D domain-containing polypeptides and uses thereof

Abstract

Provided herein are D domain-containing polypeptides that specifically bind to a target of interest, such as nucleic acids encoding the D domain-containing polypeptides, vectors containing the nucleic acids, and host cells containing the nucleic acids and vectors. Also provided herein are methods for producing and using D domain-containing polypeptides, nucleic acids, vectors, and host cells, for example, but not limited to, in diagnostic and therapeutic applications. Also provided herein are multifunctional chimeric antigen receptor (CAR)-based compositions and adapters, and their use in methods for directing an immune response against target cells. In some embodiments, the method includes the use of CAR-expressing cells in combination with an adapter. The adapter confers the ability to modulate, alter, and / or direct the cell-mediated immune response of the CAR-expressing cells in vitro and in vivo.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 385,333, filed November 29, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is hereby incorporated by reference in its entirety. The XML file, created on November 14, 2023, is named 48104-710_601_SL.xml and is 120,395 bytes in size.

[0003] FIELD OF THE INVENTION The field of the invention relates generally to D domain-containing polypeptides, including multifunctional chimeric antigen receptors and D domain-containing adaptors, and their use in methods of treatment, for example, by directing an immune response against target cells. [Background technology]

[0004] background Adoptive transfer of genetically modified T cells is a rapidly evolving, innovative treatment for cancer. Chimeric antigen receptor (CAR)-engineered T cells are renewable agents capable of providing sustained functional immunity. Clinical efficacy has been demonstrated with CD19 CAR T in a range of hematological cancers, and promising early clinical data have been reported for other genetically modified CAR T in solid tumors. However, significant challenges must be addressed before CAR technology can more fully realize its substantial potential. Summary of the Invention [Means for solving the problem]

[0005] Abstract There remains a substantial unmet need for new target-binding compositions, particularly for such agents containing alternative binding scaffolds (e.g., non-antibody scaffolds). Agents of particular interest may be characterized, for example, by substantially reduced production costs and / or comparable or superior reagent, diagnostic, and / or therapeutic properties compared to antibodies. The present disclosure provides novel target-binding D-domain (DD) polypeptides based on non-antibody structural scaffolds. In some embodiments, the D-domain polypeptides (DDpp) are characterized by high target binding affinity and by the non-antibody structural scaffold. In some embodiments, the DDpp are target-specific binding polypeptides that can be advantageously used to target therapeutic agents (e.g., immune cells) to specific cells (e.g., diseased cells), thereby reducing or eliminating off-target effects. In some embodiments, the provided DDpps are used as therapeutic agents to bind to cells or soluble factors involved in disease.

[0006] In one aspect, provided herein is a protein comprising a D domain (DD) target binding domain (DDpp), wherein the DD specifically binds to CS1. In some embodiments, the CS1 is human CS1 or a fragment thereof (e.g., SEQ ID NO: 1). In some embodiments, the DDpp is monovalent or multivalent. In some embodiments, the DDpp is monospecific or multispecific. In further embodiments, the DDpp is monospecific and multivalent. In other embodiments, the DDpp is multispecific and multivalent. Fusion proteins comprising one or more DDpps are also provided, as are methods of making and using the fusion proteins. Nucleic acids encoding DDpps, and vectors and host cells containing the nucleic acids, are also provided. Non-limiting examples of such uses include, but are not limited to, target analysis, and diagnostic and therapeutic applications. In some embodiments, the DDpp comprises a CS1-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-106. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 60.

[0007] In one aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising a target binding domain comprising a DD that binds to CS1 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the CAR comprises a target binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR further comprises a second target binding domain having the same or a different target as the DD target binding domain. In some embodiments, the CAR is expressed in an immune cell. In some embodiments, the immune cell is an autologous cell. In some embodiments, the immune cell is an allogeneic cell. In some embodiments, the immune cell is an immune effector cell. In some embodiments, the immune cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell). In some embodiments, the cell is an autologous immune cell. In some embodiments, the cell is an autologous T cell (CAR-T cell) or an autologous natural killer (NK) cell. In some embodiments, the cells are allogeneic immune cells. In some embodiments, the cells are allogeneic T cells (CAR-T cells) or allogeneic natural killer (NK) cells. In some embodiments, the CAR is expressed in immune cells derived from human embryonic stem cells (CAR-hESC) or induced pluripotent stem cells (CAR-iPSC).

[0008] Nucleic acids encoding the disclosed DDpp (e.g., DDpp fusion proteins, CARs, or adapters) are also provided. Additionally, vectors (e.g., plasmids, viral vectors, and non-viral vectors) containing nucleic acids encoding DDpp (e.g., DDpp fusion proteins, CARs, or adapters), as well as host cells containing the nucleic acids and vectors, are provided. In some embodiments, the vector comprises a nucleotide sequence that regulates expression of a polypeptide encoded by the nucleic acid molecule. In further embodiments, the vector comprises an inducible promoter sequence. In additional embodiments, the vector comprises one or more additional standard components for expression of a protein encoded by the nucleic acid (e.g., a promoter, packaging components, etc.). In some embodiments, the vector is a lentiviral vector.

[0009] In one aspect, the present disclosure also provides a host cell comprising a nucleic acid molecule encoding a target-binding DDpp disclosed herein. In some embodiments, the host cell (e.g., a cell of a cell line) is engineered to express a protein containing a DD disclosed herein (e.g., a DD having an amino acid sequence of SEQ ID NOs: 41-106). In some embodiments, expression of a DDpp (e.g., a DDpp fusion protein, or an adapter) by the host cell allows for production and isolation of the DDpp. In some embodiments, expression results in a DDpp (e.g., a CAR) that is expressed on the surface and / or integrated into the membrane of the host cell. In some embodiments, the host cell is a bacterial, yeast, fungal, or plant cell. In other embodiments, the host cell is a mammalian cell. In further embodiments, the mammalian cell is an immune cell. In one embodiment, the host cell is a human immune cell. In some embodiments, the human immune cell is a T cell. In other embodiments, the human immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is an autologous cell. In some embodiments, the immune cell is an allogeneic cell. In some embodiments, the human immune cells display a DDpp (e.g., a CAR) on their cell surface.

[0010] In one aspect, the present disclosure further provides a host cell expressing a protein comprising a DD disclosed herein. In some embodiments, the host cell expresses a chimeric antigen receptor (CAR) comprising a DD disclosed herein. In some embodiments, the CAR comprises a target binding domain and a transmembrane domain comprising a DD comprising an amino acid sequence selected from SEQ ID NOs: 41-106. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the CAR further comprises an intracellular domain (including a signaling domain). In some embodiments, the CAR immune cell is a T cell. In some embodiments, the CAR immune cell is an NK cell. In some embodiments, the CAR immune cell is not a T cell or an NK cell. In some embodiments, the CAR immune cell is an autologous immune cell. In some embodiments, the CAR immune cell is an allogeneic immune cell. In some embodiments, the host cell is an immune effector cell that further comprises a second CAR polypeptide having a DD or other binding domain (e.g., an scFv) that specifically binds to the same or a different target as the first CAR expressed by the host immune cell (e.g., a different epitope of the same target, or a second target of interest expressed by a cancer cell).

[0011] Pharmaceutical compositions containing the proteins comprising the DDs disclosed herein, the nucleic acids encoding the proteins disclosed herein, the vectors disclosed herein containing the nucleic acids, the viruses encoding the proteins, and the host cells containing the nucleic acids and / or vectors disclosed herein are also provided, as are kits (e.g., therapeutic kits, diagnostic kits, kits for research use, etc.) containing one or more of the disclosed target-binding DDpps (e.g., DDpp fusion proteins, e.g., DD-Fc and DD-CAR, or adapters), nucleic acid molecules, vectors, and host cells.

[0012] The DDpp provided herein possess activities that include, but are not limited to, the ability to specifically bind to CS1 in vitro or in vivo, and the ability to serve as a reactive site for linking or associating a protein, e.g., a DDpp fusion protein, with one or more additional moieties (e.g., solid supports) and / or other modifications. The DDpp provided herein may also possess additional desirable properties and / or functionalities useful in manufacturing, formulation, and biological, diagnostic, and therapeutic applications.

[0013] Methods of using DDpp in diagnostic and therapeutic applications are also provided. In one embodiment, the present disclosure provides a method of treating a disease or disorder, comprising administering a therapeutically effective amount of a DDpp (e.g., a DDpp fusion protein, a CAR, and / or an adapter) that specifically binds to CS1 to a subject in need of disease or disorder treatment. In some embodiments, the disease or disorder is cancer, a B-cell malignancy (e.g., multiple myeloma), a disease or disorder of the immune system, or an infectious disease. Also provided is a method of treating a disease or disorder, comprising co-administering an additional therapeutic agent together with the disclosed DDpp. In some embodiments, the disease or disorder is myeloma. In some embodiments, the disease or disorder is multiple myeloma. [Brief explanation of the drawings]

[0014] [Figure 1] Luciferase-based cytotoxicity assay in MM1S-GFP-luc cells using AFPDD101-CAR cells and CS1-targeting adaptor proteins containing the CS1DD006 or CS1DD020 CS1-specificity D domain. Shown is the % lysis of MM1S-GFP-luc cells.

[0015] [Figure 2] CS1 expression by multiple myeloma cell lines.

[0016] [Figure 3]Adapters containing CS1DD006 and CS1DD020 induce cytokine release by AFPDD101-CAR cells in the presence of CS1-expressing target cells. IFN-gamma release in the presence of L363, MM1S, U266, OPM2, and H929 multiple myeloma cells.

[0017] [Figure 4] CS1 and BCMA expression by MM1S and H929 multiple myeloma cell lines.

[0018] [Figure 5] Bispecific targeting responses vary with antigen density. % cytolysis of MM1S-GFP-luc and H929-GFP-luc cells is shown. % cytolysis by AFPDD101-CAR cells in the presence of CS1, BCMA, or CS1 x BCMA-specific adapters, and CS1-expressing MM1S (panel A) and H929 (panel B) target cells.

[0019] [Figure 6] Bispecific targeting responses vary with antigen density. Figure 1 shows IFN-gamma and IL-2 release by AFPDD101-CAR cells in the presence of CS1DD020 or BCMADDXX containing monovalent or bivalent adapters and CS1-expressing MM1S and H929 target cells.

[0020] [Figure 7] In vivo characterization of CS1-targeting bivalent adaptors, showing proliferation of MM1S-GFP-luc cells in mice after various treatments.

[0021] [Figure 8] Degranulation assay using AFPDD101-CAR T cells and CS1-targeting adaptor protein with or without MM1S target cells. % CD107a+ cells among CD3+FLAG+CD4+ cells are shown.

[0022] [Figure 9]Degranulation assay using AFPDD101-CAR T cells and CS1-targeting adaptor protein with or without MM1S target cells. % CD107a+ cells among CD3+FLAG+CD8+ cells are shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description The section headings used herein are for organizational purposes only and should in no way be construed as limiting the subject matter described. Definition of Terms

[0024] Whenever an embodiment is described herein using the word "comprising," it is understood that other similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of." However, when used in the claims as transitional phrases, each should be interpreted separately, in the appropriate legal and factual context (e.g., "comprising" is considered more open-ended, while "consisting of" is more exclusive, and "consisting essentially of" achieves intermediate status).

[0025] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0026] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0027] "About," as the term is used herein, when referring to measurable values ​​such as amounts, time periods, and other measurable values ​​known in the art, is meant to encompass variations of ±20%, or in some embodiments, ±10%, or in some embodiments, ±5%, or in some embodiments, ±1%, or in some embodiments, ±0.1% from the stated value, where such variations are appropriate for performing the disclosed methods.

[0028] "Chimeric antigen receptor" or "CAR(s)," as used herein, refers to an engineered receptor that transfers antigen or target specificity onto a cell (e.g., a T cell, e.g., a naive T cell, a central memory T cell, an effector memory T cell, an NK cell, an NKT cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0029] The term "adapter," as used herein, refers to a multidomain soluble protein comprising an antigenic determinant (AD) and an antigenic determinant binding domain (ADBD), where the ADBD binds to a second AD. In addition to the AD and ADBD, the adapter can comprise additional ADs, additional ADBDs, and / or other additional domains.

[0030] The term "antigenic determinant binding domain" or "ADBD," as used herein, refers to a sequence of a polypeptide (e.g., an adapter or CAR) that is sufficient to confer recognition and specific binding to a target antigenic determinant (AD). In some embodiments, the ADBD is an antigen-binding antibody fragment, scFv, or an antigen-binding peptide that is not based on an antibody or antibody fragment sequence (e.g., a D domain or affibody). In some embodiments, the ADBD comprises a non-antibody-based binding scaffold (e.g., a D domain, affibody, fibronectin domain, nanobody, lipocalin domain, ankyrin domain, maxibody, protein A domain, or affilin domain). In some embodiments, the ADBD is a D domain. In some embodiments, the ADBD is an antibody-based binding sequence. In some embodiments, the ADBD is an scFv or domain antibody (dAb). In some embodiments, the ADBD is capable of binding to a target antigen on the surface of a cell. In some embodiments, the ADBD is capable of binding to a target antigen on the surface of an immune effector cell. In some embodiments, the ADBD has the ability to bind to a growth factor receptor, an immunoregulatory receptor, or a hormone receptor.

[0031] In certain embodiments, the ADBD is a non-antibody scaffold-based polypeptide sequence sufficient to confer recognition and specific binding to a target antigenic determinant. In some embodiments, the non-antibody-based ADBD is a polypeptide capable of binding to a target antigen on the surface of a cell. In some embodiments, the non-antibody-based ADBD is capable of binding to a growth factor receptor, an immunoregulatory receptor, or a hormone receptor. In some embodiments, the ADBD is a D-domain-based polypeptide. In certain embodiments, the ADBD is a D-domain-based polypeptide sufficient to confer recognition and specific binding to a target antigenic determinant. In some embodiments, the ADBD is a D-domain-based polypeptide capable of binding to a target antigen on the surface of a cell. In some embodiments, the ADBD is a D-domain-based polypeptide capable of binding to a growth factor receptor, an immunoregulatory receptor, or a hormone receptor. In some embodiments, the ADBD is a D-domain-based polypeptide capable of binding to a target antigen on a serum protein.

[0032] The term "D domain" refers to a target-binding polypeptide that shares a specific sequence and certain structural features of the reference scaffold sequence: MGSWAEFKQRLAAIK TRLEALGGSEAELAAFEKEIAAFESELQAYKGKGNPEVEALRKEAAAIRDELQAYRHN (SEQ ID NO: 5) (see WO2016 / 164305 and WO2016 / 164308, each of which is incorporated herein by reference in its entirety). The reference scaffold is a variant of a non-naturally occurring and untargeted antiparallel three-helix bundle reference polypeptide that was originally engineered as an attempt at protein folding (see Walsh et al., PNAS 96: 5486-5491 (1999)), which is incorporated herein by reference in its entirety). Although the reference scaffold has no known target-binding activity, it has been discovered that polypeptides containing modifications of the reference scaffold having the amino acid sequence of SEQ ID NO: 5 can specifically bind to a target of interest. Therefore, D domains, or molecules containing D domains, can specifically (non-randomly) bind to target molecules. While not wishing to be bound by theory, it is believed that structural constraints on surface-exposed residues (which can be modified) when designing a D domain confer the ability of the surface-exposed residues to specifically bind to a target of interest. In some embodiments, a D domain generally consists of 70-75 amino acid residues. In some embodiments, a D domain contains an amino acid sequence in which up to 20 substitutions differ (e.g., due to amino acid modifications) from that of a reference scaffold having the sequence of SEQ ID NO: 5. In certain embodiments, a D domain does not contain the sequence LAAIKTRLQ (SEQ ID NO: 6).

[0033] The terms "protein" and "polypeptide" are used interchangeably herein to refer to biological polymers comprising units derived from amino acids linked through peptide bonds, and a protein may be composed of two or more polypeptide chains.

[0034] The terms "antibody" or "immunoglobulin," as used interchangeably herein, include full-length antibodies and antibody fragments containing any functional domain of an antibody, e.g., an antigen-binding fragment or single chain thereof, an effector domain, a salvage receptor-binding epitope, or portions thereof. A typical antibody comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, Cl. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FWs). Each VH and VL is composed of three CDRs and four FWs, arranged from the amino terminus to the carboxyl terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can 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 (C1q) of the classical complement system. Examples of antibodies of the present disclosure include typical antibodies, scFvs, and combinations thereof, in which, for example, DDpp is covalently linked (e.g., via a peptide bond or a chemical linker) to the N-terminus of either the heavy and / or light chain of a typical whole (full-length) antibody, or intercalated into the H and / or L chain of a full-length antibody.

[0035] The term "antibody fragment" refers to a portion of an intact antibody, including any functional domain of an antibody, such as an antigen-binding fragment or single chain thereof, an effector domain or portion thereof, and a salvage receptor-binding epitope or portion thereof. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), and Fv fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments. "Antibody fragment," as used herein, comprises an antigen-binding site or an epitope-binding site. In one embodiment, the DDpp fusion protein comprises an effector domain or portion thereof. In one embodiment, the DDpp fusion protein comprises a salvage receptor-binding epitope or portion thereof.

[0036] The term "single-chain variable fragment" or "scFv" antibody, as used herein, refers to a form of antibody (e.g., antibody fragment) that contains only the variable regions of the heavy and light chains connected by a linker peptide. An scFv may contain VL-linker-VH or VH-linker-VL. ScFv antibodies are generally 220-250 amino acids in length and contain a linker that is 10-25 amino acids in length. In one embodiment, a DDpp fusion protein comprises DDpp and an scFv.

[0037] As used herein, the term "Fc region" or simply "Fc" is understood to refer to the carboxyl-terminal portion of an immunoglobulin chain constant region, preferably an immunoglobulin heavy chain constant region, or a portion thereof. For example, an immunoglobulin Fc region can include (1) a CH1 domain, a CH2 domain, and a CH3 domain; (2) a CH1 domain and a CH2 domain; (3) a CH1 domain and a CH3 domain; (4) a CH2 domain and a CH3 domain; or (5) a combination of two or more domains and an immunoglobulin hinge region. Thus, in various embodiments, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may also include the J chain. For IgG, Fc includes immunoglobulin domains Cγ2 and Cγ3, and the hinge between Cγ1 and Cγ2. In a preferred embodiment, the immunoglobulin Fc region comprises at least an immunoglobulin hinge region, a CH2 domain, and a CH3 domain, and preferably lacks a CH1 domain. In one embodiment, the immunoglobulin class from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4). Other immunoglobulin classes, IgA (Igα), IgD (Igδ), IgE (Igε), and IgM (Igμ), may also be used. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or p260 at its carboxyl terminus, where numbering is according to the EU index as set forth in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, Bethesda, Md. (1991)). Fc may refer to this region in isolation or in the context of a full-length antibody, antibody fragment, or Fc fusion protein.Polymorphisms have been observed at several different Fc positions, including, but not limited to, positions 270, 272, 312, 315, 356, and 358, as numbered by the EU index; therefore, slight differences between the presented sequence and prior art sequences may exist. The selection of appropriate immunoglobulin heavy chain constant regions is discussed in detail in U.S. Pat. Nos. 5,541,087 and 5,726,044, each of which is incorporated herein by reference in its entirety. The selection of a particular immunoglobulin heavy chain constant region sequence from a particular immunoglobulin class and subclass to achieve a particular result is considered within the level of ordinary skill in the art. The portion of the DNA construct encoding the immunoglobulin Fc region preferably includes at least a portion of the hinge domain and, preferably, at least a portion of the CH3 domain of Fc gamma or a homologous domain in any of IgA, IgD, IgE, or IgM. Furthermore, it is contemplated that substitutions or deletions of amino acids within the immunoglobulin heavy chain constant region may be useful in practicing the methods and compositions disclosed herein. One example would be introducing amino acid substitutions in the upper CH2 region to create Fc variants with reduced affinity for Fc receptors (Cole, J. Immunol. 159: 3613 (1997)).

