an antigen binding fragment binds a same antigen as a reference antibody. In various embodiments, an antigen binding fragment cross-competes with the reference antibody, for example, binding to substantially the same or identical epitope as the reference antibody.

[0035] The terms "variable region" or "variable domain" are used interchangeably. 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 binding and specificity of a particular antibody for its particular antigen. The variability in the sequence is concentrated in those regions called complementaritydetermining regions (CDRs) while the more highly conserved regions in the variable domain are called framework regions (FR). Without wishing to be bound by any particular 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 the 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., non-human primate) framework regions (FRs).

[0036] 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.

[0037] 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.

[0038] A number of definitions of the CDRs are commonly in use: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between the two used by Oxford Molecular's AbM antibody modelling software. The contact definition is based on an analysis of the available complex crystal structures.

[0039] 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 functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0040] The term "heavy chain" when used in reference to an antibody can refer to any distinct type, e.g., alpha, delta, epsilon, gamma, and mu, based on the amino acid sequence of the constant domain, which give rise to IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3and IgG4.

[0041] The term "light chain" when used in reference to an antibody can refer to any distinct type, e.g., kappa or lambda based on the amino acid sequence of the constant domains. Light chain amino acid sequences are well known in the art. In specific embodiments, the light chain is a human light chain.

[0042] The term "antigen" refers to a compound, composition, or substance that may stimulate the production of antibodies or a T cell response in a human or animal, includingcompositions (such as one that includes a tumor-specific protein) that are injected or absorbed into a human or animal. An antigen reacts with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed antigens. A "target antigen" or "target antigen of interest" is an antigen that is not substantially found on the surface of other normal (desired) cells and to which a binding domain of a TCR or CAR contemplated herein, is designed to bind. A person of skill in the art would readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be endogenously expressed, i.e., expressed by genomic 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.

[0043] The term "autologous" refers to any material derived from the same individual to which it is later to be re-introduced. For example, engineered autologous cell therapy 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.

[0044] The term "binding affinity" generally refers to the strength 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 antigen). Unless indicated otherwise "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 limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KDis calculated from the quotient of koff / kon, whereas KAis calculated from the quotient of koff / kon. 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 kon and koffcan be determined by techniques known to one of ordinary skill in the art, such as BIACORETMor KinExATM.

[0045] The term "KD" (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, or the dissociation equilibrium constant of an antibody or antibody- binding fragment binding to an antigen. There is an inverse relationship between KDand binding affinity, therefore the smaller the KD value, the higher, i.e., stronger, the affinity. Thus, the terms "higher affinity" or "stronger affinity" relate to a higher ability to form an interaction and thereforea smaller KD value, and conversely the terms "lower affinity" or "weaker affinity" relate to a lower ability to form an interaction and therefore a larger KD value. In some circumstances, a higher binding affinity (KD) of a particular molecule (e.g., antibody) to its interactive partner molecule (e.g., antigen X) compared to the binding affinity of the molecule (e.g., antibody) to another interactive partner molecule (e.g., antigen Y) may be expressed as a binding ratio determined by dividing the larger KDvalue (lower, or weaker, affinity) by the smaller KD(higher, or stronger, affinity), for example expressed as 5-fold or 10-fold greater binding affinity, as the case may be.

[0046] The term "kA" refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody or antibody-binding fragment.

[0047] The term "binding" generally refers to a non-covalent association between or among two or more entities. Direct binding involves physical contact between entities or moieties. "Indirect" binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities may be assessed in any of a variety of contexts, e.g., where interacting entities or moieties are studied in isolation or in the context of more complex systems (e.g., while covalently or otherwise associated with a carrier entity and / or in a biological system such as a cell).

[0048] "Chemokines" are a type of cytokine that mediates cell chemotaxis, or directional movement. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin- 3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1.alpha. (MIP-1.alpha., MIP-1a), MIP-1.beta. (MIP-1b), gamma-induced protein 10 (IP-10), and thymus and activation regulated chemokine (TARC or CCL17).

[0049] The terms "Chimeric Antigen Receptor" or "CAR" refer to a molecule engineered to comprise a binding motif and a means of activating immune cells (for example T cells such as naïve T cells, central memory T cells, effector memory T cells or combination thereof), NK cells, and other cell types upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors or chimeric immunoreceptors. In some embodiments, a CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. A T cell that has been genetically engineered to express a chimeric antigen receptor may be referred to as a CAR T cell.

[0050] The antigen-binding domain of a CAR typically consists of an extracellular region that targets a surface antigen on tumor cells, and an intracellular domain to derive T cell activation.The antigen-binding domain of a CAR commonly comprises a targeting moiety, such as an antibody single chain variable fragment (scFv), which is directed against the target antigen. The T cell-activating domain of a CAR is intracellular and activates the T cell in response to the antigen- binding domain interacting with its target antigen. A T cell activating domain can contain one or more co-stimulatory domains, which are the intracellular domains of known activating T cell receptors. The selection and positioning of costimulatory domains within a CAR construct influence CAR T cell function and survival, as costimulatory domains have impacts on CAR-T cell, cytotoxic function, and safety profile. The extracellular antigen-binding and intracellular T cell- activating domains of CARs are linked by a transmembrane domain, hinge, and optionally, a spacer region. The hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to the target antigen on the tumor cell. It may be used alone or in conjunction with a spacer domain that locates the scFv away from the T cell surface. The optimal length of the spacer depends on the proximity of the binding epitope to the cell surface

[0051] "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that, when the polypeptide is present in a cell membrane, is understood to reside outside of the cell membrane, in the extracellular space.

[0052] The binding domain of the CAR may be followed by a "spacer," which refers to the region that moves the antigen binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding and activation. The spacer may further comprise a hinge region or domain. The hinge region is typically membrane proximal, and is between the transmembrane (TM) and the binding domain. In certain embodiments, a hinge region is an immunoglobulin hinge region and may be a wild-type immunoglobulin hinge region or an altered wild-type immunoglobulin hinge region. Other exemplary hinge regions used in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as IgG (e.g., IgG1, IgG2, IgG3, and IgG4), CD8α, CD4, CD28, 4-1BB, and CD7, which may be wild- type hinge regions from these molecules or may be altered.

[0053] The "transmembrane" region or domain is the portion of the CAR that anchors the extracellular binding portion to the plasma membrane of the immune effector cell. The transmembrane domain may be for example those obtained from CD8α, CD4, CD28, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137, CD3zeta, and CD154. In one embodiment, the transmembrane domain is the transmembrane domain of CD8α. In certainembodiments, the transmembrane domain is synthetic in which case it would comprise predominantly hydrophobic residues such as leucine and valine.

[0054] The “intracellular domain” comprises one or more costimulatory domain, and one or more intracellular signaling domains. The intracellular costimulatory domain can be from, e.g., 4- 1BB and / or CD28.

[0055] The "intracellular signaling domain" or "signaling domain" refers to the part of the chimeric antigen receptor protein that participates in transducing the message of effective CAR binding to a target antigen into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain. The term "effector function" refers to a specialized function of the cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the terms "intracellular signaling domain" or "signaling domain," are used interchangeably herein and refer to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal. The intracellular signaling domain is also known as the, "signal transduction domain," and is typically derived from portions of the human CD3 or FcRy chains. The intracellular signaling domain can be, e.g., derived from CD3z.

[0056] It is known that signals generated through the T cell receptor alone are generally insufficient for full activation of the T cell and that a secondary, or costimulatory signal, is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen dependent primary activation through the T cell receptor (primary cytoplasmic signaling sequences) and those that act in an antigen independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). Cytoplasmic signaling sequences that act in a costimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs.

[0057] Examples of ITAM containing primary cytoplasmic signaling sequences that are of particular use in the disclosure include those derived from CD3 zeta, FeR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0058] As used herein, the term, "costimulatory signaling domain," or "costimulatory domain", refers to the portion of the CAR comprising the intracellular domain of a costimulatory molecule. “Costimulatory molecules” are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. The inclusion of one or more costimulatory signaling domains may enhance the efficacy and expansion of T cells expressing CAR receptors. The intracellular signaling and costimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain. Suitable costimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), 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, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8alpha, CD8beta, CD9, CD96 (Tactile), CD1- 1a, CD1-1b, CD1-1c, CD1-1d, CDS, CEACAM1, CRT AM, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig alpha (CD79a), IL2R beta, IL2R gamma, IL7R alpha, integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), MEW class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), signaling lymphocytic activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0059] As will be appreciated, although scFv-based CARs engineered to contain a signaling domain from CD3 or FcRgamma have been shown to deliver a potent signal for T cell activation and effector function, they are not sufficient to elicit signals that promote T cell survival and expansion in the absence of a concomitant costimulatory signal. Thus, CARs containing a binding domain, a hinge, a transmembrane and the signaling domain derived from CD3zeta or FcRgammatogether with one or more costimulatory signaling domains (e.g., intracellular costimulatory domains derived from CD28, CD137, CD134 and CD278) may more effectively direct antitumor activity as well as increased cytokine secretion, lytic activity, survival and proliferation in CAR expressing T cells in vitro, and in animal models and cancer patients. See Milone et al., Molecular Therapy, 2009; 17: 1453-1464; Zhong et al., Molecular Therapy, 2010; 18: 413-420 and Carpenito et al., PNAS, 2009; 106:3360-3365).