[0038] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis (or other cytotoxic effects) of the target cells. To assess the ADCC activity of a molecule of interest, any in vitro ADCC assay known in the art can be used, such as those described in U.S. Pat. Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. PNAS 95: 652-656 (1998).

[0039] The terms "linker," "spacer," and "hinge" are used interchangeably herein to refer to a peptide or other chemical linkage located between two or more otherwise independent functional domains of a DDpp fusion protein, adapter, or CAR. For example, a linker may be located between the antigenic determinant (AD) domain and the antigenic determinant binding domain (ADBD) of an adapter. Similarly, a linker may be located between two antigenic determinant binding domains or between the antigenic binding domain and the transmembrane domain of a CAR. In some embodiments, a linker is a peptide or other chemical linkage located between DDpp and another polypeptide of a DDpp fusion protein. Suitable linkers for coupling two or more domains of an adapter are described herein and / or are otherwise apparent to one of skill in the art. In some embodiments, the linker is a peptide comprising the amino acid sequence of SEQ ID NOs: 16-20, 115-118, or 119.

[0040] The term "operably linked," as used herein, refers to two molecules attached such that each molecule retains at least some level of the functional activity that it had alone (assuming each molecule had functional activity). In embodiments, one molecule is operably linked to another molecule if the other molecule retains at least some level of its functional activity when the other molecule is absent the functional activity. Operably linked can also refer to the linkage of two non-functional molecules. Two molecules can be "operably linked" regardless of whether they are attached directly or indirectly (e.g., via a linker).

[0041] The terms "specifically bind," "having selective affinity for," "binds," or "binding" are used interchangeably to mean that a binding agent, such as DDpp, reacts or associates with an epitope, protein, or target molecule more frequently, rapidly, for a longer period of time, with higher affinity, or some combination of the above, than alternatives, including proteins unrelated to the target epitope, protein, or target molecule. Due to sequence identity between homologous proteins in different species, specific binding may, in some embodiments, include binding agents that recognize proteins or targets in more than one species. Similarly, due to homology within certain regions of the polypeptide sequences of different proteins, specific binding may include binding agents that recognize more than one protein or target. It is understood that, in certain embodiments, a binding agent that specifically binds to a first target may or may not specifically bind to a second target. Thus, "specific binding" does not necessarily require exclusive binding, e.g., binding to a single target (although it can include it). Thus, a binding agent may, in certain embodiments, specifically bind to more than one target. In certain embodiments, multiple targets may be bound by the same antigen binding site on a binding agent.

[0042] "Target" refers to any molecule or combination of molecules to which DDpp, e.g., a DDpp fusion protein, can bind via another component of the DDpp fusion protein, e.g., an antibody or antibody variable domain fragment, via an adapter or CAR, or via a component of the DDpp fusion protein, adapter or CAR, e.g., an antigenic determinant binding domain.

[0043] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to the portion of any molecule (e.g., a target of interest, e.g., CS1) that can be recognized and specifically bound by a specific binding agent (e.g., DDpp or antibody). When the recognized molecule is a polypeptide, epitopes can be formed from adjacent and non-adjacent amino acids and / or other chemically active surface groups of molecules (e.g., carbohydrates) juxtaposed by tertiary folding of the protein. Epitopes formed from adjacent amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. Epitopes typically comprise a unique spatial conformation of at least three amino acids, more usually at least five or eight to ten amino acids.

[0044] "Peptide tag," as used herein, refers to a peptide sequence that is part of or attached (e.g., by genetic engineering) to another protein, providing a function to the resulting fusion. Peptide tags are usually relatively short compared to the protein to which they are fused; for example, peptide tags are, in some embodiments, four or more amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids. In some embodiments, the DDpp is a fusion protein containing the peptide tag. In other embodiments, the DDpp specifically binds to the peptide tag. Numerous peptide tags having the uses provided herein are known in the art. Examples of peptide tags that can be components of a DDpp fusion protein or targets to which DDpp binds (e.g., DDpp fusion proteins) include, but are not limited to, HA (hemagglutinin), c-myc, herpes simplex virus glycoprotein D (gD), T7, GST, GFP, MBP, Strep-tag, His-tag, Myc-tag, TAP-tag, and FLAG® tag (Eastman Kodak, Rochester, NY). Similarly, antibodies against the tag epitope allow for detection and localization of the fusion protein using techniques known in the art, such as Western blots, ELISA assays, and cell immunostaining.

[0045] "Derived from," as the term is used herein, indicates a relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first and second molecules and does not imply or imply a limitation on the process or origin of the first molecule from which the second molecule is derived. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure so that it has the required function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or imply a limitation to a particular process for producing the intracellular signaling domain; for example, it does not mean that one must start with a CD3 zeta sequence and delete or mutate unwanted sequences to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0046] The term "naturally occurring," when used in reference to biological materials, e.g., nucleic acid molecules, polypeptides, antigenic determinants, and host cells, refers to something that is found in nature and has not been modified by humans. Conversely, "non-natural" or "synthetic," when used in reference to biological materials, refers to something that is not found in nature and has been modified by humans.

[0047] As used herein, a "modification" with respect to the sequence of a reference sequence includes substitutions, deletions, insertions, and / or additions to the sequence of corresponding amino acid positions in the reference sequence (e.g., a DD disclosed herein).

[0048] A "substitution" with respect to a sequence of a reference sequence refers to the replacement of a particular amino acid residue with a different amino acid residue at a corresponding amino acid position in the reference sequence.

[0049] A "conservative" amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine (K), arginine (R), histidine (H)), acidic side chains (e.g., aspartic acid (D), glutamic acid (E)), uncharged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), non-charged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)), and non-charged polar side chains (e.g., glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), cysteine ​​(C)). Conservative substitutions include polar side chains (e.g., alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), beta-branched side chains (e.g., threonine (T), valine (V), isoleucine (I)), and aromatic side chains (e.g., tyrosine (Y), phenylalanine (F), tryptophan (W), histidine (H)). For example, substitution of tyrosine for phenylalanine is a conservative substitution. In certain embodiments, conservative substitutions in the sequence of DDpp result in altered or unaltered specific binding of the DDpp containing the substitution to the target of interest (e.g., CS1) to which it binds. In one embodiment, conservative substitutions in the sequence of DDpp do not abolish binding of the DDpp containing the substitution to the target of interest to which it binds. Methods for identifying conservative and non-conservative nucleotide and amino acid substitutions that confer, alter, or maintain selective binding affinity are known in the art (see, e.g., Brummell, Biochem. 32: 1180-1187 (1993); Kobayashi, Protein Eng. 12(10): 879-884 (1999); and Burks, PNAS 94: 412-417 (1997)).

[0050] A "non-conservative" amino acid substitution is one in which an amino acid residue is replaced with another amino acid residue having a dissimilar side chain. In one embodiment, the non-conservative substitution in the sequence of the DDpp results in specific binding of the DDpp containing the substitution to the intended target (e.g., CS1) to which it binds. In one embodiment, the non-conservative substitution in the sequence of the DDpp does not abolish the binding of the DDpp containing the substitution to the intended target to which it binds. In one embodiment, the non-conservative substitution in the sequence of the DDpp, adapter, or CAR results in retained specific binding of the DDpp, adapter, or CAR containing the substitution to the intended target to which it binds.

[0051] The terms "unnatural amino acid," "amino acid analog," and "non-standard amino acid residue" are used interchangeably herein. Unnatural amino acids that can be substituted in the DDpps provided herein are known in the art. In one embodiment, the unnatural amino acid is 4-hydroxyproline, which can replace proline; 5-hydroxylysine, which can replace lysine; 3-methylhistidine, which can replace histidine; homoserine, which can replace serine; and ornithine, which can replace lysine. Additional examples of unnatural amino acids that can be substituted in the DDpps disclosed herein include, but are not limited to, molecules such as D-isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-aminoisobutyric acid, A-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine, and pyrrolysine-fluoroamino acids, designer amino acids such as beta-methyl amino acids, C alpha-methyl amino acids, and N alpha-methyl amino acids, or combinations of unnatural amino acids. Additional unnatural amino acids can include, for example, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids. As discussed herein, in some embodiments, the unnatural amino acids or amino acid analogs can include deletions of one or more amino acids from the sequence.

[0052] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include, but are not limited to, DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA, and / or tRNA. In some embodiments, the isolated polynucleotide is a modified mRNA containing non-naturally occurring nucleosides or nucleotides. In some embodiments, the modified mRNA contains 2-thiouridine, pseudouridine, or 1-methylpseudouridine.

[0053] The terms "vector," "cloning vector," and "expression vector," as used herein, refer to a vehicle by which a nucleic acid sequence (e.g., a disclosed DDpp, adapter, or CAR coding sequence) can be maintained or amplified in, or introduced into, a host cell (e.g., a cloning vector) so as to transform the host and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, phages, viruses, etc.

[0054] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a nucleic acid encoding the disclosed DDpp, adapter, or CAR. Host cells include, but are not limited to, bacteria, yeast, plant, animal, and mammalian cells. A host cell includes the progeny of a single host cell, which may not necessarily be completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations and / or alterations. A host cell includes cells that have been transfected or infected in vivo, in vitro, or ex vivo with a nucleic acid encoding the disclosed DDpp, adapter, or CAR. In some examples, the host cell can express and display the disclosed DDpp or CAR on its surface, such as in phage display or CAR T cells. In some embodiments, the host cell can express the adapter. In some embodiments, the host cell can express and secrete the adapter. In some embodiments, the host cell can express the CAR. In some embodiments, the host cell can express and display the CAR on its surface. "Expression" includes transcription and / or translation.

[0055] As used herein, the terms "solid support," "support," "matrix," and "resin" are used interchangeably and refer, without limitation, to any column (or column material), bead, test tube, microtiter dish, solid particle (e.g., agarose or sepharose), microchip (e.g., silicon, silicon glass, or gold chip), or membrane (e.g., biological or filtration membrane) to which DDpp, antibodies, or other proteins can be attached (e.g., coupled, linked, or adhered), either directly or indirectly (e.g., through other binding partner intermediates, such as other antibodies or protein A), or into which DDpp or antibodies can be embedded (e.g., through receptors or channels). Reagents and techniques for attaching polypeptides to solid supports (e.g., matrices, resins, plastics, etc.) are well known in the art. Suitable solid supports include, but are not limited to, chromatography resins or matrices (e.g., SEPHAROSE-4 FF agarose beads), the walls or floors of wells in plastic microtiter dishes, silica-based biochips, polyacrylamide, agarose, silica, nitrocellulose, paper, plastic, nylon, metal, and combinations thereof. DDpp and other compositions can be attached to the support material by non-covalent association or by covalent bonding using reagents and techniques known in the art. In one embodiment, DDpp is coupled to the chromatography material using a linker.

[0056] As used herein, the terms "pharmaceutically acceptable" or "physiologically tolerable," and grammatical variations thereof, when referring to compositions, carriers, diluents, and reagents, are used interchangeably and indicate that the material can be administered to or on a human without producing therapeutically prohibited undesirable physiological effects, such as nausea, dizziness, acute gastric peristalsis, and the like.

[0057] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection or infusion techniques.

[0058] "Modulate" means adjusting or regulating in magnitude, frequency, degree, or activity. In another related aspect, such modulation may be positively modulated (e.g., an increase in frequency, degree, or activity) or negatively modulated (e.g., a decrease in frequency, degree, or activity). In some embodiments, modulation in a positive or negative direction is referenced relative to the function of a cell, tissue, or organ prior to administration of the therapeutic agent. In additional embodiments, modulation in a positive or negative direction is referenced with respect to a normal, healthy cell, tissue, or organ.

[0059] An "effective amount" of a DDpp (such as a DDpp fusion protein), CAR cell, adaptor, and / or CAR cell / adaptor composition provided herein is an amount sufficient to carry out a specifically stated purpose, e.g., to cause an observable change in the level of one or more biological activities associated with the target to which the DDpp (e.g., DDpp fusion protein), CAR cell, and / or adaptor binds. In certain embodiments, the change increases the level of the target activity. In other embodiments, the change decreases the level of the target activity. An "effective amount" can be determined empirically and routinely for the stated purpose. The term "therapeutically effective amount" refers to an amount of a DDpp (such as a DDpp fusion protein), CAR cell, and / or adaptor, or other therapeutic agent effective to "treat" (e.g., reduce the symptoms of) a disease or disorder in a subject (mammal). The term "therapeutically effective amount" also refers to an amount effective, at the necessary dosages and for the necessary duration, to achieve a desired prophylactic result.

[0060] The terms "patient," "subject," "animal," and "mammal" are used interchangeably and refer to mammals, e.g., human patients and non-human primates, as well as laboratory animals, e.g., rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, e.g., chickens, amphibians, and reptiles. "Mammal," as used herein, refers to any member of the class Mammalia, including, without limitation, humans and non-human primates, e.g., chimpanzees, and other ape and monkey species; livestock animals, e.g., cows, sheep, pigs, goats, and horses; domestic mammals, e.g., dogs and cats; laboratory animals, including rodents such as mice, rats, and guinea pigs; and the like. In certain embodiments, the patient is a human. The term does not denote a particular age or sex. As such, adult and newborn subjects, regardless of male or female, as well as embryos and fetuses, are intended to be included within the scope of this term.

[0061] The terms "treat," "treatment," and "treating," as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (reduce or delay) symptoms, complications, or biochemical manifestations of a disease, condition, or disorder, to alleviate the symptoms of a disease, condition, or disorder, or to halt or inhibit its further development. Treatment can be prophylactic (to prevent or delay the onset of a disease or to prevent the manifestation of clinical or asymptomatic symptoms thereof), or to therapeutically suppress or alleviate symptoms after the manifestation of the target pathology of a disease, condition, or disorder, to prevent the pathology, to seek or obtain a beneficial outcome, or to reduce the chance that an individual will develop a condition, even if treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition, as well as those prone to have the condition, or those in whom the condition is to be prevented. Treatment can be with a DDpp fusion protein, CAR cell, adapter, and / or CAR cell / adapter composition, alone or in combination with an additional therapeutic agent. In some embodiments, the terms "treat," "treatment," and "treating" are used herein to refer to therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (reduce or delay) symptoms, complications, or biochemical manifestations of a proliferative disorder, or to alleviate one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder. In specific embodiments, the terms "treat," "treatment," and "treating" refer to the alleviation of at least one measurable physical parameter of a proliferative disorder, e.g., tumor growth, although not necessarily discernible by the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to the inhibition of progression of a proliferative disorder, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physical parameter, or both. In other embodiments, the terms "treat," "treatment," and "treating" refer to the reduction or stabilization of tumor size, tumor cell growth or survival, or cancerous cell number.

[0062] "Cancer," "tumor," or "malignant tumor" are used synonymously and refer to any of several diseases characterized by uncontrolled, abnormal cell growth, the ability of affected cells to spread (metastasize) locally or through the bloodstream and lymphatic system to other parts of the body, and any of several characteristic structural and / or molecular features. "Tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. A "cancerous tumor," or "malignant cell," is understood to be a cell that has specific structural properties, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated using the DDpp fusion proteins, CAR cells, adapters, and / or CAR cell / adapter compositions provided herein include, without limitation, myeloma, multiple myeloma, light-chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma, IgD myeloma, IgE myeloma, and IgM myeloma. Other types of cancers and tumors that can be treated using the DDpp fusion proteins, CAR cells, adapters, and / or CAR cell / adapter compositions are described herein or otherwise known in the art. Reference to a particular "type" of cancer, tumor, or tumor cell is understood to mean a cancer, tumor, or tumor cell characterized by a specific disease.

[0063] The term "tumor antigen" refers to an antigen common to a specific hyperproliferative disorder, such as cancer. The terms "tumor antigen" or "cancer antigen" are used interchangeably herein. In some embodiments, the cancer is myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is myeloma, multiple myeloma, light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma, IgD myeloma, IgE myeloma, and IgM myeloma.

[0064] Tumor and cancer antigens can be further defined as "tumor-specific antigens (TSAs)," "cancer-specific antigens (CSAs)," "tumor-associated antigens (TAAs)," or "cancer-associated antigens (CAAs)." TSAs are antigens that are unique to tumor cells and not present in other cells in the body. TAAs are antigens found in both tumors and some normal cells. TAAs can be expressed in normal cells under conditions that do not allow for the induction of a state of immunological tolerance to the antigen. Expression of TAAs in tumors can occur under conditions that allow the immune system to respond to the antigen. TAAs can be expressed in normal cells during fetal development, when the immune system is immature and unable to respond, or can be normally present at very low levels in normal cells, but are expressed at much higher levels in tumor cells. Due to the dynamic nature of tumors, in some instances, tumor cells may express unique antigens at certain stages, and in others, may also express antigens that are also expressed in non-tumor cells. Therefore, including a particular marker as a TAA does not preclude it from being considered a TSA.

[0065] The term " autoimmune disease " as used herein is defined as the disorder resulting from autoimmune response.Autoimmune disease is the result of inappropriate and excessive response to autoantigen.The example of autoimmune disease includes but is not limited to Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis etc.

[0066] The term "target cell," as used herein, refers to a cell or cells involved in a disease and can be targeted by a DDpp-containing composition or by a CAR, adaptor, and / or CAR / adaptor composition provided herein. Target cells include any cell in a subject (e.g., a human or animal) that can be targeted by a DDPP, CAR, adaptor, and / or CAR / adaptor composition. Target cells can be cells that express or overexpress a target (e.g., CS1) to which a CAR, adaptor, and / or CAR / adaptor composition specifically binds. Target cells can be cells that express or overexpress a target (e.g., CS1) to which a DDpp fusion protein, CAR, adaptor, and / or CAR / adaptor composition specifically binds. In some embodiments, target cells are cells that express CS1.

[0067] "Autologous," as the term is used herein, refers to any material derived from the same individual that is later reintroduced.

[0068] "Allogeneic," as the term is used herein, refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species is sufficiently genetically distinct to be antigenically interactive.

[0069] The term "effector cell," as used herein, refers to a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcgRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; in certain embodiments, PBMCs and NK cells are preferred. Effector cells can be isolated from their native source, for example, from blood or PBMCs, as described herein or otherwise known in the art. In a specific embodiment, the effector cells are human effector cells.

[0070] The term "effector function" refers to a specialized immune function of a differentiated cell. Effector functions of T cells can be, for example, cytolytic or helper activity, including the secretion of cytokines.

[0071] The term "immune cell," as used herein, refers to a cell of a mammalian immune system, including, but not limited to, antigen-presenting cells, B cells, basophils, cytotoxic T cells, dendritic cells, eosinophils, granulocytes, helper T cells, leukocytes, lymphocytes, macrophages, mast cells, memory cells, monocytes, natural killer cells, neutrophils, phagocytes, plasma cells, and T cells.

[0072] The terms "T cells" and "T lymphocytes" are interchangeable and are used synonymously herein. Examples include, but are not limited to, naive T cells, central memory T cells, effector memory T cells, or combinations thereof.

[0073] The term "immune response," as used herein, refers to immunity, including, but not limited to, innate immunity, humoral immunity, cellular immunity, immunity, inflammatory response, acquired (adaptive) immunity, autoimmunity, and / or hyper-reactive immunity.

[0074] The term "transduction," as used herein, refers to the introduction of foreign nucleic acid into a cell using a viral vector. "Transfection," as used herein, refers to the introduction of foreign nucleic acid into a cell using recombinant DNA technology. The term "transformation" refers to the introduction of a "foreign" (e.g., exogenous, extracellular, or otherwise non-endogenous) nucleic acid (DNA or RNA) sequence into a host cell, such that the host cell expresses the introduced nucleic acid to produce a desired substance, e.g., a protein or enzyme encoded by the introduced coding sequence. The introduced nucleic acid sequence may also be referred to as a "cloned" or "foreign" gene or sequence and may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by the cell's genetic machinery. The nucleic acid sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced nucleic acid (e.g., DNA or RNA) has been "transformed" and is a "transformant" or "clone." The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species, or can be non-naturally occurring.