[0060] 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, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In certain embodiments, one or more amino acid residues within a CDR(s) or within a framework region(s) of an antibody or antigen-binding molecule thereof can be replaced with an amino acid residue with a similar side chain. In general, two sequences are generally considered to be "substantially similar" if they contain a conservative amino acid substitution in corresponding positions. For example, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and / or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type may be considered a conservative substitution.

[0061] "Combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic moieties). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combinationcomposition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0062] The "control elements" or "regulatory sequences" present in an expression vector are those non-translated regions of the vector-origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine Dalgarno sequence or Kozak sequence), introns, a polyadenylation sequence, 5' and 3' untranslated regions - which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous promoters and inducible promoters maybe used.

[0063] The term "dosing regimen" may be used to refer to a set of one or more unit doses that are administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, a dosing regimen comprises a plurality of doses and consecutive doses are separated from one another by time periods of equal length; in some embodiments, a dosing regimen comprises a plurality of doses and consecutive doses are separated from one another by time periods of at least two different lengths. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen is periodically adjusted to achieve a desired or beneficial outcome.

[0064] An "epitope" refers to a localized region of an antigen to which an antibody can specifically bind. An epitope can be, for example, contiguous amino acids of a polypeptide (linear or contiguous epitope) or an epitope can, for example, come together 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 scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization may be accomplished using any of the known methods in the art (e.g., Giege R et al., (1994) ActaCrystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen N E (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 Wyckoff H W et al.; U.S. 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 C W; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies may be accomplished using any method known to one of skill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270: 1388-1394 and Cunningham BC & Wells JA (1989) Science 244: 1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0065] "Endogenous" with reference to a gene, protein, and / or nucleic acid refers to the natural presence of that gene, protein, and / or nucleic acid in a cell, such as an immune cell.

[0066] "Exogenous" refers to an introduced agent, such as a nucleic acid, gene, or protein, into a cell, for example from an outside source. A nucleic acid introduced into a cell is exogenous even if it encodes a protein which is naturally found in the cell. Such exogenous introduction of a nucleic acid encoding a protein can be used to increase the expression of the protein over the level that would naturally be found in the cell under similar conditions, e.g., without the introduction of the exogenous nucleic acid.

[0067] A "fragment" or "portion" of a material or entity as described herein has a structure that comprises a discrete portion of the whole, e.g., of a physical entity or abstract entity. In some embodiments, a fragment lacks one or more moieties found in the whole. In some embodiments, a fragment consists of or comprises a characteristic structural element, domain or moiety found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the wholepolymer (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). The whole material or entity may in some embodiments be referred to as the "parent" of the fragment.

[0068] The term "fusion polypeptide" or "fusion protein" generally refers to a polypeptide comprising at least two segments. Generally, a polypeptide containing at least two such segments is considered to be a fusion polypeptide if the two segments are moieties that (1) are not comprised in nature in the same peptide, and / or (2) have not previously been linked or connected to one another in a single polypeptide, and / or (3) have been linked or connected to one another through action of the hand of man. In embodiments, a CAR is a fusion protein.

[0069] A "T cell receptor" or "TCR" refers to antigen-recognition molecules present on the surface of T cells. During normal T cell development, each of the four TCR genes, alpha, beta, gamma, and delta, may rearrange leading to highly diverse TCR proteins.

[0070] 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 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.

[0071] The term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided polypeptide sequences are known. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences may be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which may need to be introduced for optimal alignment of the two sequences. Comparison or alignment of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm, such as BLAST (basic local alignment search tool). In some embodiments, polymeric molecules are considered to be "homologous" to one another iftheir sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95- 100%).

[0072] The terms "improve," "increase," "inhibit," and "reduce" indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may comprise a measurement in certain system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) an agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may comprise a measurement in comparable system known or expected to respond in a comparable way, in presence of the relevant agent or treatment.

[0073] 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, eosinophils, 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 infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0074] The term "immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapies or NK cell therapies. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, and allogeneic T cell transplantation.

[0075] One of skill in the art will recognize techniques to enhance the effectiveness of cell therapy using, e.g., preconditioning techniques such as those found in U.S. Patent Nos. 9,855,298 and 10,322,146, the contents of which are hereby incorporated by reference in their entirety.

[0076] The T cells of the immunotherapy can come from 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 (PBMCs), 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 bloodcollected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTMseparation and / or apheresis. Additional methods of isolating T cells for a T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is herein incorporated by references in its entirety.

[0077] The term "in vitro" refers to events occurring in an artificial environment, e.g., in a test tube, reaction vessel, cell culture, etc., rather than within a multi-cellular organism. 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 term "in vivo" refers to events that occur within a multi-cellular organism, such as a human or a non-human animal.

[0078] The term "isolated" refers to a substance that (1) has been separated from at least some components with which it was associated at an earlier time or with which the substance would otherwise be associated, and / or (2) is present in a composition that comprises a limited or defined amount or concentration of one or more known or unknown contaminants. An isolated substance, in some embodiments, may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85- 90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) of other non-substance components with which the substance was associated at an earlier time, e.g., other components or contaminants with which the substance was previously or otherwise would be associated. In certain instances, a substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of molecules of a same or similar type. For instance, in certain instances, a nucleic acid, DNA, or RNA substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of non-substance nucleic acid, DNA, or RNA molecules. For instance, in certain instances, a polypeptide substance is isolated if it is present in a composition that comprises a limited or reduced amount or concentration of non-substance polypeptide molecules. In certain embodiments, an amount may be, e.g., an amount measured relative to the amount of a desired substance present in a composition. In certain embodiments, a limited amount may be an amount that is no more than 100% of the amount of substance in a composition, e.g., no more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the amount of substance in a composition (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). In certain instances, a composition is pure or substantially pure with respect to a selected substance. In some embodiments, an isolated substance is about 80%, about 85%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%). A substance is "pure" if it is substantially free of other components or of contaminants. In some embodiments, a substance may still be considered "isolated" or even "pure," after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without comprising such carriers or excipients.

[0079] The term "lymphocyte" includes natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell toxic) lymphocyte that represent a component of the inherent immune system. NK cells reject tumors and cells infected by viruses. It works through 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 role in cell-mediated-immunity (no antibody involvement). Its T cell receptors (TCR) differentiate themselves from other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible 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 TSCMcells, like naïve cells, are CD45RO-, CCR7+, CD45RA+, CD62L+(L-selectin), CD27+, CD28+ and IL-7R alpha+, but they also express large amounts of CD95, IL-2RB, CXCR3, and LFA-1, and show numerous functional attributes distinctive of memory cells); (ii) central memory T.sub.CM cells express L-selectin and the CCR7, they secrete IL-2, but not IFN-gamma or IL-4, and (iii) effector memory TEM cells, however, do not express L-selectin or CCR7 but produce effector cytokines like IFN-gamma and IL-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 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.

[0080] The term "neutralizing" refers to an antigen binding molecule, scFv, antibody, or a fragment 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 ligand's ability to bind through indirect means (such as structural or energetic alterations in the ligand). In some embodiments, the antigenbinding molecule, scFv, antibody, or a fragment thereof prevents the protein to which it is bound from performing a biological function.

[0081] The term "nucleic acid" refers to any polymeric chain of nucleotides. A nucleic acid may be DNA, RNA, or a combination thereof. In some embodiments, a nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, a nucleic acid comprises of one or more nucleic acid analogs. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide.

[0082] The term "operably linked" refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. For example, a control element "operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element.

[0083] The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide 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 herein, the term refers to both short chains, which also commonly are referred 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. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0084] In other embodiments, a vector for use in practicing the embodiments described herein including, but not limited to expression vectors and viral vectors, will include exogenous, endogenous, or heterologous sequences such as promoters and / or enhancers. An "endogenous" control sequence is one which is naturally linked with a given gene in the genome. An "exogenous" control sequence is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. A "heterologous" sequence is an exogenous sequence that may be from a different protein of the same species or a different species than the protein or cell being genetically manipulated.

[0085] The term "promoter" as used herein refers to a recognition site of a polynucleotide (DNA or RNA) to which an RNA polymerase binds. An RNA polymerase initiates and transcribes polynucleotides operably linked to the promoter. In some embodiments, promoters operative in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and / or another sequence found 70 to 80 bases upstream from the start of transcription, a CNCAAT region where N may be any nucleotide.

[0086] The term "enhancer" refers to a segment of DNA which contains sequences capable of providing enhanced transcription and in some instances may function independent of their orientation relative to another control sequence. An enhancer may function cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA which contains sequences capable of providing both promoter and enhancer functions.

[0087] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not deleterious to the recipient thereof, or that any deleterious effect is outweighed by a benefit to the recipient thereof. With respect to a carrier, diluent, or excipient used to formulate a composition as disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof, or any deleterious effect must be outweighed by a benefit to the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one portion of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulationand not deleterious to the patient, or any deleterious effect must be outweighed by a benefit to the recipient. Some examples of materials which may serve as pharmaceutically acceptable carriers comprise: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0088] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, a pharmaceutical composition may be formulated for administration in solid or liquid form, comprising, without limitation, a form adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0089] "Regulatory T cells" ("Treg", "Treg cells", or "Tregs") refer to a lineage of CD4+T lymphocytes that participate in controlling certain immune activities, e.g., autoimmunity, allergy, and response to infection. Regulatory T cells may regulate the activities of T cell populations, and may also influence certain innate immune system cell types. Tregs may be identified by the expression of the biomarkers CD4, CD25 and Foxp3, and low expression of CD127. Naturally occurring Treg cells normally constitute about 5-10% of the peripheral CD4+T lymphocytes.However, Treg cells within a tumor microenvironment (i.e., tumor-infiltrating Treg cells), Treg cells may make up as much as 20-30% of the total CD4+T lymphocyte population.

[0090] "Single chain variable fragment", "single-chain antibody variable fragments" or "scFv" antibodies refer to forms of antibodies comprising the variable regions of only the heavy and light chains, connected by a linker peptide.

[0091] The phrase "therapeutic agent" may refer to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms or human subjects. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, in accordance with presence or absence of a biomarker, etc. In some embodiments, a therapeutic agent is a substance that may be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent is an agent that has been or is required to be approved by a government agency before it may be marketed for administration to humans. In some embodiments, a therapeutic agent is an agent for which a medical prescription is required for administration to humans.

[0092] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" 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 disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration 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 evaluated using a variety of methods 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.

[0093] The terms "transduction" and "transduced" refer to the process whereby foreign DNA is introduced 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, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr viralvector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adenovirus associated vector, a lentiviral vector, or any combination thereof.

[0094] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, 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" includes a partial remission. In another embodiment, "treatment" or "treating" includes a complete remission. In some embodiments, treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. In some embodiments, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0095] The term "vector" or “lentiviral vector” refers to a recipient nucleic acid molecule modified to comprise or incorporate a provided nucleic acid sequence. One type of vector is a "plasmid," which refers to a circular double stranded DNA molecule into which additional DNA may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors comprise sequences that direct expression of inserted genes to which they are operatively linked. Such vectors may be referred to herein as "expression vectors." Standard techniques may be used for engineering of vectors, e.g., as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference.

[0096] A "binding protein" is a protein that is able to bind non-covalently to another molecule. A binding protein can bind to, for example, a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein) and / or a protein molecule (a protein-binding protein).In the case of a protein-binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more molecules of a different protein or proteins. A binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA-binding, RNA-binding and protein-binding activity.

[0097] The term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double- stranded. The term "donor sequence" refers to a nucleotide sequence that is inserted into a genome. A donor sequence can be of any length, for example between 2 and 10,000 nucleotides in length (or any integer value therebetween or thereabove), preferably between about 100 and 1,000 nucleotides in length (or any integer therebetween), more preferably between about 200 and 500 nucleotides in length.

[0098] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Illustrative retroviruses suitable for use in some embodiments, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and lentivirus.

[0099] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type-1, and HIV type-2); visna-maedi virus (VMV) virus; the caprine arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0100] In some embodiments, compositions contemplated herein comprise an effective amount of an expanded modified T cell composition, alone or in combination with one or more therapeutic agents. Thus, the T cell compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The compositions may also be administered in combination with antibiotics and anti-viral agents. Such therapeutic agents may be accepted in the art as a treatment for a disease state as described herein, such as gastric cancer.

[0101] In an aspect of the present invention, it is preferred that an inflammatory effect or response is reduced relative to existing constructs and methods of treatment.

[0102] For example, the inflammatory effect or response is reduced as compared to tisagenlecleucel (Kymriah) or axicabtagene ciloleucel (Yescarta).

[0103] In certain embodiments, a construct described herein does not produce or otherwise elicit an inflammatory response.

[0104] In certain embodiments, a construct described herein produces or otherwise elicits an inflammatory response which is less than tisagenlecleucel (Kymriah) or axicabtagene ciloleucel (Yescarta).

[0105] In certain embodiments, a therapeutic method described herein does not produce or otherwise elicit an inflammatory response in a subject.

[0106] In certain embodiments, a therapeutic method described herein produces or otherwise elicits an inflammatory response in a subject which is less than tisagenlecleucel (Kymriah) or axicabtagene ciloleucel (Yescarta).

[0107] In an aspect, the inflammatory effect or response is reduced as indicated by reduced levels of interferon gamma, while maintaining substantially the same cytotoxicity.

[0108] In an aspect, the inflammatory effect or response is reduced as indicated by reduced levels of interferon gamma and IL-2, while maintaining substantially the same cytotoxicity.

[0109] In certain embodiments, the inflammatory effect or response is reduced as indicated by reduced levels of interferon gamma, as compared to the inflammatory effect or response produced by CAR-T cells made using a CD3+ T-cell population (e.g., tisagenlecleucel (Kymriah) or axicabtagene ciloleucel (Yescarta)), while maintaining substantially the same cytotoxicity.

[0110] In certain embodiments, compositions comprising T cells contemplated herein may be administered in conjunction with one or more additional therapeutic agents. The additional therapeutic agent may be an approved agent to treat cancer, e.g., nivolumab, pembrolizumab, ramucirumab, and trastuzumab. Exemplary therapeutic agents include but are not limited to an inhibitor of B-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK), an inhibitor of a phosphatidylinositol 3 -kinase (PI3K), an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K / mT0R inhibitor, an inhibitor of Bruton's tyrosine kinase (BTK), an inhibitor of Isocitrate dehydrogenase 1 (IDH1), an inhibitor of Isocitrate dehydrogenase 2 (IDH2), an inhibitor of indoleamine 2,3 -dioxygenase-1) (IDO1) (e.g., epacadostat), an inhibitor of HER3, an inhibitor ofLSD1, an inhibitor of MDM2, an inhibitor of BCL2, an inhibitor of CHK1, an inhibitor of activated hedgehog signaling pathway, an agent that selectively degrades the estrogen receptor, Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, an Hsp90 inhibitor, Ad-GM- CSF, Temazolomide, IL-2, IFNα, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, enzastaurin, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, an IL- 10 antagonist, an IL-4 antagonist, an IL- 13 antagonist, an IL-17 antagonist, an HVEM antagonist, an ICOS agonist, a treatment targeting CX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, a treatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, a Selectin agonist, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, FOLFIRI, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti -LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, and an anti-GITR antibody. Additionally or alternatively, compositions comprising T cells contemplated herein may be administered in conjunction with anti-HER2 therapy, anti-PD-1 / PDL-1 therapy, or anti-VEGF / VEFGR therapy. For example, compositions comprising T cells contemplated herein may be administered in conjunction with a PD-1 inhibitor (e.g., pembrolizumab, nivolumab, or cemiplimab) or a PD-L1 inhibitor (e.g., atezolizumab, avelumab, or durvalumab).

[0111] A variety of therapeutic agents may be used in conjunction with the compositions described herein. In one embodiment, the composition comprising T cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.

[0112] Retroviral-based gene therapy vectors (e.g., gammaretroviral, lentiviral) are the predominant choice for CAR transduction due to the stable integration of these vectors, which results in long-term expression of the CAR. Critical vector quality attributes (e.g., titer, potency,purity) are directly determine the number of copies stably integrated into the target cells, and therefore to a large extent determine the potency of the CAR T cell product.

[0113] Claudins are key structural and functional components of epithelial tight junctions, which act to regulate cell-cell permeability, maintain ion homeostasis, and support cell adhesion and polarity. Claudins are tetraspan transmembrane proteins of 22-27 kDa that multimerize within or across cell membranes to form a protective barrier. Claudin 18 (CLDN 18) was initially identified as a target gene for the transcription factor T / EBP / NKX2.1. CLDN 18 has been confirmed to localize to cellular tight junctions in mouse and human. Two isoforms of CLDN 18 are present because of alternative splicing: CLDN18.1, expressed specifically in normal lung, and CLDN18.2, expressed in differentiated cells of the gastric mucosa. Human CLDN18.2 is a 261 amino acid protein with two extracellular loops, and has about 92% sequence identity to human CLDN18.1 (FIG. 1). Unlike the second extracellular loop, the first extracellular loop of CLDN18.2 has eight amino acid differences from CLDN18.1. CLDN18.2 homology to other family members is more limited, with 29-34% overall identity to CLDN1, CLDN6 and CLDN7. CLDN18.2 is expressed in several tumor types, including gastric cancer, pancreatic cancer, esophageal cancer, mucinous ovarian cancer and non-small cell lung cancer. CLDN18.2 expression in gastric cancer includes the invasive front and metastatic sites, although absolute levels of CLDN18 are reported to be decreased in these settings.

[0114] Another challenge to successful CAR T cell immunotherapy is immunosuppression caused by the tumor microenvironment (TME) of solid tumors. For example, Transforming Growth Factor beta (TGF-β) is a pleiotropic cytokine produced by many cell types and in large amounts within cancer microenvironments (Dahmani et al., TGF-β in T Cell Biology: Implications for Cancer Immunotherapy. Cancers 2018, 10(194): 1-21). TGF-β binds to TGFβR2, which recruits and phosphorylates TGFβR1, which once phosphorylated, in turn phosphorylates receptor- regulated SMADs (R-SMADs). The phosphorylated SMADs complex with coSMADs translocate to the nucleus to help regulate gene expression. In the context of T cells, TGF-β signaling suppresses CAR-T cell therapy effectiveness by inhibiting T cell proliferation, activation, and effector functions and by favoring regulatory T-cell differentiation. Therefore, TGF-β-associated immunosuppression is a significant hurdle that must be overcome to obtain effective and persistent CAR-T cell therapy for solid tumors. To minimize the risk of immunosuppression the CAR T therapies should include CAR T cells that can persist in the immunosuppressive TME of solid tumors.EXAMPLES Materials and Methods Antibody generation

[0115] Monoclonal antibodies were generated via mice immunization with DNA, protein, and / or cell-based immunogens. To obtain monoclonal antibody against CLDN18.2, hybridoma supernatants were tested for their ability to bind to the surface of CLDN18.2 expressing engineered CHO cell line but failed to bind to CLDN18.1 expressing cells. The mouse VH and VL sequences were further determined. The mouse VH and VL sequences were further humanized with the established methods. Vector Construction

[0116] In accordance with the present disclosure, the bispecific CAR (anti-CLDN18.2 / TGF- β CAR) of the present disclosure may utilize a lentiviral backbone, the pALD, Aldevron's (Fargo, North Dakota) Lenti expression plasmid. For this, both the bispecific CAR transgene and the EF1α promoter are incorporated into the pALD plasmid. The third generation pALD lentiviral vector is a replication-incompetent and self-inactivating vector, due to the number of essential genes that have been deleted. It includes an additional number of safety features; an altered 3′ long terminal repeat (LTR) renders the vector “self-inactivating” to prevent integrated genes from being repackaged and a heterologous coat protein (e.g., VSV-G) is used in place of the native HIV-1 envelope protein. Key components of the pALD vector include the following elements.