[0075] "Co-express," as used herein, refers to the expression of two or more protein-coding sequences by the same cell or cell population. The coding sequences may be, for example, nucleic acids that each encode a single protein or a chimeric protein as a single polypeptide chain.

[0076] "Cell surface receptor" refers to molecules and complexes of molecules that can receive signals and transmit such signals across the plasma membrane of a cell. An example of a cell surface receptor provided herein is an activated integrin receptor, e.g., an activated αvβ3 integrin receptor on metastatic cells. As used herein, "cell surface receptor" also includes molecules expressed on the cell surface that contain a DDpp capable of binding to a target of interest (e.g., CS1). The term "receptor" refers to a cell-associated protein that binds to or otherwise interacts with a molecule (e.g., a ligand) and mediates the effect of the ligand on the cell. In some embodiments, the molecule that interacts with the receptor is a bioactive molecule. Membrane-bound cell surface receptors are typically characterized by a multidomain structure that includes an extracellular ligand-binding domain, a transmembrane domain, and an intracellular effector domain that typically participates in signal transduction.

[0077] "Antigen loss escape variant," as used herein, refers to a cell that exhibits reduced expression or loss of expression of the target antigen, which antigen is targeted by a CAR provided herein. A. Antigenic determinant (AD)

[0078] An antigenic determinant (AD) is an epitope that can be recognized and specifically bound by an antigenic determinant binding domain (ADBD) (e.g., an antigen-binding fragment of an antibody, or an alternative scaffold binding domain (ASBD) (e.g., a D domain)). The ADs in the adapters and on target cells provided herein can be bound by CARs, as discussed below.

[0079] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is an AD present in a naturally occurring protein or other molecule. In some embodiments, the AD is an AD that is endogenous to humans.

[0080] In some embodiments, the AD in the adaptor is an AD present on a target cell.

[0081] In some embodiments, the AD (e.g., in the adaptor and / or on the target cell) is an AD present in a transmembrane protein, e.g., an AD present in the extracellular portion of a transmembrane protein. In some embodiments, the AD is a tumor antigen. In some embodiments, the AD is a tumor-associated antigen. In some embodiments, the AD is a tumor-specific antigen.

[0082] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is a cancer antigen. In some embodiments, the AD is a cancer-associated antigen. In some embodiments, the AD is a cancer-specific antigen.

[0083] In some embodiments, the AD is an epitope of CS1. In further embodiments, the AD is an epitope of CS1 to which elotuzumab binds. In some embodiments, the AD (e.g., in the adaptor and / or on the target cell) is an epitope of BCMA. In some embodiments, the AD is an epitope of CD19. In some embodiments, the AD is an epitope of CD20. In some embodiments, the AD is an epitope of CD22. In some embodiments, the AD is an epitope of CD123. In some embodiments, the AD is an epitope of HER2. In some embodiments, the AD is an epitope of AFP. In some embodiments, the AD is an epitope of AFP p26. In some embodiments, the AD is an epitope of CD45. In some embodiments, the AD is an epitope of human CD45 to which the UCHL-1, A6, or ODP4 antibody binds. In some embodiments, the AD is an epitope of human CD45 to which the 4KB5, MB1, KiB3, 2H4, or MT2 antibody binds. In some embodiments, the AD is an epitope of CD26. In some embodiments, the AD is an epitope of CD30. In some embodiments, the AD is an epitope of CD33. In some embodiments, the AD is an epitope of CD38.

[0084] In some embodiments, the AD is an epitope of CS1. In further embodiments, the AD is the epitope of CS1 to which elotuzumab binds. In further embodiments, the AD includes 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO:1.

[0085] In some embodiments, the AD is an epitope of AFP p26. In further embodiments, the AD comprises 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 8-14 or 15. In further embodiments, the AD comprises 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 8. In further embodiments, the AD comprises the amino acid residues of SEQ ID NO: 8. In further embodiments, the AD comprises 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 10. In further embodiments, the AD comprises the amino acid residues of SEQ ID NO: 10. In a further embodiment, the AD comprises amino acid residues of SEQ ID NOs: 8-14 or 15.

[0086] In some embodiments, the AD (e.g., in the adaptor and / or on the target cell) is bound by a chimeric antigen receptor (CAR). In some embodiments, the AD is bound by a cell expressing a chimeric antigen receptor. In some embodiments, the AD (e.g., on the target cell) is bound by an adaptor. In some embodiments, the AD is bound by an scFv. In some embodiments, the AD is bound by an alternative scaffold binding domain (ASBD). In some embodiments, the AD is bound by a D domain. In some embodiments, the AD is bound by an antibody or antigen-binding fragment thereof.

[0087] Suitable ADs for use in connection with the DDpps (e.g., adapters and CARs) disclosed herein are disclosed in International Application Publication Nos. WO2016164305, WO2016164308A1, WO2019099440 and WO2019099433, U.S. Patent Nos. 10,662,248 and 10,647,775, and U.S. Patent Application Nos. 20200223934 and 20210002381, each of which is incorporated by reference herein for all purposes. B. Antigenic determinant binding domain (ADBD)

[0088] A protein domain that binds to an antigenic determinant (AD) is referred to herein as an "antigenic determinant binding domain" or "ADBD." In some embodiments, the ADBD is sufficient to confer recognition and specific binding to a target of interest. The ADBDs described herein may be present in DDpp fusion proteins, adapters, and / or chimeric antigen receptors (CARs).

[0089] In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adaptor, and / or CAR) is an antibody or antigen-binding fragment thereof. In some embodiments, the ADBD is an scFv. In some embodiments, the ADBD is an alternative scaffold binding domain. In some embodiments, the ADBD is a D domain.

[0090] Suitable ADBDs for use in connection with the DDpps (e.g., adaptors and CARs) disclosed herein are disclosed in International Application Publication Nos. WO2016164305, WO2016164308A1, WO2019099440 and WO2019099433, U.S. Patent Nos. 10,662,248 and 10,647,775, and U.S. Patent Application Nos. 20200223934 and 20210002381, each of which is incorporated herein by reference for all purposes. i. Antibody-derived antigenic determinant binding domain (ADBD)

[0091] In some embodiments, one or more ADBDs (e.g., of a DDpp fusion protein, adapter, and / or CAR) may be derived from an antibody molecule, such as a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain (VHH), for example, from human or camelid origin. In some embodiments, the ADBD is derived from the same species in which the adapter or CAR will ultimately be used, for example, for use in humans. It may be beneficial to include a human or humanized ADBD for the adapter and / or CAR. Compositions and techniques for routinely producing such ADBDs are known in the art.

[0092] In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) comprises a fragment of an antibody sufficient to confer recognition and specific binding to a target antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, or Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments.

[0093] In some embodiments, the ADBD (e.g., a DDpp fusion protein, adapter, and / or CAR) is an "scFv," which may comprise a fusion protein comprising the VL and VH chains of an antibody, where the VH and VL are linked, for example, via a short, flexible polypeptide linker, such as a linker described herein. scFvs can be routinely prepared according to methods known in the art (see, e.g., Bird et al., Science 242: 423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).

[0094] In some embodiments, the ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a single-domain antigen-binding (SDAB) molecule. SDAB molecules include molecules containing complementarity-determining regions that are part of a single-domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules that naturally lack light chains, single domains derived from traditional four-chain antibodies, engineered domains, and single-domain scaffolds other than those derived from antibodies. SDAB molecules can be derived from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. The term also includes naturally occurring single-domain antibody molecules from species other than Camelidae and sharks.

[0095] In some embodiments, the ADBD (e.g., of the DDpp fusion protein, adaptor, and / or CAR) comprises a human antibody or fragment thereof. In some embodiments, the ADBD (e.g., of the DDpp fusion protein, adaptor, and / or CAR) comprises a humanized antibody or fragment thereof.

[0096] Antibody humanization is well known in the art and is essentially the methodology of Winter and coworkers (Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al., Science 239: 1534-1536 (1988)), by replacing rodent CDRs or CDR sequences with those of the corresponding human antibody, i.e., CDR grafting (EP 239,400; International Application Publication No. WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; and 6,548,640; the contents of which are incorporated herein by reference in their entireties). Antibody humanization can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4 / 5): 489-498; Studnicka et al., Protein Engineering 7(6): 805-814 (1994); and Roguska et al., PNAS 91: 969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entireties. ii. Alternative Scaffold Binding Domains

[0097] In some embodiments, an ADBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is an alternative scaffold binding domain (ASBD). An "alternative scaffold binding domain" or "ASBD," as used herein, is an antigenic determinant binding domain derived from or corresponding to a non-antibody-based binding scaffold.

[0098] In some embodiments, the present disclosure provides a CAR comprising an ADBD that is an ASBD. In some embodiments, the present disclosure provides a cell comprising a CAR comprising an ADBD that is an ASBD. In further embodiments, an immune effector cell comprising a CAR comprising an ASBD is provided. In some embodiments, the present disclosure provides an adaptor comprising an ADBD that is an ASBD.

[0099] In further embodiments, the present disclosure provides compositions comprising an adaptor and a CAR, each comprising an ASBD.

[0100] In some embodiments, the binding of the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) to the target AD is mediated by the secondary structure of the binding scaffold, for example, an alpha helix or a beta sheet. In some embodiments, the ASBD is a three-helix bundle-based binding domain. In some embodiments, the ASBD is a D-domain-based binding domain. In other embodiments, the ASBD is a Z-domain (affibody)-based binding domain.

[0101] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adaptor, and / or CAR) is a D domain (de novo binding domain)-based AD binding domain. In some embodiments, the D domain comprises a sequence selected from the group of SEQ ID NOs: 41-105 and 106. In some embodiments, the D domain comprises the sequence of SEQ ID NO: 46. In some embodiments, the D domain comprises the sequence of SEQ ID NO: 60.

[0102] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a Z-domain scaffold (affibody)-based AD binding domain. Z-domain scaffold-based binding domains generally consist of 58 amino acid residues, with substitutions at up to 13 positions located at the first and second of three alpha helices conferring target (AD) recognition and binding specificity for the target (AD) of interest. Z-domain (affibody) scaffold-based binding domains are further described in U.S. Patent No. 5,831,012, the entire contents of which are incorporated herein by reference in their entirety.

[0103] Additional examples of ASBDs that display secondary structure-mediated target binding include DARPins, affilins, and armadillo repeat-based binding scaffolds.

[0104] In some embodiments, the ASBD (eg, of a DDpp fusion protein, adaptor and / or CAR) is a DARPin-based AD-binding domain.

[0105] In some embodiments, the ASBD is an Adnectin-based AD binding domain. The Adnectin-based binding domain is derived from the 10th domain of fibronectin type III (10Fn3). This ADBD is a 94-amino acid binding domain that generally adopts a beta-sandwich fold containing seven chains connected by six loops. The substitution of three surface-exposed loops at one end of the Adnectin domain creates a target (AD)-specific binding moiety.

[0106] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adaptor, and / or CAR) is a lipocalin-, affilin-, or anticalin-based AD-binding domain. The anticalin scaffold is composed of eight antiparallel β-strands and generally displays a conserved β-barrel structure of 160-180 amino acids. The ligand-binding pocket of anticalin-based binding scaffolds is composed of four loops, each containing up to 24 substitutions, which collectively confer target (AD) recognition and binding specificity.

[0107] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is an Avimer scaffold-based AD binding domain. Avimer scaffold-based binding domains are derived from the A domain of a cell surface receptor and are generally 35 amino acids in length. Avimer scaffold-based binding domains are further described in U.S. Application Publication Nos. 20040175756, 20050053973, 20050048512, and 20060008844, the entire contents of each of which are incorporated herein by reference in their entirety.

[0108] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is an AD-binding domain based on a phenomer scaffold. The phenomer-binding domain is generally 60-75 amino acids long and consists of a pair of antiparallel beta sheets joined by two flexible loops. Substitutions / insertions in the loops confer AD target recognition and binding specificity.

[0109] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a knottin scaffold-based AD-binding domain. The knottin scaffold-based binding domain corresponds to a 30-amino acid protein fold composed of three antiparallel β-strands connected by loops of variable length and multiple disulfide bonds.

[0110] In some embodiments, the ASBD (e.g., of a DDpp fusion protein, adapter, and / or CAR) is a Kunitz domain-based AD binding domain. Kunitz domain-based binding domains are derived from the active motif of Kunitz-type protease inhibitors and are generally approximately 60 amino acids in length. The hydrophobic core of this ADBD is composed of a twisted two-strand antiparallel β-sheet and two α-helices stabilized by three pairs of disulfide bonds. Substitutions and insertions in three loops confer AD target recognition and binding specificity. Kunitz scaffold-based binding domains are further described in International Application Publication No. WO2004063337, the entire contents of which are incorporated herein by reference in their entirety.

[0111] In some embodiments, the ASBD (eg, of a DDpp fusion protein, adaptor and / or CAR) is a WW domain-based AD-binding domain. C. Linker

[0112] A linker is a peptide or other chemical linkage positioned between two or more otherwise independent functional domains of a DDpp fusion protein, adaptor, or CAR.

[0113] Suitable linkers for operably linking two or more functional domains of a DDpp and additional components of a DDpp fusion protein, or an adaptor, in a single amino acid sequence include, but are not limited to, polypeptide linkers, such as glycine linkers, serine linkers, mixed glycine / serine linkers, glycine and serine-rich linkers, or linkers composed primarily of polar polypeptide fragments.

[0114] In one embodiment, the linker is primarily composed of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In one embodiment, one or more linkers in a DDpp fusion protein, adapter, or CAR are primarily composed of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In one embodiment, one or more linkers in a DDpp fusion protein, adapter, or CAR are primarily composed of one or more amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In another embodiment, one or more linkers in a DDpp fusion protein, adapter, or CAR are primarily composed of sterically unhindered amino acids. In another embodiment, the linker comprises a majority of amino acids that are glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycine (e.g., (Gly)5 (SEQ ID NO: 16) and (Gly)8 (SEQ ID NO: 17)), poly(Gly-Ala), and polyalanine. In some embodiments, the peptide linker contains the sequence Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 18). In some embodiments, the peptide linker contains the sequence Gly-Gly-Gly-Gly-Asp-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 19). In some embodiments, the peptide linker contains the sequence of SEQ ID NOs: 16-20, 115-118, or 119.

[0115] In one embodiment, a DDpp fusion protein, adapter, or CAR comprises an ADBD (e.g., a D domain) directly attached (i.e., without a linker) to another component of the DDpp fusion protein, adapter, or CAR, respectively. In one embodiment, a DDpp fusion protein, adapter, or CAR contains at least two, at least three, at least four, or at least five ADBDs (e.g., D domains) directly attached to another domain of the DDpp fusion protein, adapter, or CAR, respectively.

[0116] In another embodiment, an ADBD (e.g., a D domain) can be operably linked to another component of a DDpp fusion protein, adapter, or CAR through a linker. A DDpp fusion protein, adapter, or CAR can contain a single linker, multiple linkers, or no linkers. In one embodiment, a DDpp fusion protein, adapter, or CAR comprises an ADBD (e.g., a D domain) operably linked to another component of a DDpp fusion protein, adapter, or CAR, respectively, through a linker peptide. In one embodiment, a DDpp fusion protein, adapter, or CAR contains at least two, at least three, at least four, or at least five ADBDs (e.g., D domains) operably linked to another domain of a DDpp fusion protein, adapter, or CAR, respectively, through the same or different linkers.

[0117] Linkers can be of any size or composition, so long as they are capable of operably linking the functional domains of a DDpp fusion protein, adapter, or CAR in a manner that allows the functional domains to function (e.g., the ability of an antigenic determinant binding domain to bind to a target of interest). In some embodiments, the linker is about 1 to about 100 amino acids, about 1 to 50 amino acids, about 1 to 20 amino acids, about 1 to 15 amino acids, about 1 to 10 amino acids, about 1 to 5 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. It should be apparent that the length, degree of flexibility, and / or other properties of the linker can have some effect on the properties of the final polypeptide of the invention, including, but not limited to, affinity, specificity, or avidity for a target of interest or for one or more other target proteins of interest. When two or more linkers are used in a DDpp fusion protein, adapter, or CAR, these linkers can be the same or different. In the context and disclosure provided herein, one of skill in the art can routinely determine the optimal linker composition and length for the purpose of operably linking functional domains of a DDpp fusion protein, adapter, or CAR.

[0118] The linker may also be a non-peptide linker, such as an alkyl linker or a PEG linker. For example, an alkyl linker, such as -NH-(CH)sC(0)- (where s = 2 to 20), can be used. These alkyl linkers may be further substituted with any sterically unhindered group, such as lower alkyl (e.g., C1 to C6), lower acyl, halogen (e.g., Cl, Br), CN, NH, phenyl, etc. An exemplary non-peptide linker is a PEG linker. In certain embodiments, the PEG linker has a molecular weight of about 100 to 5,000 kDa, or about 100 to 500 kDa.

[0119] Suitable linkers for coupling functional domains of DDpp fusion proteins, adapters, or CARs by chemical crosslinking include, but are not limited to, homobifunctional chemical crosslinking compounds such as glutaraldehyde, imidoesters such as dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl pimelimidate (DMP), or N-hydroxysuccinimide (NHS) esters such as dithiobis(succinimidyl propionate) (DSP) and dithiobis(sulfosuccinimidyl propionate) (DTSSP). Examples of suitable linkers for coupling functional domains of DDpp fusion proteins, adapters, or CARs include, but are not limited to, crosslinkers with an amine-reactive end and a sulfhydryl-reactive moiety at the other end, or an NHS ester and an SH-reactive group (e.g., maleimide or pyridinyl) at one end.

[0120] In additional embodiments, one or more of the linkers in the DDpp fusion protein, adapter, or CAR are cleavable. Examples of cleavable linkers include, but are not limited to, peptide sequences recognized by various types of proteases (in vitro or in vivo), such as Tev, thrombin, Factor Xa, plasmin (a blood protease), metalloproteases, cathepsins (such as GFLG), and proteases found in other body compartments.

[0121] In some embodiments, the linker is a "cleavable linker" that facilitates release of the DDpp fusion protein functional domain, adapter functional domain, or cytotoxic agent in or on the cell surface. For example, an acid-labile linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (see, e.g., Chari, Can. Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020; and U.S. Application Publication No. 20090110753; the contents of each of which are incorporated herein by reference in their entirety) can be used, where the covalent bond between the DDpp or cytotoxic agent and the fusion partner is desirably cleaved intracellularly when the composition is internalized by the cell. The terms "cleaved intracellularly" and "intracellular cleavage" refer to a metabolic process or reaction within a cell in a DDpp-drug conjugate, whereby a covalent attachment, i.e., a covalent attachment linked via a linker between the DDpp and the cytotoxic agent, between the DDpp and the fusion partner, or between two DDpps, is broken, resulting in free DDpp and / or cytotoxic agent dissociated inside the cell.

[0122] In additional embodiments, one or more of the linkers in the CAR are cleavable. Examples of cleavable linkers include, but are not limited to, peptide sequences recognized by various types of proteases (in vitro or in vivo), such as Tev, thrombin, factor Xa, plasmin (a blood protease), metalloproteases, cathepsins (such as GFLG), and proteases found in other body compartments.

[0123] In some embodiments, short oligopeptide or polypeptide linkers, approximately 1 to 100 amino acids in length, are used to link together any of the domains of the CAR. The linker may be composed of flexible residues such as glycine and serine (or any other amino acid) to allow adjacent protein domains to move freely relative to one another. The amino acid sequence composition of the linker may be selected to minimize the potential immunogenicity of the CAR. Longer linkers can be used if it is desirable to ensure that two adjacent domains do not sterically interfere with one another.

[0124] In some embodiments, a length of 2 to 10 amino acids preferably forms the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. In further embodiments, the linker is 10 to 15 amino acids in length, or 15 to 20, or 20 to 30, or 30 to 60, or 60 to 100 amino acids in length (or any range between those listed). In further embodiments, the linker is a glycine-serine doublet sequence. In some embodiments, the extracellular spacer domain (ESD) corresponds to the human T-cell surface glycoprotein CD8 alpha chain ESD region (e.g., amino acid residues 138-182 of the CD8 alpha chain; Swiss-Prot Acc. No. P01732). In some embodiments, the CD8 alpha ESD comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the ESD corresponds to a CD8 ESD region further modified by amino acid substitutions to improve expression function or immunogenicity. In a further embodiment, the ESD corresponds to a sequence containing the CD28 ESD or a modification of the CD28 ESD that confers improved expression function or immunogenicity.

[0125] Linker optimization can be assessed using techniques described herein and / or otherwise known in the art. In some embodiments, the linker does not disrupt the ability of the DDpp fusion protein, adapter, or CAR to bind to a target antigenic determinant and / or another adapter or CAR functional domain to function properly (e.g., the ability of an effector functional domain in an adapter to elicit effector function or the ability of an FcRn-binding domain in an adapter to bind FcRn). D domain polypeptide (DDpp)

[0126] According to various embodiments, the present disclosure provides a DDpp that specifically binds to CS1. In some embodiments, the DDpp comprises a D domain (DD) that specifically binds to CS1 and comprises the amino acid sequence of SEQ ID NO: 41-105 or 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60. Proteins comprising variants of the D domain that retain the ability to specifically bind to their respective targets are also provided.

[0127] In some embodiments, DDpp is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a full-length antibody or an antibody fragment. In some embodiments, DDpp is fused to the amino terminus of a full-length antibody heavy chain; the amino terminus of a full-length antibody light chain; the carboxyl terminus of a full-length antibody heavy chain; or the carboxyl terminus of a full-length antibody light chain. In other embodiments, DDpp is fused to an Fc antibody fragment. In additional embodiments, the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane-associated domain; (iii) human serum albumin or a fragment thereof; (iv) AFP or a fragment thereof; (v) AFP p26 or a fragment thereof; (vi) an extracellular domain of a receptor or a fragment thereof; and (vii) an extracellular domain of an intracellular receptor (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the protein contains a heterologous polypeptide comprising an extracellular domain or a fragment of the extracellular domain of CS1 (SEQ ID NO: 1), BCMA (SEQ ID NO: 2), CD123 (SEQ ID NO: 3), or CD19. In some embodiments, the protein contains a heterologous polypeptide comprising an antigenic portion of a serum protein (e.g., AFP and AFP p26). In some embodiments, the protein contains a heterologous polypeptide comprising an antigenic portion of an intracellular protein (e.g., a nucleoprotein). In some embodiments, the protein is labeled. In further embodiments, the label is selected from the group consisting of an enzymatic label, a fluorescent label, a luminescent label, a bioluminescent label, and a biotin moiety. In additional embodiments, the protein is conjugated to a therapeutic or cytotoxic agent. In some embodiments, the protein contains a heterologous polypeptide that binds to one or more major histocompatibility complex (MHC) class I or class II complexes.

[0128] In some embodiments, the DD of the DDpp is a variant of a CS1-binding DD reference sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106 that retains the ability to specifically bind to CS1. In some embodiments, the sequence of the variant DD comprises a variant amino acid sequence having at least 75%, 80%, 85%, 87%, 89%, 90%, 92%, 94%, 96% or 98% sequence identity to a reference DD sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106, and wherein the variant DD retains the ability to specifically bind to CS1.

[0129] In certain embodiments, the identity between a variant DD (query) sequence and a reference DD sequence, also referred to as global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. Comp. App. Biosci. 6: 237-245 (1990). The preferred parameters used in FASTDB amino acid alignment are: matrix=PAM 0, k-tuple=2, mismatch penalty=1, joining penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or the shorter of the length of the target amino acid sequence. According to this embodiment, if the reference DD sequence is shorter than the variant DD query sequence due to N- or C-terminal deletions, not due to internal deletions, the result is manually corrected to take into account the fact that the FASTDB program does not take into account the N- and C-terminal truncations of the reference DD sequence when calculating global identity percentage. For reference sequences that are truncated at the N- and C-terminus, the percent identity compared to the query sequence is corrected by calculating the number of residues in the query sequence at the N- and C-terminus of the reference sequence that are not matched / aligned with the corresponding target residues as a percentage of the total bases of the query sequence. The determination of whether a residue is matched / aligned is determined by the results of FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the above FASTDB program using the specified parameters to arrive at a final percent identity score. This final percent identity score is the one that is used for the purposes of this embodiment.

[0130] In some embodiments, the disclosed DDpp (e.g., DDpp fusion protein) is labeled. Labels that can be used to label a DDpp include, but are not limited to, enzymatic labels, fluorescent labels, luminescent labels, and bioluminescent labels. In some embodiments, the label is a biotin moiety. In some embodiments, the label is a streptavidin moiety. In some embodiments, the label is a His-tag or a FLAG tag. In some embodiments, the label is luciferase, green fluorescent protein, red fluorescent protein, or other similar agent. In some embodiments, the DDpp comprises a CS1-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 60.

[0131] In other embodiments, the DDpp fusion protein is attached to a solid support. In some embodiments, the solid support is selected from the group consisting of beads, glass slides, chips, gelatin, and agarose. In some embodiments, the DDpp comprises a CS1-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 60.

[0132] In some embodiments, the DDpp (e.g., a DDpp fusion protein) is associated with a liposome. In some embodiments, the DDpp is associated with a liposome through a covalent bond. In some embodiments, the DDpp is a fusion protein. In further embodiments, the DDpp is a CAR. In additional embodiments, the DDpp is associated with a liposome through an ionic bond rather than a covalent bond. In some embodiments, the DDpp comprises a CS1-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 60.

[0133] In some embodiments, the target-binding DDpp is conjugated to a therapeutic or cytotoxic agent (e.g., a chemotherapeutic or radiotherapeutic agent). In some embodiments, the DDpp comprises a CS1-binding DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 60. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0134] In some embodiments, the present disclosure provides compositions comprising one or more of the DD sequences disclosed in Table 1. In other embodiments, the present disclosure provides compositions comprising one or more DDs comprising sequences having 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, or 95-99% homology (and overlapping ranges) to a sequence disclosed in Table 1. In some embodiments, DDs with such homology are functionally similar or identical compared to their respective reference sequences in Table 1. In some embodiments, the present disclosure provides polypeptides comprising one or more DDs that compete (in whole or in part) with one or more of the DD sequences (reference sequences) disclosed in Table 1 for their respective targets. The ability of a polypeptide to compete with a reference polypeptide for binding to its respective target can be routinely determined using standard competition assays known in the art. In some embodiments, competition does not require that the polypeptide compete for the same epitope as polypeptide (DD) of Table 1, but rather the polypeptide can compete by binding to a sterically hindering epitope, an overlapping epitope, etc. A.CS1 binding DDpp

[0135] In some embodiments, the present disclosure provides a protein that specifically binds to CS1 (SEQ ID NO: 1) and comprises a D domain (DD) target binding domain (DDpp) comprising the amino acid sequence of SEQ ID NO: 41-105 or 106. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the DDpp comprises a DD comprising the amino acid sequence of SEQ ID NO: 60.

[0136] In some embodiments, the DD of the DDpp specifically binds to CS1. In further embodiments, the DD specifically binds to CS1 having the amino acid sequence of SEQ ID NO:1. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:41-105 and 106. In other embodiments, the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:41-105 and 106. In some embodiments, the DDpp specifically binds to CS1 (SEQ ID NO:1) and comprises the amino acid sequence of SEQ ID NO:46 or 60. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO:46. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO:60.

[0137] In other embodiments, the CS1-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-binding DDpp comprises multiple target-binding domains that bind to a single target (e.g., a dimer, trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that specifically bind to CS1 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that have the same sequence. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that specifically bind to different epitopes of CS1 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 and further comprises two, three, four, five, or more than five additional, different DDs, or a target-binding binding domain (e.g., an scFv) that specifically binds to CS1 or a different target antigen. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106) and further comprises one or more additional DDs or other target-binding binding domains that bind to one or more antigens expressed on the surface of B-lineage cells. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106) and further comprises one or more additional DDs or other target-binding binding domains that bind to one or more cancer antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106) and specifically binds to two, three, four, five, or more than five different targets.In further embodiments, the DDpp comprises a DD that specifically binds to CS1 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106) and specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of different cancer cells. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 60.

[0138] In some embodiments, the DDpp comprises a variant of a CS1-binding DD (reference DD) disclosed herein that retains the ability to specifically bind to CS1. In some embodiments, the sequence of the CS1-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions compared to a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions compared to a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions compared to a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 60.

[0139] In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 1-22, 29-46, and 52-72 of a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative substitutions at positions corresponding to amino acid residues 1-22, 29-46, and 52-72 of a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions at positions corresponding to amino acid residues 1 to 22, 29 to 46, and 52 to 72 of a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41 to 105 and 106. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 60.

[0140] In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 2 to 6, 8 to 10, 12, 13, 15 to 17, 19, 20, 29, 30, 32 to 34, 36, 37, 39 to 41, 43, 44, 52 to 55, 57 to 59, 61, 62, 64 to 66, and 68 to 70 of a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 conservative substitutions at positions corresponding to amino acid residues 2-6, 8-10, 12, 13, 15-17, 19, 20, 29, 30, 32-34, 36, 37, 39-41, 43, 44, 52-55, 57-59, 61, 62, 64-66, and 68-70 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions at positions corresponding to amino acid residues 2 to 6, 8 to 10, 12, 13, 15 to 17, 19, 20, 29, 30, 32 to 34, 36, 37, 39 to 41, 43, 44, 52 to 55, 57 to 59, 61, 62, 64 to 66, and 68 to 70 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 60.

[0141] In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative or non-conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference CS1-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the sequence of the CS1-binding DD variant contains a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1 to 3, 1 to 5, or 1 to 10 non-conservative substitutions at positions corresponding to amino acid residues 7, 11, 14, 18, 21, 28, 31, 35, 38, 42, 45, 53, 56, 60, 63, and 67 of a reference DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 60.

[0142] In some embodiments, the present disclosure provides a CS1-binding DDpp that completely or partially (e.g., overlaps with an epitope) blocks binding of a reference DD to CS1, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 41-105 and 106. In other embodiments, the present disclosure provides a CS1-binding DDpp that binds to the same epitope of CS1 as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs: 41-105 and 106. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the reference CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 60.

[0143] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to CS1. In some embodiments, the DD of the DDpp fusion protein specifically binds to CS1 having the amino acid sequence of SEQ ID NO:1. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, DDpp comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the DDpp fusion protein comprises a full-length antibody or a portion (fragment) of an antibody. In some embodiments, the DDpp fusion protein comprises a full-length IgG antibody (e.g., IgG1, IgG2, IgG2, or IgG4). In further embodiments, the DDpp fusion protein comprises a full-length antibody that specifically binds to a cancer antigen. In further embodiments, the DDpp comprises a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN882). In other embodiments, the CS1-binding DDpp is an Fc-fusion protein.

[0144] In some embodiments, the DDpp is a fusion protein comprising a CS1-binding DD operably linked to a serum protein. In some embodiments, the CS1-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the DDpp comprises the amino acid sequence of SEQ ID NO: 60. In other embodiments, the CS1-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-binding DDpp fusion protein comprises all or a portion of human serum albumin. In some embodiments, the DDpp fusion protein comprises AFP (SEQ ID NO: 7), or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises AFP p26 (SEQ ID NO: 8), or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 8-14 or 15. In some embodiments, the DDpp fusion protein contains a fragment of a serum protein or an antigenic fragment of a serum protein (e.g., AFP and AFP p26). In some embodiments, the DDpp fusion protein comprises a fragment of a serum protein consisting of 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 10 to 50 amino acids. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 60.

[0145] In some embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-binding DDpp comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-binding DDpp comprises the amino acid sequence of SEQ ID NO: 60. In other embodiments, the CS1-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In further embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of CS1 (SEQ ID NO: 1), or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, NKG2D, and gp96, or a fragment thereof.

[0146] In some embodiments, the CS1-binding DDpp fusion protein contains a fragment of the extracellular domain of a cell surface receptor consisting of 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 10 to 50 amino acids. In some embodiments, the DDpp fusion protein contains a fragment of the extracellular domain of BCMA (SEQ ID NO: 2). In some embodiments, the DDpp fusion protein contains a fragment of the extracellular domain of CS1 (SEQ ID NO: 1). In some embodiments, the DDpp contains a fragment of the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.

[0147] In additional embodiments, the CS1-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-binding DDpp comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-binding DDpp comprises the amino acid sequence of SEQ ID NO: 60. In other embodiments, the CS1-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-binding DDpp fusion protein comprises a fragment of 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 10 to 50 amino acid residues of an intracellular protein (e.g., a nuclear protein).

[0148] Nucleic acids encoding DDpp and vectors containing the nucleic acids are also provided. Host cells containing the nucleic acids and vectors (including viral particles) containing the nucleic acids are also provided. In some embodiments, the host cell is a prokaryotic or eukaryotic organism that displays the variant DD on its surface. In some embodiments, the host cell displays the variant DD on its surface. In further embodiments, the host cell is a phage that displays the variant DD on its surface. In further embodiments, the host cell is a human immune cell that expresses the variant DD fusion protein on its surface.

[0149] A DDpp agonist refers to a DDpp that, in some ways, increases or enhances the biological activity of a DDpp target (e.g., CS1), or has biological activity equivalent to that of a known agonist of the DDpp target. In another embodiment, the DDpp is an antagonist of the target to which it binds (e.g., CS1). A DDpp antagonist refers to a DDpp that completely or partially blocks, or in some ways prevents, the biological activity of a DDpp target protein, or has biological activity equivalent to that of a known antagonist or inhibitor of the DDpp target protein. DDpp fusion protein

[0150] DDpp fusion proteins are provided herein. "Fusion proteins," "chimeric polypeptides," "chimeric proteins," "chimeric antigens," and DDpps containing / comprising heterologous polypeptides are polypeptides composed of at least two polypeptides, produced, for example, by recombinant processes, and optionally a linker that operably links the two polypeptides into one continuous polypeptide. The two polypeptides can be operably attached directly or indirectly.

[0151] A "DDpp fusion protein" as provided herein comprises at least one DDpp disclosed herein that specifically binds to a target of interest (e.g., CS1 (SEQ ID NO: 1), or a fragment thereof). In one embodiment, the DDpp fusion protein contains one DDpp.

[0152] In some embodiments, the DDpp fusion protein is a soluble protein comprising one or more target-binding DDpp and a p26 protein (e.g., having a sequence of SEQ ID NO: 8-15 or 16). In some embodiments, the soluble DDpp fusion protein has an in vivo plasma half-life of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or longer. In some embodiments, the soluble fusion protein has an in vivo plasma half-life in mice of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or longer than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours. In some embodiments, the soluble DDpp fusion protein has an in vivo plasma half-life in humans of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or greater than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours.

[0153] In some embodiments, the present disclosure provides methods for modifying the in vivo half-life (e.g., in mice or humans) of a soluble fusion protein comprising a p26 protein (e.g., having a sequence of SEQ ID NO: 8-15 or 16). In some embodiments, the soluble p26 fusion protein comprises one or more target-binding DDpp. In some embodiments, the half-life of the p26 soluble fusion protein is increased or decreased by substituting or deleting one or more amino acid residues normally found in human p26 protein, or by inserting one or more amino acid residues not normally found in human p26 protein. In another embodiment, the p26 sequence of the soluble fusion protein is modified by substitution (conservative and / or non-conservative substitutions), deletion, and / or insertion of 1, 2, 3, 4, 5, 10, or 1-20, 1-10, 3-10, or 3-5 amino acids to increase or decrease the in vivo half-life of the soluble fusion protein. In a specific embodiment, the amino acid residue corresponding to glutamine (Gln, Q) at position 217 of SEQ ID NO: 8 in p26 is substituted with another amino acid residue. In a further embodiment, the substitution is Gln217Pro. In another embodiment, the p26 sequence of the soluble fusion protein is modified by the deletion of 1 to 150, 1 to 100, 1 to 50, 1 to 25, or 1 to 10 amino acid residues to increase or decrease the in vivo half-life of the soluble fusion protein. In additional embodiments, the p26 sequence of the soluble fusion protein is modified by the substitution (conservative and / or non-conservative substitutions), deletion, and / or insertion of 1, 2, 3, 4, 5, 10, or 1 to 20, 1 to 10, 3 to 10, or 3 to 5 amino acids to increase or decrease the interaction of the soluble fusion protein with FcRn. A. Multimeric DDpp fusion protein

[0154] In one embodiment, a DDpp fusion protein comprises two or more DDpps, where the two or more DDpps have the same or different specificities. In additional embodiments, the DDpp fusion protein comprises tandem repeats of the same or different DDpps, allowing the DDpp fusion protein to bind to multiple targets and / or repeated or different epitopes on the same target. In some embodiments, a DDpp fusion protein comprises at least two, three, four, or five, or more than five, DDpps. In some embodiments, a DDpp fusion protein contains one to three, one to four, one to five, or more than five different DDpps. In some embodiments, a DDpp fusion protein contains at least two, three, four, or five, or more than five different DDpps. Thus, a DDpp fusion protein can be a monomeric DDpp (i.e., containing one DDpp) or a multimeric DDpp (i.e., containing two or more DDpps in tandem, optionally operably connected by a linker). In some embodiments, the use of multimeric DDpp provides enhanced (e.g., synergistic) target binding. In additional embodiments, multimeric DDpp allow targeting of two or more targets using a single DDpp construct (e.g., bispecific, trispecific, etc.). Linking two or more identical DDpp results in multivalent molecules that offer distinct advantages over monovalent compositions (e.g., increased binding avidity, target clustering, and receptor activation). Linking two or more different DDpp results in multivalent and multispecific molecules that have the potential to bind to two or more target antigens, either independently or simultaneously.

[0155] Multimeric DDpp fusion proteins can be DDpp homomultimers (i.e., containing two or more identical DDpps in tandem, optionally connected by a linker) (e.g., homodimers, homotrimers, homotetramers, etc.), or DDpp heteromultimers (i.e., containing two or more DDpps, where at least two different DDpp proteins are present). The number of monomeric DDpps included within a multimeric composition can vary depending on the embodiment and can be defined, at least in part, by the expression system in which the DDpp is produced. In some embodiments, however, the fusion protein can comprise a multimer of about 5 to about 10 DDpp subunits, about 10 to about 15 subunits, about 15 to about 20 subunits, about 20 to about 25 subunits, or about 25 to about 30 subunits (including numbers and endpoints between the listed numbers). Additionally, the multiple tandem components of a DDpp fusion protein can contain the same or different DDpps. In some DDpp fusions, DDpp exists as a monomer or in a homo- or hetero-multimer, eg, a homo- or hetero-dimer, homo- or hetero-trimer, homo- or hetero-tetramer.

[0156] DDpp fusion proteins can be "monospecific" or "multispecific." A DDpp fusion protein that is "multispecific" (e.g., bispecific, trispecific, or of higher order multispecificity) recognizes and binds to two or more different epitopes present on one or more different molecules (e.g., proteins, solid support structures, etc.).

[0157] In some embodiments, two or more DDs are fused together as a multivalent DDpp. The DDs of a multivalent DDpp can be the same or different. Thus, the present disclosure provides DDpp homodimers (i.e., DDpps containing two identical DDs), DDpp homomultimers (i.e., DDpps containing three or more identical DDs), DDpp heterodimers (i.e., DDpps containing two different DDs), and DDpp heteromultimers (i.e., DDpps containing three or more DDs, where at least two of the DDs are different), which include any of the DDs described herein, optionally attached by one or more linkers.