[0117] CMV promoter: The cytomegalovirus (CMV) promoter replaces the U3 LTR in SIN vectors and drives transcription of viral ribonucleic acid (RNA) in packaging cells. This RNA is then packaged into live virus.

[0118] 5' LTR-ΔU3: This is a deleted version of the HIV-15' long terminal repeat. The LTRs carry both promoter and polyadenylation function. In the pALD plasmid, 5' LTR-ΔU3 is deleted for safety. This does not affect the production of viral RNA during packaging because the promoter function is supplemented by the CMV promoter engineered upstream of 5'LTR-ΔU3 LTR.

[0119] Ψ: HIV-1 packaging signal required for the packaging of viral RNA into virus.

[0120] RRE: HIV-1 Rev Response Element (RRE). RRE permits the nuclear export of viral RNA by the viral Rev protein during viral packaging.

[0121] cPPT: HIV-1 Central Polypurine Tract (cPPT). cPPT creates a "DNA flap" that increases nuclear import of the viral genome during target cell infection. This improves vector integration into the host genome, resulting in higher transduction efficiency.

[0122] Incorporation of a cPPT and a posttranscriptional regulatory element (PRE) into lentivirus vectors provides increased transduction efficiency and transgene expression.

[0123] Hybrid EF1a / HTLV1 promoter: The hEF1a-HTLV promoter is a composite promoter comprising the human Elongation Factor-1α (EF-1α) core promoter and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat. The EF-1α promoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The promoter drives the expression of the anti-CLDN18.2 / TGF-β CAR proteins.

[0124] Kozak: Kozak consensus sequence The Kozak consensus sequence is placed in front of the start codon of the opening reading frame (ORF) of interest to facilitate translation initiation in eukaryotes.

[0125] Bispecific CAR ORF: The open reading frame of the bispecific anti- CLDN18.2 / TGF-β CAR proteins. The ORF optionally may encode a bispecific CAR containing a mIgK signal peptide (METDTLLLWVLLLWVPGSTG: SEQ ID NO:76) at the N-terminus.

[0126] WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The WPRE enhances viral RNA stability in packaging cells, leading to higher titer of packaged lentiviral particles. Here, a mutation was introduced into the translation initiation (ATG) site of the X protein to eliminate any potential translation through the WPRE promoter sequence.

[0127] 3' LTR-ΔU3: A truncated version of the HIV-13' long terminal repeat that deletes the U3 region. This leads to the self-inactivation of the promoter activity of the 5' LTR upon viral vector integration into the host genome (since the 3' LTR is copied onto 5' LTR during viral integration). The polyadenylation signal contained in 3' LTR-ΔU3 serves to terminates all upstream transcripts produced both during viral packaging and after viral integration into the host genome.

[0128] pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

[0129] Spacer and transmembrane domain. In the bispecific CAR of the present disclosure, the non-signaling Extracellular Spacer Domain may be derived from the IgG4 Hinge region, while the transmembrane domain can be derived from human CD28 or CD8α. The spacer provides a flexible link between the scFv and the transmembrane domains. It allows the antigen-binding domain to accommodate different orientations to facilitate antigen recognition. The transmembrane domain provides a physical link between the spacer and intracellular signaling domains. The length and topology of the spacer and transmembrane domains are critical in providing an appropriate steric orientation for specific antigen recognition and subsequent T cell activation.

[0130] Intracellular signaling domains. The CAR intracellular signaling domains play crucial roles in T cell activation, persistence, and effector functions. Although the CD3ζ chain is adequate for T cell activation, one or more costimulatory domains are also needed to fully activate T cells and to promote CAR T cell persistence (Milone et al., Mol. Ther. 17(8):1453-64 (2009) ). These domains are typically derived from the intracellular domains of costimulatory proteins such as 4-1BB (CD137). Differences in costimulatory domain function may impact product safety and activity by affecting CAR T cell cytokine production, expansion, cytotoxicity and persistence after administration. The intracellular signaling domains of the bispecific CAR of the present disclosure may be derived from human 4-1BB and CD3ζ.

[0131] Plasmid DNA can be prepared from a vial of the plasmid construct according to written procedures at Aldevron. Details of the plasmid production process will be provided in the IND with a letter of cross-reference to the Aldevron Drug Master File (DMF). Lentiviral Vector Manufacturing

[0132] Production of the lentiviral vector is as follows. Using the current serum-free suspension manufacturing process, viral vector can be manufactured by transient transfection using Lentigen’s four plasmid system, which includes research grade transfer plasmid and Lentigen’s three helper plasmids. The downstream process may include clarification, Benzonase® treatment, tangential flow filtration, and column chromatography. After purification, the Vector will be concentrated approximately 100-fold, formulated in formulation buffer, sterile filtered, and filled into 1 mL vials. In preparation for GMP production of the lentiviral batch, one 2 ml Research- grade batch and 4L non-GMP batch was prepared by Lentigen. CAR T cell manufacturing and characterization

[0133] CAR T cell manufacturing can be performed with previously established methods, for example, the methods disclosed in PCT / US2023 / 071707, the entirety of which is incorporated by reference. A non-limiting example is provided below.

[0134] CD62L T cell Isolation and activation On Day 1, Naïve / Memory T cells from donors were isolated from leukopaks by diluting the leukapheresis 1:1 with PBS and 2% FBS and added to a tube containing Ficoll. PBMC were isolated and CD62L cells were further enriched using Miltenyi CD62L selection beads, LS columns, and MACS magnetic separation following Miltenyi protocol. Isolated CD62L cells were isolated post-enrichment and activated with TransAct at a final dilution of 1:100 from the total culture volume. Optimizer media, with Optimizer supplement, Optimizer Glutamax, 1% P / S, 50 IU / mL Interleukin-2 (IL-2) and 0.5 ng / mL Interleukin-15 (IL-15).

[0135] CAR T cell generation Two days after activation, cells were transduced with the lentivirus harboring the anti-CLDN18.2 / TGF-β CAR construct at a multiplicity of infection of 10, calculated from titer provided by Vector Builder. Culture medium, IL-2, and IL-15 cytokines were added / replenished on days 5, 8, and 10. On Day 8, CAR T cells were enriched using an anti- FLAG-APC antibody, anti-APC Miltenyi MACS beads, LS columns, and MACS Magnet following MACS Miltenyi protocol. After enrichment, isolated CAR T cells were added to fresh culture medium and expanded until Day 12 when cells were collected for assays or frozen in CS10 for storage.

[0136] Target cell coculture On Day 11, AGS GFP-FFLUC, AGS CLDN18.2 GFP- FFLUC, AGS CLDN18.2 GFP-FFLUC, and / or GSU GFP-FFLUC were counted using AOPI cell counting dye and counted using the Luna® to determine viable cell concentrations. One thousand fifteen hundred target cells (AGS GFP-FFLUC, AGS CLDN18.2 GFP-FFLUC, AGS CLDN18.2 GFP-FFLUC, and / or GSU GFP-FFLUC) were seeded in a white 384-well flat-bottom plate at 2,000 cells per well and placed in an incubator overnight to allow adherent cells to attach.

[0137] On Day 12, T cells were counted using AOPI cell counting dye and counted using the Luna® to determine viable cell concentrations. Transgene positive CAR-T cells were normalized with untransduced cells to normalize the % transfection to a similar transduction range. The CAR-T groups or untransduced cells were titrated 2-fold at a starting ratio of 20:1 effector:target and co-cultured with target cells for 48 hrs.

[0138] After 48h, plates were removed from the incubator and Bio-GLO luciferase reagent was added per manufacturer’s protocol (Promega, Catalog: G7940) to obtain the Relative Luminescence Units released from the GFP-FFLUC positive target cells. Percent cytotoxicity was calculated using a target cell-only well.