[0158] In some embodiments, two or more DDs are linked by a multimerization domain or attached via chemical linkage to create a multivalent DD complex. The DDs of the multivalent DD complex can be the same or different. Therefore, the present disclosure provides DD homodimeric complexes (i.e., DD complexes containing two identical DDs), DD homomultimeric complexes (i.e., DD complexes containing three or more identical DDs), DD heterodimeric complexes (i.e., DD complexes containing two different DDs), and DD heteromultimeric complexes (i.e., DD complexes containing three or more DDs, where at least two of the DDs are different), which include any of the DDs described herein, optionally attached by one or more linkers.

[0159] In one embodiment, a multispecific DDpp fusion protein contains at least two DDpps that bind to at least two different epitopes on a single target of interest (e.g., CS1 and BCMA). In a further embodiment, the DDpp fusion is bispecific and specifically binds to two different targets expressed on the surface of two different cell types. In one embodiment, a bispecific DDpp fusion protein specifically binds to a target on a cancer cell and a target on an immune effector cell. In one embodiment, a bispecific DDpp fusion protein specifically binds to a target expressed on a cancer cell (e.g., CS1) and a target expressed on the surface of a T lymphocyte (e.g., CD3). In one embodiment, a bispecific DDpp fusion protein specifically binds to CS1 and BCMA. In some embodiments, a bispecific DDpp fusion protein specifically binds to CS1 and BCMA and comprises a BCMA-binding DD, optionally wherein the BCMA-binding DD comprises the amino acid sequence of SEQ ID NOs: 25-39 or 40, and optionally wherein the BCMA-binding DD comprises the amino acid sequence of SEQ ID NO: 34, 35, or 39. In some embodiments, the bispecific DDpp fusion protein further comprises a CS1-binding DD. In one embodiment, the bispecific DDpp fusion protein specifically binds to CS1 and CD3.

[0160] In additional embodiments, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope on a target of interest and at least one other domain or sequence (e.g., an antibody fragment or domain, e.g., an scFv) that confers the function of specifically binding to a different epitope on the same target of interest. In one embodiment, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope on a target of interest and at least one domain or sequence, e.g., an antibody fragment or domain (e.g., an scFv), that confers the function of specifically binding to an epitope on a different target of interest. In one embodiment, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope on a target of interest and at least one domain or sequence that specifically binds to an epitope on a different target on the same cell. In other embodiments, the DDpp fusion protein comprises at least one DDpp and at least one other DDpp or domain sequence, e.g., an antibody fragment or domain, that confers the function of specifically binding to a solid support.

[0161] In further embodiments, multimeric DDpp fusions containing two or more DDpps are then fused to other heterologous proteins (or subdomains thereof), thereby conferring multivalent and multispecific properties to the fusion partner. Examples of DDpp fusion partners include, but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, T cell receptor (TCR) alpha chains, T cell receptor beta chains, cell surface receptor subdomains, peptides, peptide tags (e.g., FLAG or myc), fibronectin type III repeats, z-domains, and elastin-like polypeptides. The number and location of DDpps, as well as their respective positions within the fusion protein, can vary. For example, DDpps can be located at one or all termini of the fusion partner and / or interspersed within heterologous subunits within the DDpp fusion partner.

[0162] In additional embodiments, the DDpp fusion protein comprises a polypeptide sequence containing DDpp and an additional domain. In some embodiments, the DDpp fusion protein comprises DDpp and a member selected from an antibody, an antibody fragment (e.g., an antigen-binding domain or portion thereof (e.g., scFv), an effector domain or portion thereof, an FcRn-binding domain or portion thereof, and an Fc or portion thereof), a serum protein (e.g., albumin or a portion thereof), a cytokine, a growth factor, a hormone, an imaging agent, a labeling agent, and a peptide tag. In some embodiments, the DDpp fusion protein comprises an Fc domain of an immunoglobulin (e.g., a human Fc domain) or portion thereof. In further embodiments, the Fc domain is a variant human Fc domain.

[0163] In some embodiments, DDpp is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide comprises a full-length antibody or an antibody fragment. In some embodiments, DDpp is fused to the amino terminus of a full-length antibody heavy chain; the amino terminus of a full-length antibody light chain; the carboxyl terminus of a full-length antibody heavy chain; or the carboxyl terminus of a full-length antibody light chain. In other embodiments, DDpp is fused to an Fc antibody fragment. In additional embodiments, the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane-associated domain; (iii) human serum albumin or a fragment thereof; (iv) AFP or a fragment thereof; (v) AFP p26 or a fragment thereof; (vi) an extracellular domain of a receptor or a fragment thereof; and (vii) an extracellular domain of an intracellular receptor (e.g., a nuclear protein) or a fragment thereof. In some embodiments, DDpp comprises a heterologous polypeptide comprising the extracellular domain or a fragment of the extracellular domain of a cell surface receptor.

[0164] In some embodiments, the DDpp of the DDpp fusion protein is incorporated into a larger multidomain molecular complex (e.g., a monomeric or multimeric DDpp fusion protein), thereby conferring the functional properties of the incorporated DDpp to the resulting fusion protein. In some embodiments, the DDpp fusion protein comprises DDpp and a polypeptide sequence derived from an antibody, an antibody fragment, a serum protein (e.g., human serum albumin) or serum protein fragment, or a cell surface receptor, the alpha chain of a T cell receptor (TCR), the beta chain of a T cell receptor, a cytokine, a growth factor, a hormone, or an enzyme, or a fragment thereof. Incorporation of a DD into a multidomain and / or multifunctional complex can be routinely achieved by recombinant fusion to another polypeptide, conjugation to another chemical moiety, and covalent chemical linkage to another polypeptide (or other desired chemical compound) using techniques known in the art. The DDpp fusion protein can additionally contain other optional components, such as linkers and other components described herein. B. Adapter

[0165] In some embodiments, the DDpp fusion proteins described herein are adapter proteins. The adapters comprise an antigenic determinant (AD) and an antigenic determinant binding domain (ADBD). The adapters can further comprise additional ADs, additional ADBDs, and / or other additional domains. In some embodiments, the adapters provided herein comprise at least one ADBD that comprises a D domain.

[0166] In some embodiments, the adaptor provided herein comprises (a) a D domain (DD) that binds to CS1 and (b) an antigenic determinant (AD). In some embodiments, the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the adaptor is a monovalent adaptor comprising a single D domain that binds to CS1. In some embodiments, the adaptor is a bivalent adaptor comprising two D domains that bind to CS1. In some embodiments, the two D domains that bind to CS1 are the same. In some embodiments, the two D domains that bind to CS1 are different. In some embodiments, the adaptor is a bivalent adaptor comprising a first D domain that binds to CS1 and a second D domain that binds to a second AD. In some embodiments, the second AD is BCMA. In some embodiments, the monovalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the monovalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the bivalent adaptor comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the bivalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the bivalent adaptor comprises two identical D domains comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the bivalent adaptor comprises two identical D domains comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the adaptor comprises an AFP p26 antigenic determinant (AD). In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14 and 15. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 11.In some embodiments, the monovalent adaptor comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the monovalent adaptor comprises the amino acid sequence of SEQ ID NO: 110. In some embodiments, the bivalent adaptor comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the monovalent adaptor comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the adaptor comprises one or more linkers. In some embodiments, the adaptor can direct an immune response to cells expressing CS1 in an in vitro assay comprising the adaptor, cells expressing CS1, and immune effector cells expressing a CAR comprising an ADBD that binds to the AD included in the adaptor, e.g., a CAR comprising a D domain that binds to AFP p26.

[0167] In some embodiments, the adaptor provided herein comprises (a) a D domain that binds to CS1 and (b) an antigenic determinant binding domain (ADBD) that binds to AFP p26 AD. In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14, and 15. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105, and 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD. In some embodiments, the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the adapter comprises one or more linkers. In some embodiments, the adapter can direct an immune response to cells expressing CS1 in an in vitro assay comprising the adapter, cells expressing CS1, and immune effector cells expressing a CAR comprising AFP p26 AD.

[0168] In the adaptors provided herein, the AD may be at the N-terminus of the ADBD. Alternatively, the ADBD may be at the N-terminus of the AD. In some embodiments, the AD and the ADBD are directly fused. In some embodiments, the AD and the ADBD are fused via a linker (a protein linker or a chemical linker) or another protein domain (e.g., a functional domain). In some embodiments, the adaptors provided herein comprise at least one ADBD comprising a D domain.

[0169] In some embodiments, the adapter comprises a linker positioned between the ADBD of the adapter and another functional domain. In some embodiments, the linker is positioned between the two ADBDs of the adapter. In some embodiments, the linker is positioned between the AD and ADBD of the adapter. Suitable linkers for coupling two or more functional domains of the adapter will be apparent to those skilled in the art and may generally be any linker used in the art to link peptides, proteins, or other organic molecules. Exemplary linkers are provided herein. In certain embodiments, the linker is suitable for constructing proteins or polypeptides intended for pharmaceutical use. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain.

[0170] In addition to an AD (or multiple ADs) and an ADBD (or multiple ADBDs), the adapters provided herein can further comprise additional domain(s), e.g., a domain that confers extended half-life.

[0171] In some embodiments, the adaptor, or the ADBD in the adaptor, is deimmunized.

[0172] The adapters provided herein have uses including, but not limited to, diagnostic, analytical, and therapeutic applications. In certain embodiments, the adapters are used in combination with chimeric antigen receptors (CARs) provided herein expressed on the surface of cells, for example, to kill target cells.

[0173] Suitable adaptors for use in connection with the DDpps (e.g., adaptors and CARs) disclosed herein are disclosed in International Application Publication Nos. WO2016164305, WO2016164308A1, WO2019099440 and WO2019099433, U.S. Patent Nos. 10,662,248 and 10,647,775, and U.S. Patent Application Nos. 20200223934 and 20210002381, each of which is incorporated herein by reference for all purposes. i. Antigenic determinant (AD)

[0174] The adapters provided herein comprise at least one antigenic determinant (AD). In some embodiments, the adapter comprises a single AD. In some embodiments, the adapter comprises two or more ADs. When the adapter comprises two or more ADs, the ADs can be the same or different.

[0175] In some embodiments, the AD is an epitope of AFP p26. In further embodiments, the AD comprises 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 8. In further embodiments, the AD comprises the amino acid sequence of SEQ ID NO: 8. In further embodiments, the AD comprises 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 10. In further embodiments, the AD comprises the amino acid sequence of SEQ ID NO: 10. In further embodiments, the AD comprises amino acid residues of SEQ ID NO: 8-15 or 16.

[0176] In some embodiments, the adaptor comprises the extracellular domain of BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO: 2). In some embodiments, the adaptor comprises between 5 and 25, between 5 and 50, between 75, between 5 and 100, between 5 and 125, or between 5 and 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 2.

[0177] In some embodiments, the adaptor comprises the extracellular domain of CS1 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1). In some embodiments, the adaptor comprises between 5 and 25, between 5 and 50, between 5 and 75, between 5 and 100, between 5 and 125, or between 5 and 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 1.

[0178] In some embodiments, the adaptor comprises the extracellular domain of CD19 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 95). In some embodiments, the adaptor comprises between 5 and 25, between 5 and 50, between 5 and 75, between 5 and 100, between 5 and 125, or between 5 and 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO: 95.

[0179] In some embodiments, the AD is an epitope of AFP. In further embodiments, the AD comprises 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of SEQ ID NO:7.

[0180] In some embodiments, the adaptor comprises a p26 protein (e.g., having a sequence of SEQ ID NO: 8-15 or 16). In some embodiments, the adaptor has an in vivo plasma half-life of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or longer. In some embodiments, the adaptor has an in vivo plasma half-life in mice of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or longer than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours. In some embodiments, the adapter has an in vivo plasma half-life in humans of at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 16 hours, at least 32 hours, at least 64 hours, or greater than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours.

[0181] In some embodiments, the present disclosure provides methods for modifying the in vivo half-life (e.g., in mice or humans) of an adaptor comprising a p26 protein (e.g., having the sequence of SEQ ID NOs: 8-15 or 16). In some embodiments, the adaptor comprises one or more target-binding DDpps. In some embodiments, the half-life of the adaptor is increased or decreased by substituting or deleting one or more amino acid residues normally found in human p26 proteins, or by inserting one or more amino acid residues not normally found in human p26 proteins. In other embodiments, the p26 sequence of the adaptor is modified by substitution (conservative and / or non-conservative substitutions), deletion, and / or insertion of 1, 2, 3, 4, 5, 10, or 1-20, 1-10, 3-10, or 3-5 amino acids to increase or decrease the in vivo half-life of the adaptor. In certain embodiments, the amino acid residue corresponding to glutamine (Gln, Q) at position 217 of p26 (SEQ ID NO: 8) is substituted with another amino acid residue. In a further embodiment, the substitution is Gln217Pro. In another embodiment, the p26 sequence of the adapter is modified by the deletion of 1 to 150, 1 to 100, 1 to 50, 1 to 25, or 1 to 10 amino acid residues to increase or decrease the in vivo half-life of the adapter. In additional embodiments, the p26 sequence of the adapter is modified by the substitution (conservative and / or non-conservative substitutions), deletion, and / or insertion of 1, 2, 3, 4, 5, 10, or 1 to 20, 1 to 10, 3 to 10, or 3 to 5 amino acids to increase or decrease the interaction of the adapter with FcRn.

[0182] In some embodiments, the AD (e.g., in the adapter and / or on the target cell) is an AD present in a naturally occurring protein or other molecule. In some embodiments, the AD is an AD that is endogenous to humans.

[0183] In some embodiments, the AD is an epitope of a human intracellular protein. In further embodiments, the AD is an epitope of a human intracellular protein selected from Tyk2, Jak1, Jak2, Jak3, LCK, ZAP-70, and GRB2. In further embodiments, the AD includes 5 to 25, 5 to 50, 5 to 75, 5 to 100, 5 to 125, or 5 to 150 amino acid residues, more than 150 amino acid residues, or all of the amino acid residues of the intracellular protein.

[0184] In some embodiments, the target of interest to which the ADBD of an adaptor specifically binds is itself the AD of another adaptor having a different sequence. ii. Antigenic determinant binding domain (ADBD)

[0185] The adapters provided herein comprise at least one antigenic determinant binding domain (ADBD). In some embodiments, the adapter contains one ADBD. In some embodiments, the adapter contains at least two, three, four, or five, or more than five ADBDs. In some embodiments, the adapter contains one to three, one to four, one to five, or more than five different ADBDs. In some embodiments, the adapter contains at least two, three, four, or five, or more than five different ADBDs. As such, the adapter can comprise a monomeric ADBD (i.e., containing one antigenic determinant binding domain) or a multimeric ADBD (i.e., containing two or more antigenic determinant binding domains in tandem, optionally operably connected by a linker). In some embodiments, the use of multimeric adapters provides enhanced (e.g., synergistic) target binding. In additional embodiments, the use of multimeric adapters allows for targeting of two or more targets using a single adapter construct (e.g., bispecific, trispecific, etc.). In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0186] Multimeric adapters can be homomultimeric (i.e., containing two or more identical ADBDs, optionally connected by a linker) (e.g., homodimers, homotrimers, homotetramers, etc.) or adapter heteromultimeric (i.e., containing two or more antigenic determinant binding domains, where at least two different antigenic determinant binding domains are present). The number of ADBDs contained in any particular adapter can vary depending on the embodiment and can be defined, at least in part, by the expression system in which the adapter is produced. In some embodiments, however, the fusion protein can comprise a multimer of about 5 to about 10 ADBDs, about 10 to about 15 ADBDs, about 15 to about 20 ADBDs, about 20 to about 25 ADBDs, or about 25 to about 30 ADBDs (including numbers and endpoints between the listed ones). Also, the multiple domains of an adapter can contain the same or different ADBDs. In some embodiments, two, three, four, five, or more than five domains are in tandem. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0187] In one embodiment, the adapter comprises two or more operably linked ADBDs. In one embodiment, the adapter comprises two ADBDs that bind to the same or different ADs on the target antigen. Linking two or more identical ADBDs that bind to the same target antigen results in a multivalent molecule that offers distinct advantages (e.g., increased binding avidity, target clustering, and receptor activation) over compositions containing only one ADBD for a target antigen. In another embodiment, the adapter comprises two ADBDs that bind to different antigens. In some embodiments, the adapter comprises two ADBDs that bind to different antigens on the same cell. In some embodiments, the adapter comprises two ADBDs that bind to different antigens on different cells. Linking two or more ADBDs results in a multivalent and multispecific adapter that has the potential to bind to two or more target antigens, either independently or simultaneously. In some embodiments, the multivalent adapter can simultaneously bind to the same target antigen. In some embodiments, the multivalent adapter can simultaneously bind to different target antigens. In some embodiments, the adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5 to 500, 5 to 400, 10 to 300, 5 to 200, 50 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 75, 100 to 500, 100 to 400, 100 to 300, 100 to 200, or 100 to 150 amino acids in length. In a further embodiment, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant, hi a further embodiment, the adapter comprises two or more operably linked ADBDs separated by an AFP P26 antigenic determinant.In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0188] The ADBD in the adapters provided herein can bind to any AD. In some embodiments, the ADBD binds to CS1 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1). In some embodiments, the ADBD binds to AFP p26 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 8-15 or 16, preferably SEQ ID NO: 8 or 9). In some embodiments, the ADBD binds to BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO: 2). In some embodiments, the ADBD binds to BCMA and comprises a BCMA-binding DD, optionally comprising the amino acid sequence of SEQ ID NO: 25-39 or 40, optionally comprising the amino acid sequence of SEQ ID NO: 34, 35, or 39. In some embodiments, the ADBD binds to CD3. Adapters can be "monospecific" or "multispecific." Adapters that are "multispecific" (e.g., bispecific, trispecific, or higher order multispecific) recognize and bind to two or more different epitopes present on one or more different molecules. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0189] In some embodiments, the adaptor comprises a domain (e.g., the extracellular domain) of CS1 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1). In some embodiments, the adaptor comprises a domain (e.g., the extracellular domain) of BCMA (e.g., a polypeptide comprising the sequence of SEQ ID NO: 2). In some embodiments, the adaptor comprises a fragment of a domain. In some embodiments, the adaptor comprises a fragment of a domain that is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5 to 500, 5 to 400, 10 to 300, 5 to 200, 50 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, 10 to 50, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100, 50 to 75, 100 to 500, 100 to 400, 100 to 300, 100 to 200, or 100 to 150 amino acids in length. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0190] In some embodiments, the adapter contains at least two ADBDs that bind and bridge one or more target antigens and / or complexes containing the target antigens to which the ADBDs bind. In some embodiments, the bridged antigens are on the same cell. In some embodiments, the bridged antigens are on different cells. In some embodiments, the adapter comprises two or more operably linked ADBDs separated by an antigenic determinant (e.g., a domain described above). In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CS1 antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP p26 antigenic determinant. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0191] In some embodiments, the adapter contains at least two of the same ADBDs (i.e., is multivalent). In some embodiments, the multivalent adapter can simultaneously bind to two or more of the same target antigens. In some embodiments, the adapter is multivalent and can simultaneously bind to the same target antigen. In some embodiments, the multi-multivalent adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CS1 antigenic determinant. In further embodiments, the multivalent adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the multivalent adapter comprises two or more operably linked ADBDs separated by an AFP p26 antigenic determinant. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0192] In some embodiments, the adapter contains at least two ADBDs that bind to different antigens (i.e., is multispecific). In some embodiments, the multispecific adapter can simultaneously bind to different target antigens. In some embodiments, the adapter is also multivalent and can simultaneously bind to the same target antigen. In some embodiments, the multispecific adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CS1 antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP p26 antigenic determinant. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0193] In one embodiment, the multispecific adapter contains at least two ADBDs that bind to at least two different epitopes on a single target of interest (e.g., CS1). In additional embodiments, the multispecific adapter comprises at least one ADBD that specifically binds to one epitope on the target of interest and at least one other ADBD that specifically binds to a different epitope on the same target antigen. In one embodiment, the multispecific adapter comprises at least one ADBD that specifically binds to an epitope on a first target antigen and at least one ADBD that specifically binds to an epitope on a second antigen. In some embodiments, the adapter comprises at least one ADBD that specifically binds to an epitope on a first target antigen on a cell and at least one ADBD that specifically binds to an epitope on a second antigen on the same cell. In some embodiments, the adapter comprises at least one ADBD that specifically binds to an epitope on a first target antigen on a cell and at least one ADBD that specifically binds to an epitope on a second antigen on a different cell. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0194] In further embodiments, the adapter comprises two or more ADBDs operably linked to other heterologous proteins (or subdomains thereof), thereby conferring the multivalency, multispecificity, and / or functional properties (e.g., increased pharmacokinetics, e.g., half-life or pharmacodynamics, e.g., increased function) of the fusion partner to the adapter fusion protein. Examples of adapter fusion partners include, but are not limited to, antibodies, antibody subdomains (e.g., scFv or Fc domains), serum albumin, serum albumin subdomains, cell surface receptors, T cell receptor (TCR) alpha chains, T cell receptor beta chains, cell surface receptor subdomains, peptides, peptide tags (e.g., FLAG or myc). The number and location of ADBDs and their respective positions within the adapter can vary. For example, ADBDs can be located at one or all termini of the fusion partner and / or interspersed within heterologous subunits within the adapter fusion partner. In some embodiments, the adapter comprises two or more ADBDs separated by a heterologous protein (e.g., an antigenic determinant). In some embodiments, the heterologous protein is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the heterologous protein is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs that comprise a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0195] In one embodiment, the adapter is bispecific and contains an ADBD that specifically binds to two different target antigens. In a further embodiment, the bispecific adapter specifically binds to two different target antigens expressed on the surface of two different cell types. In a further embodiment, the bispecific adapter specifically binds to two different target antigens expressed on the surface of a tumor cell. In a further embodiment, the bispecific adapter specifically binds to two different target antigens (e.g., CS1 and BCMA) expressed on the surface of a myeloma cell. In some embodiments, the bispecific adapter binds to CS1 and BCMA and comprises a BCMA-binding DD, optionally wherein the BCMA-binding DD comprises the amino acid sequence of SEQ ID NOs: 25-39 or 40, and optionally wherein the BCMA-binding DD comprises the amino acid sequence of SEQ ID NO: 34, 35, or 39. In some embodiments, the bispecific adapter further comprises a CS1-binding DD. In one embodiment, the bispecific adapter binds to target antigens expressed on different cells. In a further embodiment, the bispecific adapter binds to target antigens expressed on different cells of a tumor. In another embodiment, the bispecific adapter binds to target antigens expressed on different cells within the tumor vasculature or tumor microenvironment. In one embodiment, the bispecific adapter specifically binds to a cancer cell target and an immune effector cell target. In one embodiment, the bispecific adapter specifically binds to a target expressed on a cancer cell (e.g., CS1) and a target expressed on the surface of a T lymphocyte (e.g., CD3). In some embodiments, the bispecific adapter can simultaneously bind to different target antigens. In some embodiments, the bispecific adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CS1 antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP P26 antigenic determinant. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0196] In some embodiments, an adapter comprises two or more ADBDs, the ADBDs can be any of the types of ADBDs discussed herein. For example, the ADBDs can be an antibody, an antigen-binding fragment thereof, an ScFv, an alternative scaffold binding domain, a D domain, a T cell receptor, or an antigen-binding fragment thereof. In some embodiments, an adapter provided herein comprises at least one ADBD comprising a D domain. In some embodiments, an adapter provided herein comprises two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of an adapter provided herein comprise a D domain.