[0139] TGF- β1 coculture On Day 12, CAR T cells were counted using AOPI cell counting dye and counted using the Luna® to determine viable cell concentrations. TGF-β was titrated 2-fold starting at a concentration of 20 nanogram per mL and a fixed number of T cells were seeded. After overnight incubation, supernatants were collected and analyzed with Biolegend CBA quantification assay to analyze IFN- γ secretion, manufacturer’s protocol was followed (BD Bioscience, Catalog: 558269). EXAMPLE 1: Construction of CAR Expressing Vectors

[0140] Exemplary tandem targeting constructs for anti-CLDN18.2 / TGF-β CAR as described herein, as well as various monospecific CAR constructs, were generated with the exemplary protocol described above. The constructs are summarized in the table below:Table 1. Summary of the exemplary CAR constructs Construct Name CAR Promoter CLDN18.2 scFv scFv Bispecific Format scFv Orientation Linker Linker CT041_CARsGen Single EF1a CT041 VH-VL (G4S)3 NA CD28 CT041 Single EF1a Hz.CT041 VL-VH Whitlow NA Single Input CAR 1 Single EF1a B3 / VH3-21 VL-VH Whitlow NA Single Input CAR 2 Single EF1a B3 / VH3-7 VL-VH Whitlow NA Single Input CAR 3 Single EF1a O11 / VH3- VL-VH Whitlow NA 21 Single Input CAR 4 Single EF1a O11 / VH3-7 VL-VH Whitlow NA Bispecific CAR 1 Bispecific EF1a B3 / VH3-21 VL-VH Whitlow (G4S)3 (SEQ ID NO: 40) Bispecific CAR 2 Bispecific EF1a B3 / VH3-7 VL-VH Whitlow (G4S)3 (SEQ ID NO: 42) Bispecific CAR 3 Bispecific EF1a O11 / VH3- VL-VH Whitlow (G4S)3 (SEQ ID NO: 44) 21 Bispecific CAR 4 Bispecific EF1a O11 / VH3-7 VL-VH Whitlow (G4S)3 (SEQ ID NO: 46) Bispecific CAR 5 Bispecific EF1a B3 / VH3-21 VL-VH Whitlow (G4S)3 (SEQ ID NO: 48) TGF-β Single EF1a NA NA (G4S)3 NA Construct Name TGF-β scFv Hinge Trans- Costim Signaling scFv Orientation membrane Domain Domain CT041_CARsGen NA NA CD8a CD28 CD28 CD3z CD28 CT041 NA NA CD8a CD8a 41BB CD3z Single Input CAR 1 NA NA CD8a CD8a 41BB CD3z Single Input CAR 2 NA NA CD8a CD8a 41BB CD3z Single Input CAR 3 NA NA CD8a CD8a 41BB CD3z Single Input CAR 4 NA NA CD8a CD8a 41BB CD3z Bispecific CAR 1 TGF-β VH-VL IgG4 short CD28 41BB CD3z (SEQ ID NO: 40) Bispecific CAR 2 TGF-β VH-VL IgG4 short CD28 41BB CD3z (SEQ ID NO: 42) Bispecific CAR 3 TGF-β VH-VL IgG4 short CD28 41BB CD3z (SEQ ID NO: 44) Bispecific CAR 4 TGF-β VH-VL IgG4 short CD28 41BB CD3z (SEQ ID NO: 46) Bispecific CAR 5 TGF-β VH-VL IgG4 short CD8a 41BB CD3z (SEQ ID NO: 48) TGF-β TGF-β VH-VL IgG4 short CD28 41BB CD3z

[0141] A murine monoclonal antibody against CLDN18.2 having a VH (SEQ ID NO: 2) and a VL(SEQ ID NO: 4) was obtained from Accurus Biosciences, Inc. Each of the VHand VLwassubject to humanization of the framework region to create eleven combinations. The combinations were first selected as single-inputs to evaluate their potency.

[0142] To construct the bispecific CAR, an anti-TGF-β scFv having a VH(SEQ ID NO: 18) and a VL (SEQ ID NO: 20) has been used.

[0143] Single-input constructs also include CARsgen’s CT041 and an anti-TGF-β CAR. Single Input CAR 1 comprises SEQ ID NO: 14 as VLand SEQ ID NO: 6 as VH. Single Input CAR 2 comprises SEQ ID NO: 14 as VLand SEQ ID NO: 10 as VH. Single Input CAR 3 comprises SEQ ID NO: 16 as VL and SEQ ID NO: 6 as VH. Single Input CAR 4 comprises SEQ ID NO: 16 as VL and SEQ ID NO: 10 as VH. Example 2. Characterization of various CAR constructs and CAR T cells.

[0144] Various constructs were evaluated based upon (1) cytotoxic potency; (2) response to TGF-β; and (3) level of cross-reactivity to CLDN18.1. Among about eighty constructs for anti- CLDN18.2 / TGF-β CAR, five lead constructs (Bispecific CARs 1-5) were selected for further characterization.

[0145] To evaluate cytotoxicity, untransduced or transgene positive T cells were cocultured with target cells at an E:T titration range starting at 10:1 transduced CAR T cells to target cells. The Single Input CAR T cells and Bispecific CAR T cells effectively killed AGS gastric cancer target cells overexpressing Claudin 18.2 antigen (FIG. 2A and FIG. 2B, respectively). The cytotoxicity appeared identical as compared with a CARsgen-derived construct (FIGs. 2A and 2B).

[0146] These constructs were also compared in a gastric cell line, GSU, which not only expresses Claudin 18.2 antigen but are also known to secrete TGF-β1, verified by using a SMAD2 / 3 reporter cell line (data not shown). The Single Input CARs and the Bispecific CAR T cells kill GSU gastric cancer target cells endogenously expressing Claudin 18.2 antigen (FIG. 2C and FIG. 2D, respectively). The TGF-β CAR T cells likewise, kill at a higher E:T ratio of GSU, which secretes detectable levels of TGF-β in culture (FIGs. 2C and 2D).

[0147] To verify functionality of the TGF-β CAR portion of the bispecific constructs, T cells expressing Bispecific CARs 1-5 and anti-TGF-β CAR were titrated and IFN-γ was quantified from the co-culture supernatants to compere the bispecific constructs against a single input anti- TGF-β CAR (FIG. 3). Bispecific CAR 1, Bispecific CAR 3, and Bispecific CAR 5 constructs showed potency similar to, or better than the single input anti-TGF-β CAR when comparing the EC50 of the human TGF-β1 protein titration (Table 2).Table 2. EC50 from Target cell and TGF-β cultures (summarized from FIGs. 2A-D and 3) AGS CLDN 18.2 GSU TGF-β CT041 0.2862 2.01E-10 NA Single Input CAR 1 0.3765 2.15E-11 NA Single Input CAR 2 0.3066 1.46E-12 NA Single Input CAR 3 0.3171 6.84E-13 NA Single Input CAR 4 0.2237 9.54E-12 NA Bispecific CAR 1 0.2319 0.0000763 3.656 Bispecific CAR 2 0.262 1.2 4.419 Bispecific CAR 3 0.2159 2.36E-11 5.64 Bispecific CAR 4 0.1339 9.61E-12 8.75 Bispecific CAR 5 0.07513 0.0883 2.16 TGF-β 104.5 6.168 4.996 Untransduced 7875 Unstable UnstableSEQUENCES SEQ ID NO: 1 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGT GCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGG CTGGAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGA TTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAG GACACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO: 2 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGR FTISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSS SEQ ID NO: 3 GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGC TGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAG AAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGAT CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGAC CTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTG GAGCTGAAA SEQ ID NO: 4 DIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPD RFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK SEQ ID NO: 5 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTCAGTAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG CTGGAGTGGGTCTCATCCATTAGTGGTGGTGGTAGTACCTACTACCCAGACTCAGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAG GACACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCA SEQ ID NO: 6 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISGGGSTYYPDSVKGR FTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSS SEQ ID NO: 7 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTCAGTAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG CTGGAGTGGGTCGCATCCATTAGTGGTGGTGGTAGTACCTACTACCCAGACTCAGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAG GACACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCA SEQ ID NO: 8 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISGGGSTYYPDSVKGR FTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSSSEQ ID NO: 9 GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGT GCAGCCTCTGGATTCACCTTTAGTAGCTATGCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGG CTGGAGTGGGTGGCCAGCATAAGCGGAGGTGGAAGTACGTACTATCCTGACTCTGTGAAGGGCCGA TTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAG GACACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTGTCACAGTCTCCTCA SEQ ID NO: 10 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISGGGSTYYPDSVKGR FTISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSS SEQ ID NO: 11 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAAC TGCAAGTCCAGCCAGAATCTTTTAAACAGCTCCAACCAGAAGAACTACTTAGCTTGGTACCAGCAG AAACCAGGACAGCCTCCTAAGCTGCTCATTTACTTTGCATCTACCCGGGAATCCGGGGTCCCTGAC CGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGAT GTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAA SEQ ID NO: 12 DIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLIYFASTRESGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIK SEQ ID NO: 13 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAAC TGCAAGTCCAGCCAGAATCTTTTAAACAGCTCCAACCAGAAGAACTACTTAGCTTGGTACCAGCAG AAACCAGGACAGCCTCCTAAGCTGCTCGTTTACTTTGCATCTACCCGGGAATCCGGGGTCCCTGAC CGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGAT GTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAA SEQ ID NO: 14 DIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIK SEQ ID NO: 15 GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCC TGCAAGTCTAGTCAGAACCTCTTGAATAGTAGTAATCAGAAAAACTATTTGGCCTGGTACCTGCAG AAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTTGCTTCCACTCGGGAGTCTGGAGTCCCAGAC AGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGAT GTTGGAGTTTATTACTGCCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAA SEQ ID NO: 16 DIVMTQTPLSLPVTPGEPASISCKSSQNLLNSSNQKNYLAWYLQKPGQSPQLLIYFASTRESGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTKLEIKSEQ ID NO: 17 CAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGC AAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCCCCAGGACAGGGA CTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAGCGGTTTAAGGGT CGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGCTCATTGCGCAGC GAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCCATGGATTATTGG GGACAGGGAACCTTGGTGACAGTGTCCTCT SEQ ID NO: 18 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKG RVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSS SEQ ID NO: 19 GAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGTTTGTCCCCCGGGGAGAGGGCGACACTGTCA TGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTGGCATGGTATCAACAAAAGCCAGGGCAGGCC CCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCACCCGGTATACCGGACAGGTTCTCCGGATCC GGCAGTGGAACCGACTTCACACTCACGATAAGCCGGCTTGAGCCCGAAGACTTCGCTGTGTATTAC TGCCAGCAATATGCCGACAGCCCTATCACATTCGGACAGGGGACCCGGCTTGAGATTAAG SEQ ID NO: 20 ETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAPGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIK SEQ ID NO: 21 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGT GCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGG CTGGAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGA TTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAG GACACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAAGCACATCTGGTAGTGGAAAGCCTGGC TCAGGAGAAGGTAGCACAAAGGGCGACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCA GTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAG AACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACC ATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA SEQ ID NO: 22 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGR FTISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSSGSTSGSGKPG SGEGSTKGDIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK SEQ ID NO: 23 GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGC TGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAG AAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGAC CTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTG GAGCTGAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAA GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCA GCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTG GAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGATTC ACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAGGAC ACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO: 24 DIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPD RFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGSTSGSGKPGSGEGSTKGE VKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGRF TISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSS SEQ ID NO: 25 CAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGC AAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCCCCAGGACAGGGA CTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAGCGGTTTAAGGGT CGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGCTCATTGCGCAGC GAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCCATGGATTATTGG GGACAGGGAACCTTGGTGACAGTGTCCTCTGGTGGCGGAGGTTCTGGAGGCGGAGGCTCAGGGGGA GGGGGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGTTTGTCCCCCGGGGAGAGGGCG ACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTGGCATGGTATCAACAAAAGCCA GGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCACCCGGTATACCGGACAGGTTC TCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGGCTTGAGCCCGAAGACTTCGCT GTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGACAGGGGACCCGGCTTGAGATT AAG SEQ ID NO: 26 QVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKG RVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGGGGSGGGGSGG GGSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAPGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIK SEQ ID NO: 27 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGT GCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGG CTGGAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGA TTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAG GACACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAAGCACATCTGGTAGTGGAAAGCCTGGC TCAGGAGAAGGTAGCACAAAGGGCGACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCA GTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAG AACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACC ATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGAA ACCGTACTGACGCAGAGCCCTGGGACCCTTAGTTTGTCCCCCGGGGAGAGGGCGACACTGTCATGT AGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTGGCATGGTATCAACAAAAGCCAGGGCAGGCCCCT CGGCTCTTGATCTACGGTGCCTCTTCAAGAGCACCCGGTATACCGGACAGGTTCTCCGGATCCGGC AGTGGAACCGACTTCACACTCACGATAAGCCGGCTTGAGCCCGAAGACTTCGCTGTGTATTACTGC CAGCAATATGCCGACAGCCCTATCACATTCGGACAGGGGACCCGGCTTGAGATTAAGGGCAGTACT AGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCCAGGTGCAGCTTGTGCAAAGT GGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTC TCATCCAACGTGATTTCTTGGGTGAGACAGGCCCCAGGACAGGGACTGGAGTGGATGGGCGGAGTC ATCCCAATCGTAGACATCGCTAATTATGCTCAGCGGTTTAAGGGTCGAGTCACAATCACTGCTGAT GAGAGCACATCTACGACCTACATGGAACTGAGCTCATTGCGCAGCGAAGATACCGCCGTGTATTAC TGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCCATGGATTATTGGGGACAGGGAACCTTGGTGACA GTGTCCTCTGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATCTACATCTGGGCGCCCTTG GCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAGAGGACGAAAG AAACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGTACAGACCACGCAGGAGGAAGATGGG TGTAGCTGTCGCTTCCCTGAGGAAGAGGAAGGTGGATGCGAGTTGAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGA GAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGG AAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAG ATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACA GCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGA SEQ ID NO: 28 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGR FTISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSSGSTSGSGKPG SGEGSTKGDIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGGGGSGGGGSE TVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAPGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSQVQLVQS GAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKGRVTITAD ESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSESKYGPPCPPCPIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 29 ACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGCT GCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAGA AACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGATC GCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACC TGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTGG AGCTGAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAAG TGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAG CCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGG AGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGATTCA CCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAGGACA CGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTACT GGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGG CAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCCCCAGGACAGGGACTGG AGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAGCGGTTTAAGGGTCGAG TCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGCTCATTGCGCAGCGAAG ATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCCATGGATTATTGGGGAC AGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTG GGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGTTTGTCCCCCGGGGAGA GGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTGGCATGGTATCAACAAA AGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCACCCGGTATACCGGACA GGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGGCTTGAGCCCGAAGACT TCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGACAGGGGACCCGGCTTG AGATTAAGGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATCTACATCTGGGCGCCCTTGG CCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAAAGAGGACGAAAGA AACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGTACAGACCACGCAGGAGGAAGATGGGT GTAGCTGTCGCTTCCCTGAGGAAGAGGAAGGTGGATGCGAGTTGAGAGTGAAGTTCAGCAGGAGCG CAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGA AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGA TTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAG CCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTGA SEQ ID NO: 30 DIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPD RFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGSTSGSGKPGSGEGSTKGE VKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGRF TISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSSGGGGSGGGGSQ VQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKGR VTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGSTSGGGSGGGSG GGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAPGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCPPCPIYIWAPL AGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 31 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGT GCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGG CTGGAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGA TTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAG GACACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAAGCACATCTGGTAGTGGAAAGCCTGGC TCAGGAGAAGGTAGCACAAAGGGCGACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCA GTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAG AACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACC ATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGC GGAGGGGGCTCTGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGTTTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTGGCATGGTATCAACAAAAG CCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCACCCGGTATACCGGACAGG TTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGGCTTGAGCCCGAAGACTTC GCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGACAGGGGACCCGGCTTGAG ATTAAGGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCCAGGTG CAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCA AGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCCCCAGGACAGGGACTGGAG TGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAGCGGTTTAAGGGTCGAGTC ACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGCTCATTGCGCAGCGAAGAT ACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCCATGGATTATTGGGGACAG GGAACCTTGGTGACAGTGTCCTCTGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC AAAAGAGGACGAAAGAAACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGTACAGACCACG CAGGAGGAAGATGGGTGTAGCTGTCGCTTCCCTGAGGAAGAGGAAGGTGGATGCGAGTTGAGAGTG AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCG GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC TGA SEQ ID NO: 32 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGR FTISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSSGSTSGSGKPG SGEGSTKGDIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGGGGSGGGGSG GGGSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAPGIPDR FSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKGSTSGGGSGGGSGGGGSSQV QLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQRFKGRV TITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSESKYGPPCPPCPIY IWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 33 GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGC TGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAG AAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGAT CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGAC CTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTG GAGCTGAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAA GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCA GCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTG GAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGATTC ACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAGGAC ACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGC GGAGGGGGCTCTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCC CCAGGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAG CGGTTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGC TCATTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCC ATGGATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACTAGCGGTGGTGGCTCC GGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGT TTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTG GCATGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCA CCCGGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGG CTTGAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGA CAGGGGACCCGGCTTGAGATTAAGGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC AAAAGAGGACGAAAGAAACTGCTTTATATATTCAAGCAACCTTTCATGCGCCCCGTACAGACCACG CAGGAGGAAGATGGGTGTAGCTGTCGCTTCCCTGAGGAAGAGGAAGGTGGATGCGAGTTGAGAGTG AAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTC AATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGG GGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCG GAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTAC CAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC TGA SEQ ID NO: 34 DIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPD RFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGSTSGSGKPGSGEGSTKGE VKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGRF TISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSSGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQ RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGSTSGGGS GGGSGGGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRA PGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCPPCPIY IWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 35 GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGT GCAGCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGG CTGGAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGA TTCACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAG GACACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGAC TACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAAGCACATCTGGTAGTGGAAAGCCTGGC TCAGGAGAAGGTAGCACAAAGGGCGACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCA GTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAG AACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCC ACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACC ATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTC ACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGTGGAGGCGGCAGTGGCGGAGGTGGGTCCGGA GGGGGCGGTAGCGGTGGCGGGGGATCTGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGTTTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTGGCA TGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCACCC GGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGGCTT GAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGACAG GGGACCCGGCTTGAGATTAAGGGCAGTACTAGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGC GGCAGCAGCCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAG GTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCCCCA GGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAGCGG TTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGCTCA TTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCCATG GATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGAATCTAAGTACGGACCGCCCTGCCCC CCTTGCCCTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTT ATCACCCTTTACTGCAAAAGAGGACGAAAGAAACTGCTTTATATATTCAAGCAACCTTTCATGCGC CCCGTACAGACCACGCAGGAGGAAGATGGGTGTAGCTGTCGCTTCCCTGAGGAAGAGGAAGGTGGA TGCGAGTTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGG GACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTGA SEQ ID NO: 36 EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGR FTISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSSGSTSGSGKPG SGEGSTKGDIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGGGGSGGGGSG GGGSGGGGSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRAP GIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKGSTSGGGSGGGSGGG GSSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQR FKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSESKYGPPCP PCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 37 GACATTGTGATGACCCAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGC TGCAAGTCCAGTCAGAACCTTTTAAATAGTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAG AAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGAT CGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGAC CTGGCAGTTTATTACTGTCAGCAACATTATAGCACTCCGCTCACGTTCGGTGCTGGGACCAAGCTG GAGCTGAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAA GTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCA GCCTCTGGATTCACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTG GAGTGGGTCGCATCCATTAGTGGTGGTGGTAGCACCTACTATCCAGACAGTGTGAAGGGCCGATTC ACCATCTCCAGAGATAATGCCAGGAACATCCTGTACCTGCAAATGAGCTATCTGAGGTCTGAGGAC ACGGCCGTGTATTACTGTGCAAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGTGGAGGCGGCAGTGGCGGAGGTGGGTCCGGA GGGGGCGGTAGCGGTGGCGGGGGATCTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCT TGGGTGAGACAGGCCCCAGGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATC GCTAATTATGCTCAGCGGTTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACC TACATGGAACTGAGCTCATTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCT TTCGTGCTGGACGCCATGGATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACT AGCGGTGGTGGCTCCGGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGC CCTGGGACCCTTAGTTTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTG GGCTCCTCTTATCTGGCATGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGT GCCTCTTCAAGAGCACCCGGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACA CTCACGATAAGCCGGCTTGAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGC CCTATCACATTCGGACAGGGGACCCGGCTTGAGATTAAGGAATCTAAGTACGGACCGCCCTGCCCC CCTTGCCCTATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTT ATCACCCTTTACTGCAAAAGAGGACGAAAGAAACTGCTTTATATATTCAAGCAACCTTTCATGCGC CCCGTACAGACCACGCAGGAGGAAGATGGGTGTAGCTGTCGCTTCCCTGAGGAAGAGGAAGGTGGA TGCGAGTTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAG CTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGG GACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAG AAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGG CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGCTGA SEQ ID NO: 38 DIVMTQSPSSLAMSVGQKVTMSCKSSQNLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPD RFTGSGSGTDFTLTISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELKGSTSGSGKPGSGEGSTKGE VKLVESGGGLVKPGGSLKLSCAASGFTFSSYAMSWVRQTPEKRLEWVASISGGGSTYYPDSVKGRF TISRDNARNILYLQMSYLRSEDTAVYYCARGGNIYDGYPYYFDYWGQGTTLTVSSGGGGSGGGGSG GGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDI ANYAQRFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGST SGGGSGGGSGGGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYG ASSRAPGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCP PCPIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 39 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAAC TGCAAGTCCAGCCAGAATCTTTTAAACAGCTCCAACCAGAAGAACTACTTAGCTTGGTACCAGCAG AAACCAGGACAGCCTCCTAAGCTGCTCATTTACTTTGCATCTACCCGGGAATCCGGGGTCCCTGAC CGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGAT GTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAG GTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCA GCCTCTGGATTCACCTTCAGTAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GAGTGGGTCTCATCCATTAGTGGTGGTGGTAGTACCTACTACCCAGACTCAGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTGTCACAGTCTCCTCAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGC GGAGGGGGCTCTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCC CCAGGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAG CGGTTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGC TCATTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCC ATGGATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACTAGCGGTGGTGGCTCC GGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGT TTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTG GCATGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCA CCCGGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGG CTTGAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGA CAGGGGACCCGGCTTGAGATTAAGGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTC TGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATC ATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT GAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTG TACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGAC CCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAA GACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCAC GACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCC CTGCCCCCAAGGTGATAAACCCAGCTTTCTTGTACAAAGTGGTGA SEQ ID NO: 40 DIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLIYFASTRESGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIKGSTSGSGKPGSGEGSTKGE VQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISGGGSTYYPDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSSGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQ RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGSTSGGGS GGGSGGGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRA PGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCPPCPMF WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 41 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAAC TGCAAGTCCAGCCAGAATCTTTTAAACAGCTCCAACCAGAAGAACTACTTAGCTTGGTACCAGCAG AAACCAGGACAGCCTCCTAAGCTGCTCATTTACTTTGCATCTACCCGGGAATCCGGGGTCCCTGAC CGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGAT GTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAG GTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCA GCCTCTGGATTCACCTTTAGTAGCTATGCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GAGTGGGTGGCCAGCATAAGCGGAGGTGGAAGTACGTACTATCCTGACTCTGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTGTCACAGTCTCCTCAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGC GGAGGGGGCTCTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCC CCAGGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAG CGGTTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGC TCATTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCC ATGGATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACTAGCGGTGGTGGCTCC GGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGT TTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTG GCATGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCA CCCGGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGG CTTGAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGA CAGGGGACCCGGCTTGAGATTAAGGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTC TGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATC ATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT GAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTG TACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGAC CCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAA GACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCAC GACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCC CTGCCCCCAAGGTGA SEQ ID NO: 42 DIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLIYFASTRESGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIKGSTSGSGKPGSGEGSTKGE VQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISGGGSTYYPDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSSGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQ RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGSTSGGGS GGGSGGGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRA PGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCPPCPMF WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 43 GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCC TGCAAGTCTAGTCAGAACCTCTTGAATAGTAGTAATCAGAAAAACTATTTGGCCTGGTACCTGCAG AAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTTGCTTCCACTCGGGAGTCTGGAGTCCCAGAC AGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGAT GTTGGAGTTTATTACTGCCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAG GTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCA GCCTCTGGATTCACCTTCAGTAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GAGTGGGTCTCATCCATTAGTGGTGGTGGTAGTACCTACTACCCAGACTCAGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTGTCACAGTCTCCTCAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGC GGAGGGGGCTCTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCC CCAGGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAG CGGTTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGC TCATTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCC ATGGATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACTAGCGGTGGTGGCTCC GGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGT TTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTG GCATGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCA CCCGGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGG CTTGAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGA CAGGGGACCCGGCTTGAGATTAAGGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTC TGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATC ATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT GAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTG TACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGAC CCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAA GACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCAC GACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCC CTGCCCCCAAGGTGA SEQ ID NO: 44 DIVMTQTPLSLPVTPGEPASISCKSSQNLLNSSNQKNYLAWYLQKPGQSPQLLIYFASTRESGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTKLEIKGSTSGSGKPGSGEGSTKGE VQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISGGGSTYYPDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSSGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQ RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGSTSGGGS GGGSGGGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRA PGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCPPCPMF WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 45 GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCC TGCAAGTCTAGTCAGAACCTCTTGAATAGTAGTAATCAGAAAAACTATTTGGCCTGGTACCTGCAG AAGCCAGGGCAGTCTCCACAGCTCCTGATCTATTTTGCTTCCACTCGGGAGTCTGGAGTCCCAGAC AGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGAT GTTGGAGTTTATTACTGCCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAG GTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCA GCCTCTGGATTCACCTTTAGTAGCTATGCGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GAGTGGGTGGCCAGCATAAGCGGAGGTGGAAGTACGTACTATCCTGACTCTGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTGTCACAGTCTCCTCAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGC GGAGGGGGCTCTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCC CCAGGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAG CGGTTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGC TCATTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCC ATGGATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACTAGCGGTGGTGGCTCC GGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGT TTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTG GCATGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCA CCCGGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGG CTTGAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGA CAGGGGACCCGGCTTGAGATTAAGGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATGTTC TGGGTGCTGGTGGTGGTCGGAGGCGTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATC ATCTTTTGGGTGAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCA GTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGT GAACTGCGGGTGAAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTG TACAACGAGCTGAACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGAC CCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAA GACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCAC GACGGCCTGTATCAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCC CTGCCCCCAAGGTGA SEQ ID NO 46 DIVMTQTPLSLPVTPGEPASISCKSSQNLLNSSNQKNYLAWYLQKPGQSPQLLIYFASTRESGVPD RFSGSGSGTDFTLKISRVEAEDVGVYYCQQHYSTPLTFGQGTKLEIKGSTSGSGKPGSGEGSTKGE VQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVASISGGGSTYYPDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSSGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQ RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGSTSGGGS GGGSGGGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRA PGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCPPCPMF WVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC ELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 47 GACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAAC TGCAAGTCCAGCCAGAATCTTTTAAACAGCTCCAACCAGAAGAACTACTTAGCTTGGTACCAGCAG AAACCAGGACAGCCTCCTAAGCTGCTCATTTACTTTGCATCTACCCGGGAATCCGGGGTCCCTGAC CGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGAT GTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGCTCACGTTCGGTCAGGGGACCAAGCTG GAGATTAAAGGAAGCACATCTGGTAGTGGAAAGCCTGGCTCAGGAGAAGGTAGCACAAAGGGCGAG GTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCA GCCTCTGGATTCACCTTCAGTAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTG GAGTGGGTCTCATCCATTAGTGGTGGTGGTAGTACCTACTACCCAGACTCAGTGAAGGGCCGATTC ACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC ACGGCTGTGTATTACTGTGCGAGAGGAGGGAATATCTATGATGGTTACCCGTACTACTTTGACTAC TGGGGCCAAGGCACCACTGTCACAGTCTCCTCAGGCGGCGGAGGATCTGGCGGAGGCGGAAGTGGC GGAGGGGGCTCTCAGGTGCAGCTTGTGCAAAGTGGCGCAGAGGTGAAGAAACCAGGTTCCAGCGTAAAGGTGAGCTGCAAGGCAAGCGGTTATACCTTCTCATCCAACGTGATTTCTTGGGTGAGACAGGCC CCAGGACAGGGACTGGAGTGGATGGGCGGAGTCATCCCAATCGTAGACATCGCTAATTATGCTCAG CGGTTTAAGGGTCGAGTCACAATCACTGCTGATGAGAGCACATCTACGACCTACATGGAACTGAGC TCATTGCGCAGCGAAGATACCGCCGTGTATTACTGTGCTCTTCCCAGAGCTTTCGTGCTGGACGCC ATGGATTATTGGGGACAGGGAACCTTGGTGACAGTGTCCTCTGGCAGTACTAGCGGTGGTGGCTCC GGGGGCGGTTCCGGTGGGGGCGGCAGCAGCGAAACCGTACTGACGCAGAGCCCTGGGACCCTTAGT TTGTCCCCCGGGGAGAGGGCGACACTGTCATGTAGGGCAAGTCAGAGTCTGGGCTCCTCTTATCTG GCATGGTATCAACAAAAGCCAGGGCAGGCCCCTCGGCTCTTGATCTACGGTGCCTCTTCAAGAGCA CCCGGTATACCGGACAGGTTCTCCGGATCCGGCAGTGGAACCGACTTCACACTCACGATAAGCCGG CTTGAGCCCGAAGACTTCGCTGTGTATTACTGCCAGCAATATGCCGACAGCCCTATCACATTCGGA CAGGGGACCCGGCTTGAGATTAAGGAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCTATCTAC ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACT CAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGCGGGTG AAGTTCAGCAGAAGCGCCGACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTG AACCTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGACCCTGAGATGGGC GGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTATAACGAACTGCAGAAAGACAAGATGGCC GAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTAT CAGGGCCTGTCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCAAGG TGA SEQ ID NO: 48 DIVMTQSPDSLAVSLGERATINCKSSQNLLNSSNQKNYLAWYQQKPGQPPKLLIYFASTRESGVPD RFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPLTFGQGTKLEIKGSTSGSGKPGSGEGSTKGE VQLVESGGGLVKPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSISGGGSTYYPDSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARGGNIYDGYPYYFDYWGQGTTVTVSSGGGGSGGGGSG GGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFSSNVISWVRQAPGQGLEWMGGVIPIVDIANYAQ RFKGRVTITADESTSTTYMELSSLRSEDTAVYYCALPRAFVLDAMDYWGQGTLVTVSSGSTSGGGS GGGSGGGGSSETVLTQSPGTLSLSPGERATLSCRASQSLGSSYLAWYQQKPGQAPRLLIYGASSRA PGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYADSPITFGQGTRLEIKESKYGPPCPPCPIY IWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 49 GSTSGSGKPGSGEGSTKG SEQ ID NO: 50 GGGGS SEQ ID NO: 51 GGGGSGGGGS SEQ ID NO: 52 GGGGSGGGGSGGGGS SEQ ID NO: 53SEQ ID NO: 54 GSTSGGGSGGGSGGGGSS SEQ ID NO: 55 ESKYGPPCPPCP SEQ ID NO: 56 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 57 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP SEQ ID NO: 58 SGQVLLESNIKVLPTWSTPVQP SEQ ID NO: 59 SQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 60 RPAAGGAVHTRGLDFACD SEQ ID NO: 61 EPKSCDKTHTCP SEQ ID NO: 62 EPKSCDTPPPCP SEQ ID NO: 63 VPCRVPPPPPCCHP SEQ ID No: 64 FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 65 IYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 66 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 67 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 68 SYAMSSEQ ID NO: 69 SISGGGSTYYPDSVKG SEQ ID NO: 70 GGNIYDGYPYYFDY SEQ ID NO: 71 KSSQNLLNSSNQKNYLA SEQ ID NO: 72 FASTRES SEQ ID NO: 73 QQHYSTPLT SEQ ID NO: 74 MSTTTCQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPY FTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCA IAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRG LAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV SEQ ID NO: 75 MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGY FTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCA IAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRG LAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV SEQ ID NO: 76 METDTLLLWVLLLWVPGSTG