[0197] In some embodiments, when an adapter comprises two or more ADBDs, these ADBDs can be the same type of antigen-binding molecule or can be different. For example, an adapter can comprise two ADBDs that are D domains. The two ADBDs that are D domains can be the same or different. An adapter can also comprise an ADBD that is a D domain and an ADBD that is an scFv. An adapter can also comprise an ADBD that is a T cell receptor or its antigen-binding fragment, and an ADBD that is an scFv. In some embodiments, an adapter comprises two or more operably linked ADBDs separated by an antigenic determinant. In some embodiments, the antigenic determinant is at least 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length. In some embodiments, the antigenic determinant is 5-500, 5-400, 10-300, 5-200, 50-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, 10-50, 50-500, 50-400, 50-300, 50-200, 50-100, 50-75, 100-500, 100-400, 100-300, 100-200, or 100-150 amino acids in length. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a CS1 antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by a BCMA antigenic determinant. In further embodiments, the adapter comprises two or more operably linked ADBDs separated by an AFP P26 antigenic determinant. In some embodiments, the adapters provided herein comprise at least one ADBD comprising a D domain. In some embodiments, the adapters provided herein comprise two or more ADBDs comprising a D domain. In some embodiments, all of the ADBDs of the adapters provided herein comprise a D domain.

[0198] In some embodiments, the ADBD of the adaptor is deimmunized. In some embodiments, the ADBD of the adaptor is deimmunized by replacing one or more amino acid residues in a predicted T cell epitope to reduce binding to a host MHC molecule.

[0199] In some embodiments, the adapter comprises an ABDB that binds to an antigen target containing an AD of interest and has no discernible effect on the function of the target. Alternatively, in some embodiments, the adapter comprises an ADBD that binds to an antigen target containing an AD of interest and completely or partially inhibits, antagonizes, agonizes, blocks, increases, stimulates, or prevents the biological activity of the target. Binding can be identified as agonistic or antagonistic and can be determined using, or by routinely modifying, assays, bioassays, and / or animal models known in the art for assessing such activity.

[0200] An adapter agonist refers to an adapter that, in some ways, increases or enhances the biological activity of the adapter target, or has biological activity equivalent to a known agonist of the adapter target. In another embodiment, the adapter is an antagonist of the target to which it binds. An adapter antagonist refers to an adapter that completely or partially blocks, or in some ways prevents, the biological activity of the adapter target, or has biological activity equivalent to a known antagonist or inhibitor of the adapter target.

[0201] In one embodiment, the adaptor specifically binds to a target of interest that is a serum protein. In one embodiment, the adaptor specifically binds to a serum protein selected from serum albumin (e.g., human serum albumin (HSA)), thyroxine-binding protein, transferrin, fibrinogen, and immunoglobulins (e.g., IgG, IgE, and IgM). Without being bound by theory, it is believed that binding of the adaptor to a carrier protein confers an improved pharmacodynamic profile to the adaptor, including, but not limited to, improved tumor targeting, tumor invasion, intratumoral diffusion, and enhanced therapeutic activity, compared to an adaptor lacking the carrier protein binding sequence (see, e.g., WO01 / 45746, the contents of which are incorporated herein by reference in their entirety). iii. Adaptor functional domains

[0202] In some embodiments, the adapter comprises a first antigenic determinant (AD), a second antigenic determinant-binding domain (ADBD), and further comprises a functional domain that confers one or more additional desirable properties (e.g., improved manufacturability) and / or pharmacokinetic or pharmacodynamic properties (e.g., improved half-life). The adapter's functional domain may be located between the AD and the ADBD. The adapter may also be located at the N-terminus of both the AD and the ADBD, or at the C-terminus of both the AD and the ADBD. In some embodiments, when the adapter comprises two or more ADs, the adapter's functional domain may be located between the two or more ADs, at the N-terminus of the two or more ADs, or at the C-terminus of the two or more ADs. In some embodiments, when the adapter comprises two or more ADBDs, the adapter's functional domain may be located between the two or more ADBDs, at the N-terminus of the two or more ADBDs, or at the C-terminus of the two or more ADBDs.

[0203] In some embodiments, the adapter comprises a functional domain selected from Fc or a variant Fc (e.g., a human Fc or a variant Fc domain) or a fragment thereof, a serum protein (e.g., human serum albumin) or a fragment thereof; an FcRn-binding domain; a serum protein-binding domain; a cytokine, a growth factor, a hormone, or an enzyme; an imaging agent; a labeling agent; and a peptide tag.

[0204] The functional domain of an adapter can be naturally derived or can be the result of recombinant engineering (e.g., phage display, xenomouse, or synthetic). In certain embodiments, the functional domain of an adapter enhances half-life, increases or decreases antibody-dependent cellular cytotoxicity (ADCC), and / or increases or decreases complement-dependent cytotoxicity (CDC) activity.

[0205] In some embodiments, the adapter comprises a functional domain selected from Fc or a variant Fc (e.g., a human Fc or a variant human Fc domain) or a fragment or derivative thereof, a serum protein (e.g., human serum albumin) or a fragment or derivative thereof (e.g., a serum protein binding domain); an FcRn binding domain; and a serum protein binding domain.

[0206] In one embodiment, the adaptor comprises a functional domain comprising an antibody effector domain or a derivative of an antibody effector domain that confers one or more effector functions to the adaptor, e.g., the ability to bind to one or more Fc receptors. In some embodiments, the functional domain comprises one or more CH2 and / or CH3 domains of an antibody having an effector function provided by the CH2 and CH3 domains. In some embodiments, the functional domain comprises one or more derivatives of the CH2 and / or CH3 domains of an antibody having an effector function provided by the CH2 and CH3 domains. Other sequences that can be included in an adaptor to provide effector function and are encompassed by the present invention will be apparent to those of skill in the art and can be routinely selected and designed to become an adaptor encompassed herein based on the desired effector function.

[0207] In one embodiment, the adaptor comprises a functional domain that increases antibody-dependent cellular cytotoxicity (ADCC) conferred by the adaptor (e.g., Bruhns et al., Blood 113: 3716-3725 (2009); Shields et al., J. Biol. Chem. 276: 6591-6604 (2001); Lazar et al., PNAS 103: 4005-4010 (2006); Stavenhagen et al., Cancer Res. 67: 8882-8890 (2007); Horton et al., Cancer Res. 68: 8049-8057 (2008); Zalevsky et al., Blood 113: 3735-3743 (2009); Bruckheimer, Neoplasia 11: 509-517 (2009); WO2006 / 020114; Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and WO2004 / 074455, each of which is incorporated herein by reference in its entirety. Examples of modifications that manipulate fragments of the Fc portion that confer effector function, contained in the functional domain of the adaptor that increase ADCC, include one or more modifications corresponding to IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L, where the numbering of residues in the Fc region corresponds to Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, The figures are from the EU Index (1991 Fifth edition).

[0208] Thus, in some embodiments, the adapter comprises a functional domain comprising an antibody fragment that confers the biological or biochemical properties of an immunoglobulin to the adapter. In some embodiments, the antibody fragment confers a property selected from the ability to non-covalently dimerize, the ability to localize at tumor sites, and increased serum half-life when compared to the adapter without the antibody fragment. In certain embodiments, the adapter is at least as stable as the corresponding antibody fragment without the adapter. In certain embodiments, the adapter is more stable than the corresponding antibody fragment without the adapter. The stability of the adapter protein can be measured using established methods, including, for example, ELISA techniques. In some embodiments, the adapter is stable in whole blood (in vivo or ex vivo) at 37° C. for at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 48 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 65 hours, at least about 70 hours, at least about 72 hours, at least about 75 hours, at least about 80 hours, at least about 85 hours, at least about 90 hours, at least about 95 hours, or at least about 100 hours (including any time in between those listed). In one embodiment, the adapter contains an immunoglobulin effector domain or half-life-affecting domain corresponding to an immunoglobulin domain or fragment, wherein at least a small portion of one or more of the constant region domains has been altered to provide a desired biological property, such as reduced or increased effector function, ability to non-covalently dimerize, increased ability to localize at tumor sites, reduced serum half-life, or increased serum half-life, when compared to an immunoglobulin fragment having a corresponding unaltered immunoglobulin sequence. These constant region domain alterations can be amino acid substitutions, insertions, or deletions.

[0209] In one embodiment, the adaptor comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers antibody-dependent cellular cytotoxicity (ADCC) to the adaptor. In additional embodiments, the adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase ADCC (e.g., Bruhns, Blood 113: 3716-3725 (2009); Shields, J. Biol. Chem. 276: 6591-6604 (2001); Lazar, PNAS 103: 4005-4010 (2006); Stavenhagen, Cancer Res. 67: 8882-8890 (2007); Horton, Cancer Res. 68: 8049-8057 (2008); Zalevsky, Blood 113: 3735-3743 (2009); Bruckheimer, Neoplasia 11: 509-517 (2009); WO06 / 020114; Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and WO04 / 074455, the contents of each of which are incorporated herein by reference in their entireties.Examples of modifications to engineer immunoglobulin fragments contained in amino acid sequences in the adaptor that increase ADCC include immunoglobulin effector domain sequences having one or more modifications corresponding to IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L, where the numbering of residues in the Fc region is as described in Kabat et al. (Kabat et al., Sequences of The information is from the EU Index of Proteins of Immunological Interest, 1991 Fifth edition.

[0210] In additional embodiments, the adaptor comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers antibody-dependent cellular phagocytosis (ADCP) to the adaptor. In additional embodiments, the adapter comprises a sequence of an immunoglobulin effector domain that has been modified to increase antibody-dependent cellular phagocytosis (ADCP) (e.g., Shields et al., J. Biol. Chem. 276: 6591-6604 (2001); Lazar et al., PNAS 103: 4005-4010 (2006); Stavenhagen et al., Cancer Res. 67: 8882-8890 (2007); Richards et al., Mol. Cancer Ther. 7: 2517-2527 (2008); Horton et al., Cancer Res. 68: 8049-8057 (2008); Zalevsky et al., Blood 113: 3735-3743 (2009); Bruckheimer et al., Neoplasia 11: 509-517 (2009); WO06 / 020114; Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and WO04 / 074455, the contents of each of which are incorporated herein by reference in their entireties.Examples of modifications that manipulate immunoglobulin fragments contained in the amino acid sequence in the adaptor that increase ADCP include IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, IgG1-F243L, R292P, Y300L, P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; and IgG1-G236A, S239D, I332E, where the numbering of the residues is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

[0211] In additional embodiments, the adaptor comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers complement-dependent cytotoxicity (CDC) to the adaptor. In additional embodiments, the adaptor comprises the sequence of an immunoglobulin effector domain that has been modified to increase complement-dependent cytotoxicity (CDC) (see, e.g., Idusogie et al., J. Immunol. 166: 2571-2575 (2001); Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Natsume et al., Cancer Res. 68: 3863-3872 (2008), the contents of each of which are incorporated by reference in their entirety). By way of example, the adapter may contain an antibody fragment or domain containing one or more of the following modifications that increase CDC: IgG1-K326A, E333A; IgG1-K326W, E333S, IgG2-E333S, where the residue numbering is that of the EU index of Kabat et al. (Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

[0212] In a further embodiment, the adapter comprises a functional domain comprising the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain, which confers to the adapter the ability to bind to the Fc gamma RIIb receptor. In a further embodiment, the adapter comprises the sequence of an immunoglobulin effector domain that has been modified to increase inhibitory binding to the Fc gamma RIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45: 3926-3933 (2008)). An example of a modification engineered immunoglobulin fragment contained in the amino acid sequence of the adapter that increases binding to the inhibitory Fc gamma RIIb receptor is IgG1-S267E, L328F.

[0213] The half-life of IgG is mediated by its pH-dependent binding to the fetal receptor FcRn. In certain embodiments, the adaptor contains a functional domain comprising the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers on the adaptor the ability to bind to the fetal receptor FcRn. In certain embodiments, the adapter contains a functional domain comprising the sequence of an immunoglobulin FcRn-binding domain that has been modified to enhance binding to FcRn (e.g., Petkova et al., Int. Immunol. 18: 1759-1769 (2006); Dall'Acqua et al., J. Immunol.169: 5171-5180 (2002); Oganesyan et al., Mol. Immunol. 46: 1750-1755 (2009); Dall'Acqua et al., J. Biol. Chem. 281: 23514-23524 (2006); Hinton et al., J. Immunol. 176: 346-356 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35: 86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282: 1709-1717 (2007); WO06 / 130834; Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Yeung et al., J. Immunol. 182: 7663-7671 (2009), the contents of each of which are incorporated herein by reference in their entireties.

[0214] In additional embodiments, the adapter comprises a functional domain comprising the sequence of an immunoglobulin effector domain that has been modified to have selective affinity for FcRn at pH 6.0 but not at pH 7.4. By way of example, the adapter functional domain can contain an antibody fragment or domain containing one or more of the following half-life-increasing modifications: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L; IgG1-H433K, N434Y; IgG1-N434A; and IgG1-T307A, E380A, N434A, where the residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference).

[0215] According to another embodiment, the adapter is 238, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, and a functional domain comprising an amino acid sequence corresponding to an immunoglobulin effector domain, modified to contain at least one substitution in its sequence corresponding to a position in the Fc region (e.g., Fc gamma) selected from 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439, wherein the numbering of residues in the Fc region is according to the EU numbering system of Kabat et al. (Sequences of proteins of Immunological Interest, 1991 Fifth edition, incorporated herein by reference). In a specific embodiment, the adapter contains a functional domain comprising the sequence of an immunoglobulin effector domain derivative, wherein at least one residue corresponding to position 434 is a residue selected from A, W, Y, F, and H. According to another embodiment, the adapter comprises the sequence of an immunoglobulin effector fragment derivative having the following respective substitutions: S298A / E333A / K334A. In an additional embodiment, the adapter comprises an immunoglobulin effector domain derivative having a substitution corresponding to K322A. In another embodiment, the adapter comprises the sequence of an immunoglobulin effector domain derivative having one or any combination of the following substitutions: K246H, H268D, E283L, S324G, S239D, and I332E. According to yet another embodiment, the adapter comprises the sequence of an immunoglobulin effector domain derivative having substitutions corresponding to D265A / N297A.

[0216] In certain embodiments, the adapter comprises a functional domain comprising the sequence of an immunoglobulin effector domain that has been glycoengineered or mutated to increase effector function using techniques known in the art. For example, inactivation (by point mutation or other means) of the constant region domain sequence contained in the adapter can reduce Fc receptor binding of the circulating adapter, thereby increasing tumor localization. In other cases, constant region modifications consistent with certain embodiments of the present invention may alleviate complement binding, thus reducing serum half-life and nonspecific association of conjugated cytotoxins. Still other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability, and other biochemical effects of the modifications, such as tumor localization, biodistribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0217] Adapters useful for practicing the provided methods can be produced using a variety of standard techniques for chemical synthesis, semi-synthesis, and recombinant DNA methodologies known in the art. In some embodiments, the overall production scheme for producing an adapter involves obtaining a reference protein scaffold and identifying a number of residues within the scaffold for modification. Depending on the embodiment, the reference scaffold may include one or more alpha-helical regions or protein structures with other tertiary structures. Once identified, the number of residues can be modified, for example, by amino acid substitution. In some embodiments, the substitutions are conservative, while in other embodiments, non-conservative substitutions are made. In some embodiments, a natural amino acid (e.g., one of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine) is substituted into the reference scaffold at the target position for modification. In certain embodiments, the modifications do not involve substituting either cysteine ​​or proline. After modifications have been made at all specified positions desired in a particular embodiment, the resulting modified polypeptides (e.g., candidate adapters) can be recombinantly expressed, for example, in a plasmid, bacteria, phage, or other vector (e.g., to increase the number of each modified polypeptide). The modified polypeptides can then be purified and screened to identify modified polypeptides with specific binding to a particular target of interest. In some embodiments, certain modified polypeptides exhibit enhanced binding specificity for a target of interest relative to a reference scaffold, which in some embodiments may exhibit little or no binding to a given target of interest.In additional embodiments, depending on the target of interest, the reference scaffold may exhibit some interaction (e.g., non-specific interaction) with the target of interest, but certain modified polypeptides exhibit an increase in binding specificity for the target of interest of at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 50-fold, or at least about 100-fold (or higher). If desired, the reference sequence and / or modified polypeptide (e.g., adapter) can be deimmunized. For example, potentially immunogenic residues or motifs can be identified and modified to reduce or eliminate potential immune responses to the adapter. Additional details regarding various embodiments of adapter production, selection, and isolation are provided in more detail below. [Table 2-1] [Table 2-2]

[0218] In some embodiments, the present disclosure provides compositions comprising one or more of the adaptors disclosed in Table 2. In other embodiments, the present disclosure provides compositions comprising one or more adaptors comprising sequences having 60-70%, 70-75%, 75-80%, 80-85%, 85-90%, 95-99% homology (and overlapping ranges) to a sequence disclosed in Table 2. In some embodiments, adaptors with such homology are functionally similar or identical compared to their respective reference sequences in Table 2. In some embodiments, the present disclosure provides polypeptides comprising one or more adaptors that compete (in whole or in part) with one or more of the adaptors (reference sequences) disclosed in Table 2 for CS1 binding. The ability of a polypeptide to compete with a reference polypeptide for binding to its respective target can be routinely determined using standard competition assays known in the art. In some embodiments, competition does not require the adaptor to compete for the same epitope as the adaptor in Table 2; rather, the polypeptide can compete by binding to a sterically hindering epitope, an overlapping epitope, etc. C. Chimeric Antigen Receptor

[0219] Also provided herein is a chimeric antigen receptor (CAR) comprising an extracellular domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR disclosed herein comprises an extracellular domain comprising at least one D domain (DD) disclosed herein to confer binding specificity. CAR can be expressed by any cell type.

[0220] In some embodiments, a chimeric antigen receptor (CAR) disclosed herein comprises a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain. In some embodiments, the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 108.

[0221] In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising at least one D domain (DD). In some embodiments, the D domain can recognize an antigenic determinant (AD) (e.g., CS1) on a target cell. In some embodiments, a CAR comprising a DD capable of binding to an AD on a target cell can be expressed on the surface of a cell (e.g., an immune cell or immune effector cell) and used, for example, to kill target cells expressing an AD. In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising at least one CS1-binding D domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 60.

[0222] In some embodiments, a CAR disclosed herein comprises an extracellular domain comprising an ADBD. In some embodiments, the ADBD is composed, at least in part, of a target-binding polypeptide (e.g., a D domain) disclosed herein. In some embodiments, the ADBD can recognize an antigenic determinant (AD) (e.g., CS1) on a target cell or an AD contained in an adaptor (e.g., p26). In some embodiments, a CAR comprising an ADBD capable of binding to an AD on a target cell can be expressed on the surface of a cell (e.g., an immune cell or immune effector cell) and used, for example, to kill target cells expressing an AD. In some embodiments, a CAR comprising a first ADBD capable of binding to a first AD (e.g., p26) can be expressed on the surface of a cell (e.g., an immune cell or immune effector cell) and used in combination with an adaptor comprising the first AD and a second ADBD (e.g., a D domain) that binds to a second AD on the target cell, for example, to kill target cells. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NO: 8-15 or 16. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, p26 comprises the amino acid sequence of SEQ ID NO:10.

[0223] The present invention further provides a means for producing cell-associated DDpps composed of at least one DDpp designed to confer binding specificity to the membrane-bound fusion protein. The DDpp receptor can be expressed by any cell type. In one embodiment, the DDpp-receptor fusion protein comprises a chimeric antigen receptor (CAR), or DDpp-CAR, which comprises an extracellular targeting domain, a transmembrane domain, and an intracellular signaling domain. In another embodiment, the DDpp-CAR is composed of an extracellular targeting domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain comprises the signaling domain. In a further embodiment, the DDpp-CAR extracellular domain comprises one or more DDpps, each of which constitutes a specific binding domain with the same or different specificity. In some embodiments, the target-specific domain is directed to one or more of the cancer or tumor antigens disclosed herein, such as, for example, non-limiting examples, CS1 and BCMA. In some embodiments, the target-specific domain is directed to AFP p26.

[0224] In some embodiments, the intracellular signaling domain or fragment thereof is selected from the group consisting of a human CD3 zeta domain, a 41BB domain, a CD28 domain, and any combination thereof. In some embodiments, the intracellular signaling domain contains the sequence of SEQ ID NO: 123, 124, or a combination thereof. In some embodiments, the intracellular signaling domain contains the sequence of SEQ ID NO: 125. Depending on the embodiment, the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFAl), CD2, CD7, LIGHT, NKG2C, B7H3, a ligand that specifically binds with CD83, and any combination thereof.

[0225] In some embodiments, the CAR comprises a fusion protein comprising an additional target-binding polypeptide.

[0226] In some embodiments, the ADBD of the CAR comprises at least one alternative scaffold binding domain (e.g., a D domain or an affibody) designed to confer binding specificity to the membrane-bound CAR. Receptors comprising alternative scaffold binding domains can be expressed by any cell type.

[0227] In one embodiment, a CAR is composed of the following elements: an extracellular domain, a transmembrane domain, and a cytoplasmic domain, wherein the cytoplasmic domain comprises a signaling domain. In another embodiment, a CAR is composed of an extracellular domain and a transmembrane domain. In a further embodiment, a CAR is composed of an extracellular domain composed of one or more ADBDs (e.g., D domains) with the same or different specificities. In one embodiment, the intracellular domain (e.g., cytoplasmic domain) of a CAR comprises the intracellular domain of a CD3 zeta chain. In another embodiment, the intracellular signaling domain of a CAR is composed of a portion of the intracellular domain of a CD3 zeta chain. In a further embodiment, the intracellular domain of a CAR comprises the intracellular domain of a CD3 zeta chain and a costimulatory signaling region. A costimulatory signaling region refers to a portion of a CAR that includes all or part of the intracellular domain of a costimulatory molecule. Costimulatory molecules and portions of these molecules that can confer costimulatory properties to a CAR are known in the art and can be routinely incorporated into CARs. In addition, truncations or mutations to these intracellular signaling and costimulatory domains may be incorporated to further enhance or reduce receptor signaling. In a preferred embodiment, the T cells are genetically modified to stably express the CAR. In such an embodiment, the cytoplasmic domain of the CAR can be designed to contain the CD28 and / or 41BB signaling domain by itself, or can be combined with any other desired cytoplasmic domain useful in the context of the present invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to further contain the signaling domain of CD3-zeta. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 123, 124, or a combination thereof. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 125. In one embodiment, the CAR comprises a cytoplasmic domain, an extracellular domain, an extracellular protein linker (e.g., found in T cells or NK cells) with a transmembrane domain that passes through the membrane of the cell, and optionally multiple signaling modules.In some embodiments, the CAR may also include an epitope tag. In some embodiments, the cytoplasmic domain of the CAR may include, but is not limited to, CD3-zeta, 41BB, and CD28 signaling modules, and combinations thereof. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 123, 124, or a combination thereof. In some embodiments, the cytoplasmic domain contains the sequence of SEQ ID NO: 125.

[0228] Also provided is an isolated nucleic acid sequence encoding a CAR that includes a target-binding polypeptide as part (or all) of its targeting region.

[0229] In some embodiments, the targeting domain of the CAR comprises multiple binding domains (e.g., a DD, or one or more DDs and scFvs) that comprise additional target-binding polypeptides.

[0230] The present disclosure also provides cells comprising a nucleic acid sequence encoding a CAR, wherein the CAR comprises, at least in part, an antigen-binding domain composed of the disclosed DDpp that binds to a target of interest (e.g., CS1), a transmembrane domain, and a signaling domain. In some embodiments, the CAR specifically binds to a tumor antigen (and thus functions to deliver CAR-expressing cells to tumors). In some embodiments, the tumor antigen is associated with a hematological malignancy. In some embodiments, the tumor antigen is CS1. In some embodiments, the CAR-expressing cells are T cells, natural killer (NK) cells, or other immune cell types. In some embodiments, the CAR-expressing cells (whether T cells, NK cells, or other cell types) exhibit anti-tumor immunity when the polypeptide binds to its corresponding tumor antigen. i. extracellular domain

[0231] The CARs provided herein comprise one or more antigenic determinant binding domains (ADBDs) (e.g., D domains) described herein. The ADBD of the CAR can be any ADBD (e.g., D domain) described herein.

[0232] Depending on the desired antigen to be targeted, the extracellular domain of the CAR can be engineered to contain one or more antigenic determinant binding domains (ADBDs) that specifically bind to the desired antigen target. For example, in one embodiment, a CAR is engineered to target CS1, and a CS1-binding ADBD (e.g., a D domain) is incorporated into the extracellular domain of the CAR. Alternatively, the extracellular domain of the CAR can contain two or more ADBDs, thereby conferring multispecificity or multivalency to the CAR.

[0233] In some embodiments, the ADBD binds to CS1 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 1). In some embodiments, the ADBD comprises a DD sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 60.

[0234] In some embodiments, the ADBD (e.g., the D domain) binds to AFP (e.g., a polypeptide comprising the sequence of SEQ ID NO: 7) or a fragment thereof. In some embodiments, the ADBD (e.g., the D domain) binds to AFP p26 (e.g., a polypeptide comprising the sequence of SEQ ID NO: 8-15 or 16). In some embodiments, the ADBD (e.g., the D domain) binds to a polypeptide comprising the sequence of SEQ ID NO: 8. In some embodiments, the DD binds to AFP p26 and comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the DD binds to AFP p26 and comprises the amino acid sequence of SEQ ID NO: 22.

[0235] In some embodiments, the CAR further comprises an ADBD that is an antibody or an antigen-binding fragment thereof. In some embodiments, the CAR comprises an ADBD that is an scFv. In some embodiments, the CAR comprises an ADBD that is an alternative scaffold binding domain. In some embodiments, the CAR comprises an ADBD that is a D domain. In some embodiments, the CAR comprises a T cell receptor or an antigen-binding fragment thereof.

[0236] Also provided herein are CARs that comprise multiple ADBDs. In some embodiments, the CARs comprise multiple identical ADBDs. In some embodiments, the CARs comprise multiple different ADBDs. In some embodiments, the CARs comprise multiple ADBDs that bind to the same antigenic determinant. In some embodiments, the CARs comprise multiple ADBDs, where the binding domains bind to different ADs. In some embodiments, the CARs comprise multiple ADBDs, where the binding domains bind to different ADs on the same cell. In some embodiments, the CARs comprise multiple ADBDs, where the binding domains bind to different ADs on different cells.

[0237] In some embodiments, a CAR comprises multiple ADBDs (e.g., D domains, affibodies, or scFvs), e.g., two, three, four, five, or more than five ADBDs, wherein each ADBD can bind to a target antigen. In one embodiment, two or more of the ADBDs of a CAR can bind to different ADs. In additional embodiments, two or more of the ADBDs of a CAR can bind to the same antigen, e.g., the same or different epitopes on the same antigen. In one embodiment, the multiple ADBDs of a CAR are linked to each other, e.g., the C-terminus of a first ADBD is linked to the N-terminus of a second ADBD. In certain embodiments, the C-terminus of a first ADBD is linked to the N-terminus of a second ADBD by a covalent bond, e.g., a peptide bond.

[0238] In some embodiments, a linker or hinge region is contained between one or more of the ADBDs; for example, the linker or hinge region is located between the C-terminus of the first ADBD and the N-terminus of the second ADBD. For example, an antigen binding member comprising two ADBDs (e.g., ADBD1 and ADBD2) can be arranged in the following configuration: [ADBD1]-[linker / hinge]-[ADBD2]. Additional ADBDs can be added in a similar manner, with a linker or hinge region located between the C-terminus of one ADBD and the N-terminus of the next ADBD, if necessary. Linkers or hinge regions suitable for use in linking multiple antigen binding members are flexible and non-cleavable, allowing each ADBD to move nearly freely independently of other ADBDs, facilitating simultaneous binding to multiple target ADs. Any flexible linker or hinge region known in the art can be used. Exemplary linkers include peptide linkers containing glycine and serine residues, such as (GGGGS)n, where n is a positive integer equal to or greater than 1, e.g., n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 20). In some embodiments, the peptide linker contains the sequence of SEQ ID NOs: 16-20, 115-118, or 119.

[0239] In some embodiments, the CAR comprises a CS1-binding DD. In some embodiments, the CAR comprises a BCMA-binding DD and a CS1-binding DD. In some embodiments, the CAR comprises a BCMA-binding DD and a CS1-binding DD, wherein the BCMA-binding DD comprises the amino acid sequence of SEQ ID NO: 25-39 or 40, and optionally the BCMA-binding DD comprises the amino acid sequence of SEQ ID NO: 34, 35, or 39.

[0240] In some embodiments, the CAR comprises two, three, four, five, or more than five DDs and / or other binding domains (e.g., scFvs) that specifically bind to a target of interest (e.g., CS1) expressed on the surface of a cancer cell. In additional embodiments, the CAR comprises two, three, four, five, or more than five DDs or other binding domains (e.g., scFvs) that specifically bind to a second, different target of interest expressed on the surface of a cancer cell. In additional embodiments, the administered CAR further comprises two, three, four, five, or more than five DDs or other binding domains (e.g., scFvs) that specifically bind to a second, different target of interest expressed by a second, different cancer cell or vascular endothelial cell. In some embodiments, the CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-binding DD comprises the amino acid sequence of SEQ ID NO: 60. ii. Extracellular spacer domain

[0241] In some embodiments, CAR comprises an extracellular spacer domain. As used herein, the term "extracellular spacer domain" or "ESD" refers to the polypeptide sequence of CAR located between the ADBD and the transmembrane domain. In some embodiments, the extracellular spacer domain allows sufficient distance from the outer surface of the cell and the ADBD, and flexibility to minimize steric hindrance between the cell and the ADBD.

[0242] In certain embodiments, the extracellular spacer domain is sufficiently short or flexible so as not to interfere with engagement of cells containing the CAR with cells bearing AD, e.g., target cells. In certain embodiments, the extracellular spacer domain is 2-20, 5-15, 7-12, or 8-10 amino acids in length. In some embodiments, the ESD domain comprises at least 50, 20, or 10 residues. In some embodiments, the ESD is 10-300, 10-250, or 10-200 residues in length.

[0243] In some embodiments, the distance the ESD extends from the cell is sufficiently short that the hinge does not interfere with the engagement of the CAR ADBD with the surface of the target cell. In some embodiments, the ESD extends less than 20, 15, or 10 nanometers from the surface of the cytotoxic cell. Therefore, the suitability of the ESD can be affected by the length of both linear chains, the number of amino acid residues, and the flexibility of the ESD. For example, an IgG4 ESD can be as long as 200 amino acids, but the distance it extends from the surface of the cytotoxic cell is smaller due to Ig domain folding. The CD8 alpha ESD is approximately 8 nm long and approximately 43 amino acids. In contrast, IgG4 C2 and C3 ESDs are approximately 200 amino acids long, but have a distance from the surface of the cytotoxic cell that is comparable to that of the CD8 alpha ESD. Without wishing to be bound by theory, the similarity of the extension is affected by flexibility.

[0244] In some embodiments, the extracellular spacer domain includes, but is not limited to, an Fc fragment of an antibody or a fragment or derivative thereof, a hinge region of an antibody or a fragment or derivative thereof, a CH2 region of an antibody, a CH3 region of an antibody, an artificial spacer sequence, or a combination thereof. Additional examples of extracellular spacer domains include, but are not limited to, a CD8a hinge and an artificial spacer composed of a polypeptide that can be as small as, for example, Gly3 or the CH1 and CH3 domains of an IgG (such as human IgG4). In some embodiments, the extracellular spacer domain is any one or more of: (i) the hinge, CH2, and CH3 regions of IgG4, (ii) the hinge region of IgG4, (iii) the hinge and CH2 of IgG4, (iv) the hinge region of CD8a, (v) the hinge, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vi) the hinge and CH2 regions of IgG1. Other extracellular spacer domains will be apparent to those of skill in the art and may be used in conjunction with the alternative embodiments provided herein.

[0245] In some embodiments, the ESD is a naturally occurring sequence. In some embodiments, the CAR ESD corresponds to an ESD derived from a human protein, a fragment thereof, or a short oligopeptide or polypeptide linker. In some embodiments, the CAR ESD corresponds to a human Ig (immunoglobulin) ESD (hinge), or a fragment thereof. In one embodiment, the ESD comprises (e.g., consists of) the amino acid sequence of an IgG4 ESD. In one embodiment, for example, the hinge comprises (e.g., consists of) the amino acid sequence of an IgD hinge. In some embodiments, the hinge can be a human CD8 hinge, or a fragment thereof. In one embodiment, for example, the hinge comprises (e.g., consists of) the amino acid sequence of a CD8 hinge.

[0246] In some embodiments, the ESD is an artificial sequence. In one embodiment, the ESD is a short oligopeptide linker containing a glycine-serine doublet.

[0247] In some embodiments, the CAR comprises a CD8a extracellular spacer domain. In some embodiments, the CD8a extracellular spacer domain comprises the amino acid sequence of SEQ ID NO: 120.

[0248] In some embodiments, the CAR does not contain an extracellular spacer domain. iii. Transmembrane domain

[0249] The term "transmembrane domain" (TMD), as used herein, refers to a region of a cell surface-expressed protein, e.g., a CAR, that spans the plasma membrane. In some embodiments, the TMD connects an extracellular sequence (e.g., an extracellular ADBD or an extracellular AD) and an intracellular sequence, e.g., an intracellular signaling domain. In some embodiments, the transmembrane domain of a CAR is a transmembrane region of a transmembrane protein (e.g., a type I transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. Other transmembrane domains will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present invention. In some embodiments, the extracellular ADBD is a CS1-binding ADBD (e.g., a D domain). In some embodiments, the extracellular ADBD comprises the amino acid sequence of SEQ ID NO: 46 or 60.

[0250] CARs can be designed to contain a transmembrane domain fused to the extracellular domain of a receptor. As described above, fusion of the extracellular and transmembrane domains can be achieved with or without a linker. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In a specific embodiment, the transmembrane domain in the CAR is a CD8a transmembrane domain. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 122. In some instances, the transmembrane domain of a CAR comprises a CD8a hinge domain. In some embodiments, the CD8a hinge domain, also referred to as the extracellular spacer domain (ESD), comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to promote or inhibit association with other surface membrane proteins.

[0251] The transmembrane domain may be naturally derived or synthetic. If the origin is natural, the domain may be derived from any membrane-bound or transmembrane protein. For purposes herein, the transmembrane region of particular use may be derived from (i.e., comprising at least the transmembrane region of) a member selected from the group consisting of the alpha, beta, or zeta chain of the T cell receptor; CD28, CD3 epsilon, CD45, CD4, CD5, CD8a, CD8b, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain is derived from the transmembrane region of NKR. In some embodiments, the transmembrane domain is derived from the transmembrane region of CD8a. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 122. In further embodiments, the transmembrane domain is selected from the group consisting of KIRDS2, OX40, TNFR2, LFA1 (CD11a, CD18), ICOS, 41BB, GITR, LTBR, ​​BAFFR, HVEM, NKp80 (KLRF1), IL2R beta, IL2R gamma, IL7R alpha, ITGA1, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGB7, VLA1, VLA6, IA4, ITGAX, CD11c, ITGB1 The transmembrane domain is derived from the transmembrane region of a molecule selected from the group consisting of CD27, CD29, ITGB2, CD2, CD11a, CD11b, CD11d, CD18, CD19, CD40, CD49a, CD49d, CD49f, CD84, CD96, CD100, CD103, CD160, CD162, CD226, CD229, CEACAM1, CRTAM, PSGL1, SLAM (SLAMF1), SLAMF4, SLAMF6 (NTB-A, Ly108), SLAMF7, SLAMF8, SELPLG, and PAG / Cbp. Alternatively, the transmembrane domain may be synthetic and preferably contains primarily hydrophobic residues, such as leucine and valine. In a further embodiment, the transmembrane domain contains a triplet of FWV (phenylalanine, tryptophan, and valine) at each end of the transmembrane domain.