Claims

CLAIMS What is claimed is:

1. A bispecific chimeric antigen receptor (CAR) comprising, in order: a) a first antigen-binding fragment that specifically binds to CLDN18.2; b) a second antigen-binding fragment that specifically binds to TGF-β; c) a spacer domain; d) a transmembrane domain; e) a costimulatory domain; and f) a signaling domain.

2. The bispecific CAR of claim 1, wherein each of the first antigen-binding fragment and the second antigen-binding fragment is a single-chain variable fragment (scFv), a Fab fragment, a single domain antibody, or a single domain antibody fragment.

3. The bispecific CAR of claim 1 or claim 2, wherein the first antigen-binding fragment is an anti-CLDN18.2 scFv; and wherein the second antigen-binding fragment is an anti-TGF-β scFv.

4. The bispecific CAR of claim 3, wherein the anti-CLDN18.2 scFv comprises: a variable heavy (VH) region comprising SEQ ID NO: 68 (HCDR1), SEQ ID NO: 69 (HCDR2), and SEQ ID NO: 70 (HCDR3); and a variable light (VL) region comprising SEQ ID NO: 71 (LCDR1), SEQ ID NO: 72 (LCDR2), and SEQ ID NO: 73 (LCDR3), and optionally wherein the VH region and VL region are separated by an scFv linker selected from the group consisting of a Witlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 49), (G4S)1 (SEQ ID NO: 50), (G4S)2 (SEQ ID NO: 51), (G4S)3 (SEQ ID NO: 52), and (G4S)4 (SEQ ID NO: 53).

5. The bispecific CAR of claim 4, wherein the VHregion and VLregion have been humanized.

6. The bispecific CAR of claim 4 or claim 5, wherein the VH region of the anti- CLDN18.2 scFv comprises SEQ ID NO: 2, 6, 8, or 10.

7. The bispecific CAR of claim 4 or claim 5, wherein the VL region of the anti- CLDN18.2 scFv comprises SEQ ID NO: 4, 12, 14, or 16.

8. The bispecific CAR of claim 4 or claim 5, wherein the anti-CLDN18.2 scFv has a VH-VL orientation.

9. The bispecific CAR of claim 8, wherein the anti-CLDN18.2 scFv comprises SEQ ID NO:

22.

10. The bispecific CAR of claim 4 or claim 5, wherein the anti-CLDN18.2 scFv has a VL-VH orientation.

11. The bispecific CAR of claim 10, wherein the anti-CLDN18.2 scFv comprises SEQ ID NO:

24.

12. The bispecific CAR of claim 3, wherein the anti-TGF-β scFv comprises: a variable heavy (VH) region comprising SEQ ID NO: 18; and a variable light (VL) region comprising SEQ ID NO: 20, and optionally wherein the VH region and VL region are separated by a Witlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 49) or a GS18 linker (GSTSGGGSGGGSGGGGSS, SEQ ID NO: 54).

13. The bispecific CAR of claim 12, wherein the anti-TGF-β scFv has a VH-VLorientation.

14. The bispecific CAR of claim 13, wherein the anti-TGF-β scFv comprises SEQ ID NO: 26.

15. The bispecific CAR of claim 12 or claim 13, wherein the anti-TGF-β scFv has a VL- VH orientation.

16. The bispecific CAR of claim 3, wherein the anti-CLDN18.2 scFv and the anti-TGF- β scFv are separated by a bispecific linker selected from the group consisting of a Witlow linker (GSTSGSGKPGSGEGSTKG, SEQ ID NO: 49), (G4S)1 (SEQ ID NO: 50), (G4S)2 (SEQ ID NO: 51), (G4S)3 (SEQ ID NO: 52), and (G4S)4 (SEQ ID NO: 53).

17. The bispecific CAR of claim 3, comprising a spacer domain comprising SEQ ID NO: 55, 56, 57, 58, 59, 60, 61, 62, or 63; a transmembrane domain comprising SEQ ID NO: 64 or SEQ ID NO: 65; a costimulatory domain comprising SEQ ID NO: 66; or a signaling domain comprising SEQ ID NO:

67.

18. The bispecific CAR of claim 3, comprising SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, or 48.

19. A nucleic acid encoding the bispecific CAR of claim 3.

20. The nucleic acid of claim 19, further comprising SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, or 47.

21. A viral vector comprising the nucleic acid of claim 19, optionally wherein the viral vector is a lentiviral vector.

22. A T cell comprising the nucleic acid of claim 19.

23. A method for making an engineered cell comprising transducing the nucleic acid of claim 19 into a T cell.

24. A method of treating gastric cancer in a subject in need thereof, comprising administering a therapeutically effective amount of a population of cells comprising the T cell of claim 22.

25. The method of claim 24, the population of cells is a population of autologous cells.

26. The method of claim 24, further comprising administering another therapeutic agent, optionally wherein the another therapeutic agent is nivolumab, pembrolizumab, ramucirumab, or trastuzumab.

27. The method of claim 24, further comprising administering a PD-1 inhibitor or a PD- L1 inhibitor, optionally wherein the PD-1 inhibitor is pembrolizumab, nivolumab, or cemiplimab, and optionally wherein the PD-L1 inhibitor is atezolizumab, avelumab, or durvalumab.

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