[0252] Exemplary NKR domains, e.g., transmembrane, hinge or stem, or intracellular (e.g., cytoplasmic) domains (specified by the NKR from which the domain is derived) of killer immunoglobulin KIR receptors (KIRs) include KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR 3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1, KIR2DP1, NCR:, NKp30, NKp44, NKp46, SLAM; receptors SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, CD2F-10, SLAMF6, SLAMF7; Fc-binding receptors CD16, FcgRIII, CD64, Ly49; receptor Ly49, lectin-associated NK cell receptor Ly49A, Ly49C; other NK receptors include NKG2D, CD160 (TM-containing splice variant), DNAM1, CRTAM, CD27, PSGL1, CD96, CD100, NKp80, CEACAM1, and CD244. iv. Intracellular signaling domain

[0253] Described herein are intracellular signaling domains that can be used in chimeric antigen receptors (CARs) according to the present invention.

[0254] "Intracellular signaling domain" (ISD) or "cytoplasmic domain," as used herein, refers to the portion of a CAR that transmits effector function signals and directs the cell to carry out its specialized function (e.g., cytolytic and helper activity, including cytokine secretion).

[0255] The cytoplasmic domain of a CAR (i.e., the intracellular signaling domain) is responsible for activating at least one of the normal effector functions of an immune cell engineered to express the CAR. The term "effector function" refers to a specialized function of a cell. Effector functions of T cells include, for example, cytolytic or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a CAR protein that transmits an effector function signal and directs the cell to perform a specialized function. Typically, the entire intracellular signaling domain corresponding to a naturally occurring receptor can be used, although in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. The term intracellular signaling domain is therefore meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic sequence of a T cell receptor (TCR), and also includes the sequence of a coreceptor that acts in concert to initiate signal transduction after antigen receptor engagement, or any derivative or variant of these sequences that has functional capabilities. Examples of domains that transmit effector function signals include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs (e.g., the η chain, FcsR1 γ and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc.), human CD3 zeta chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (e.g., Syk, ZAP 70, etc.), src family tyrosine kinases (e.g., Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5, and CD28.

[0256] In some embodiments, the intracellular signaling domain of the CAR generates an intracellular signal when the extracellular domain (e.g., ADBD) to which it is fused binds to the cognate AD. The intracellular signaling domain of the CAR can comprise a primary intracellular signaling domain and a costimulatory signaling domain. In one embodiment, the CAR is constructed for expression in immune cells (e.g., T or NK cells), such that the expressed CAR comprises domains, e.g., a primary intracellular signaling domain and / or a costimulatory signaling domain, derived from a polypeptide typically associated with immune cells. For example, in some embodiments, the CAR is for expression in T cells and comprises a 41BB domain and a CD3 zeta domain. In another embodiment, the CAR molecule is constructed for expression in immune cells, such that the expressed CAR comprises domains derived from a polypeptide not typically associated with immune cells. For example, in some embodiments, a CAR for expression in T cells comprises a KIR domain derived from an NK cell. In an alternative embodiment, a CAR for expression in NK cells comprises a 41BB domain and a CD3 zeta domain derived from a T cell (see, e.g., WO2013 / 033626, incorporated herein by reference).

[0257] The intracellular signaling domain of the CAR comprises a primary stimulatory molecule sequence sufficient to generate an intracellular signal, for example, when the ADBD to which it is fused binds to the cognate AD. In certain embodiments, the intracellular signal of the CAR mediates a T cell response selected from the group consisting of proliferation, cytokine secretion, killing, activation, and differentiation.

[0258] In one embodiment, the intracellular signaling region of the CAR comprises a domain containing an immunoreceptor tyrosine-based activation motif (ITAM). In a further embodiment, the CAR intracellular signaling region comprises one or more ITAM containing domains derived from a molecule selected from TCR zeta (CD3 zeta), FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (FCER1B), CD3 gamma, CD3 delta, CD3 epsilon, CD3 gamma, CD5, CD22, CD79a, CD79b, DAP10, DAP12, CD32 (Fc gamma RIIa), CD79a, and CD79b. In a specific embodiment, the intracellular signaling domain of the CAR comprises a CD3 zeta signaling domain. In another specific embodiment, the intracellular signaling domain of the CAR comprises a DAP12 signaling domain. In some embodiments, the ITAM containing signaling domain has at least 70, 75, 80, 85, 90, 95, 98, or 99% sequence identity to, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from, the corresponding residues of a naturally occurring ITAM-containing domain.

[0259] Examples of ITAM-containing primary cytoplasmic signaling sequences of particular use in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0260] In a preferred embodiment, the cytoplasmic domain of the CAR comprises a CD3-zeta signaling domain, either alone or in combination with any other desired cytoplasmic domain useful in the context of the CAR. For example, the cytoplasmic domain of the CAR can comprise a portion of the CD3 zeta chain and a costimulatory signaling region. The costimulatory signaling region refers to the portion of the CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for lymphocytes to effectively respond to antigens. Examples of such molecules include CD27, CD28, 41BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFAl), CD2, CD7, LIGHT, NKG2C, B7H3, TIM1, and LAG3.

[0261] "Costimulatory domain" (CSD), as used herein, refers to a portion of a CAR that enhances the proliferation, survival, and / or development of memory cells. A CAR may contain one or more costimulatory domains. Each costimulatory domain comprises, for example, one or more costimulatory domains of any one of members of the TNFR superfamily selected from CD28, CD137 (41BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM1, LFA1 (CD11a / CD18), Lck, TNFRI, TNFRII, Fas, CD30, and CD40, or combinations thereof. Other costimulatory domains (e.g., from other proteins) will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the invention.

[0262] In some embodiments, the intracellular domain of the CAR comprises an ITAM-containing domain and a costimulatory signaling domain comprising a functional fragment or analog of a costimulatory molecule sufficient to generate an intracellular signal when the extracellular ADBD to which it is fused binds to a cognate ligand. In some embodiments, the CAR is a CAR comprising any of the following: CD137 (41BB), OX40, LIGHT, TNFR2, TRANCE / RANKL, GITR, BAFFR, HVEM, B7H3, CDS, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, VLA1, VLA6, C49f, IA4, LFA1, CD2, CD4, CD7, CD8 alpha, CD8 beta, CD11A, CD11B, CD11C, CD11D, CD18, CD19, CD27, and a costimulatory signaling domain corresponding to that found in a molecule selected from CD28, CD29, CD30, CD40, CD49A, CD49D, CD69, CD84, CD96, CD100, CD103, CD150, CD160, CD162, CD226, CD229, CD278, ICAM1, CEACAM1, CRTAM, PSGL1, SLAMF1, SLAMF4, SLAMF6, SLAMF7, SLAMF8, LTBR, ​​LAT, GADS, PAG / Cbp, SLP76, NKG2C, NKp30, NKp44, NKp46, and NKp80.

[0263] In some embodiments, the CAR comprises a costimulatory domain corresponding to that found in a molecule selected from the group consisting of 41BB, CD28, CD27, ICOS, and OX40.

[0264] In some embodiments, the CAR comprises multiple costimulatory domains. In certain embodiments, the CAR comprises, from extracellular to intracellular, the following costimulatory signaling domains: 41BB-CD27, CD27-41BB, 41BB-CD28, CD28-41BB, OX40-CD28, CD28-OX40, CD28-41BB; or 41BB-CD28.

[0265] In some embodiments, the costimulatory signaling domain of the CAR has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from, the corresponding residues of a naturally occurring costimulatory domain.

[0266] In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or a combination thereof. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 125. In some embodiments, the CAR contains an intracellular signaling domain having a sequence with at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 123, 124, or 125. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or 125, which contains 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, or 20 insertions, deletions, or substitutions. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or 125, which contains 1, 2, 3, 4, 5, 7, 8, 9, or 10 insertions, deletions, or substitutions. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or 125, which comprises 1, 2, 3, 4, 5, 7, 8, 9, or 10 substitutions.

[0267] A polypeptide linker can be positioned between adjacent elements of the CAR. For example, the linker can be positioned between adjacent ADBDs, or between the ADBD and the transmembrane domain, or between the transmembrane domain and the cytoplasmic domain, or between adjacent cytoplasmic domains. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other randomly or in a defined order. Optionally, a short linker, preferably 2 to 10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0268] In some embodiments, the CAR comprises a target binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain. In some embodiments, the CAR transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the CAR comprises a CD8a transmembrane domain and a CD8a extracellular spacer domain. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the CD8a transmembrane domain comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the CD8a extracellular spacer domain (ESD), also referred to as the hinge domain, comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the CAR comprises an intracellular signaling domain selected from the group consisting of a human T cell receptor alpha, beta, or zeta chain domain; a human 41BB domain; a human CD28 domain; and any combination thereof. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or a combination thereof. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 125. In some embodiments, the CAR contains an intracellular signaling domain having a sequence with at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity to SEQ ID NO: 123, 124, or 125. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or 125, comprising 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, or 20 insertions, deletions, or substitutions. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or 125, comprising 1, 2, 3, 4, 5, 7, 8, 9, or 10 insertions, deletions, or substitutions. In some embodiments, the CAR contains an intracellular signaling domain having the sequence of SEQ ID NO: 123, 124, or 125, comprising 1, 2, 3, 4, 5, 7, 8, 9, or 10 substitutions.In some embodiments, the CAR intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the CAR further comprises a second target binding domain having the same or a different target as the DD target binding domain. In some embodiments, the CAR comprises a first target binding domain that binds to CS1 and a second target binding domain that binds a different target. In some embodiments, the CAR comprises first and second target binding domains that bind to CS1. In some embodiments, the CAR is expressed in immune effector cells. In some embodiments, the immune effector cells are T cells (CAR-T cells) or natural killer (NK) cells (CAR-NK cells). In some embodiments, the immune effector cells are autologous cells. In some embodiments, the immune effector cells are allogeneic cells. In some embodiments, the CAR is associated with a liposome. In some embodiments, the DD binds to CS1 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the CS1-specific DD comprises the amino acid sequence of SEQ ID NO: 60.

[0269] CARs suitable for use in connection with the DDpps (e.g., adaptors and CARs) disclosed herein are disclosed in International Application Publication Nos. WO2016164305, WO2016164308A1, WO2019099440 and WO2019099433, U.S. Patent Nos. 10,662,248 and 10,647,775, and U.S. Patent Application Nos. 20200223934 and 20210002381, each of which is incorporated herein by reference for all purposes. [Table 3-1] [Table 3-2] D. Additional DDpp Fusion Proteins

[0270] In some embodiments, the DDpp contains a heterologous polypeptide comprising a fragment of the extracellular domain of a cell surface receptor, the fragment consisting of 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 10 to 50 amino acids. In some embodiments, the DDpp contains a heterologous polypeptide comprising the extracellular domain or a fragment of the extracellular domain of BCMA (SEQ ID NO: 2) or CS1 (SEQ ID NO: 1). In some embodiments, the DDpp contains a heterologous polypeptide comprising the extracellular domain or a fragment of the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37; TSLPR, IL7R, and gp96.

[0271] In some embodiments, the protein comprises a heterologous polypeptide comprising a serum protein or an antigenic fragment of a serum protein (e.g., AFP and AFP p26). In some embodiments, the DDpp comprises a heterologous polypeptide comprising a fragment of a serum protein consisting of 5-500, 5-400, 5-300, 5-200, 5-100, 5-50, 10-500, 10-400, 10-300, 10-200, 10-100, or 10-50 amino acids. In some embodiments, the protein comprises a heterologous polypeptide comprising an intracellular protein or an antigenic portion of an intracellular protein (e.g., a nucleoprotein). In some embodiments, the DDpp contains a heterologous polypeptide comprising a fragment of an intracellular protein consisting of 5 to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 5 to 50, 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 100, or 10 to 50 amino acids. In some embodiments, the DDpp contains a heterologous polypeptide having a sequence of SEQ ID NO: 8-15 or 16.

[0272] In some embodiments, the DDpp fusion protein specifically binds to CS1 and / or AFP p26 and further binds to one or more additional targets of interest. The target of interest to which the DDpp fusion protein specifically binds can be any molecule to which it is desirable for DDpp to bind. For example, the target to which the DDpp fusion protein specifically binds can be CS1 and / or AFP p26, and additionally, any additional target of manufacturing, formulation, therapeutic, diagnostic, or prognostic relevance or value. Some exemplary additional targets are provided herein by way of example and are intended to be illustrative and not limiting. The additional target of interest to which the DDpp fusion protein binds can be naturally occurring or synthetic. The additional target of interest can be an extracellular or intracellular component, a soluble factor (e.g., enzymes, hormones, cytokines, and growth factors, toxins, venoms, pollutants, etc.), or a transmembrane protein (e.g., a cell surface receptor). In some embodiments, the target of interest to which the DDpp fusion protein binds is a human protein. In one embodiment, DDpp (e.g., DDpp fusion protein) binds to a human protein target of interest and its monkey (e.g., cynomolgus), mouse, rabbit, hamster and / or rabbit orthologs. In some embodiments, the additional target of interest is BCMA.

[0273] In one embodiment, the DDpp fusion protein specifically binds to CS1 and a serum protein. In one embodiment, the DDpp fusion protein specifically binds to a serum protein selected from serum albumin (e.g., human serum albumin (HSA)), thyroxine-binding protein, transferrin, fibrinogen, and immunoglobulins (e.g., IgG, IgE, and IgM). Without being bound by theory, it is believed that conjugation of DDpp to a carrier protein confers an improved pharmacodynamic profile to DDpp (or its fusion), including, but not limited to, improved tumor targeting, tumor penetration, intratumoral diffusion, and enhanced therapeutic activity, compared to DDpp fusion proteins lacking the carrier protein binding sequence (see, e.g., WO01 / 45746, the contents of which are incorporated herein by reference in their entirety). E. Antibody-Based DDpp Fusion Proteins

[0274] In some embodiments, the DDpp fusion protein comprises a full-length antibody or an antibody fragment or subdomain. In some embodiments, the DDpp fusion protein comprises a full-length IgG antibody (e.g., IgG1, IgG2, IgG2, or IgG4). In further embodiments, the DDpp fusion protein comprises a full-length antibody that specifically binds to a cancer antigen. In further embodiments, the DDpp comprises a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN882). In further embodiments, the DDpp comprises an antibody or antigen-binding fragment thereof that binds to the T cell receptor (TCR) complex on T cells (e.g., binds to the CD3 epsilon chain). In other embodiments, the DDpp is an Fc fusion protein. In further embodiments, the Fc protein comprises a variant human Fc domain.

[0275] In some embodiments, DDpp fusion proteins comprise full-length antibodies or antibody fragments or subdomains (e.g., IgG1 antibodies, IgG3 antibodies, antibody variable regions, CDR3s, scFvs, Fc, FcRn-binding subdomains, and other antibody subdomains). DDpp proteins can be operably linked to each other and / or to one or more termini of antibodies, antibody chains, antibody fragments, or antibody subdomains to form DDpp fusion proteins.

[0276] The antibody component of a DDpp fusion protein can be any suitable full-length immunoglobulin or antibody fragment (e.g., antigen-binding domain and / or effector domain) or fragment thereof. In one embodiment, the DDpp-antibody fusion protein retains the structural and functional properties of a traditional monoclonal antibody. Thus, in some embodiments, the DDpp-antibody fusion protein retains epitope-binding properties but also advantageously incorporates one or more additional target-binding specificities via the DDpp fusion. Antibodies that can be used in DDpp fusions include, but are not limited to, monoclonal, multispecific, human, humanized, primatized, and chimeric antibodies. The immunoglobulin or antibody molecules provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule. In a specific embodiment, the antibody is an Fc-optimized antibody. Antibodies can be from or derived from any animal origin, including birds and mammals, or can be synthetically produced. The antibody component of the DDpp-antibody fusion protein can be naturally derived or the result of recombinant engineering (e.g., phage display, xenomouse, and synthetic). In certain embodiments, the antibody component of the antibody-DDpp fusion enhances half-life, increases or decreases antibody-dependent cellular cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the antibody is a human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibody. In a specific embodiment, the antibody is human.

[0277] It is generally understood that the constant region mediates several effector functions. For example, binding of the C1 component of complement to antibodies activates the complement system. Complement activation is important in the opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to cells via their Fc region, and Fc receptor sites on the antibody Fc region bind to Fc receptors (FcRs) on cells. There are several Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of antibodies to Fc receptors on the cell surface triggers several important and diverse biological responses, including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, and translocation and control of immunoglobulin production.

[0278] In certain embodiments, the DDpp-Fc fusion protein has altered effector function, which in turn affects the biological profile of the administered DDpp-Fc fusion protein. For example, deletion or inactivation (by point mutation or other means) of constant region subdomains can reduce Fc receptor binding of circulating modified antibodies. In other cases, constant region modifications can alleviate complement binding, thus reducing serum half-life and nonspecific association of conjugated cytotoxins. Still other modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties, which allow for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region in accordance with the present disclosure can be readily made using biochemical or molecular engineering techniques known to those skilled in the art.

[0279] In some embodiments, the DDpp-Fc fusion protein does not have one or more effector functions. For example, in some embodiments, the DDpp-Fc fusion protein does not have antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the DDpp-Fc fusion protein does not bind to Fc receptors and / or complement factors. In certain embodiments, the DDpp-Fc fusion protein does not have effector functions. Examples of modifications that engineer the Fc sequence to reduce or eliminate ADCC and / or CDC activity and Fc receptor and / or complement factor binding are described herein or are otherwise known in the art, as are assays and procedures for testing same.

[0280] In some embodiments, DDpp-Fc fusion proteins are engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibody. In other constructs, a peptide spacer is inserted between the hinge region and the modified CH2 and / or CH3 domain. For example, compatible constructs can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region by a 5-20 amino acid spacer. Such a spacer can be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. Amino acid spacers may, in some cases, prove immunogenic and elicit an unwanted immune response against the construct. Therefore, in certain embodiments, any spacer added to the construct can be relatively non-immunogenic or even omitted altogether to maintain the desired biochemical qualities of the modified DDpp-Fc fusion protein.

[0281] In additional embodiments, the DDpp-Fc fusion protein is modified by partial deletion or substitution of several or even single amino acids in the constant region. For example, mutation of a single amino acid in a selected area of ​​the CH2 domain may be sufficient to substantially reduce Fc binding. Similarly, one or more constant region domains controlling effector functions (e.g., complement C1Q binding) can be completely or partially deleted. Such partial deletion of the constant region can improve selected properties of the DDpp-Fc fusion protein (e.g., serum half-life) while leaving other desirable functions associated with the corresponding constant region domain intact. In some embodiments, the constant region of the DDpp-Fc fusion protein is modified by mutation or substitution of one or more amino acids that enhance the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the structural and immunogenic profile of the modified DDpp-Fc fusion protein. The present disclosure also provides DDpp-Fc fusion proteins containing the addition of one or more amino acids to the constant region to enhance desirable properties, such as reduced or increased effector function, or providing attachment sites for one or more cytotoxins, or enhancing the labeling of carbohydrate moieties. In such embodiments, it may be desirable to insert or replace specific sequences from selected constant region domains.

[0282] In some embodiments, DDpp is operably linked to an antibody fragment or subdomain (e.g., an scFv, a diabody, EP404,097; WO93 / 111161; WO14 / 028776; and Holliger et al., PNAS 90: 6444-6448 (1993), the contents of each of which are incorporated herein by reference in their entirety). The antibody fragment or subdomain can be any fragment or domain of an antibody. See, for example, WO04 / 058820, WO99 / 42077, and WO05 / 017148, the contents of each of which are incorporated herein by reference in their entirety. For example, the DDpp fusion protein can contain an antibody effector domain or a derivative of an antibody effector domain that confers one or more effector functions to the DDpp and / or confers on the DDpp fusion protein the ability to bind to one or more Fc receptors. In some embodiments, the DDpp-antibody fusion protein contains an antigen-binding fragment of an antibody or fragment thereof. In additional embodiments, the DDpp-antibody fusion protein contains an immunoglobulin effector domain comprising one or more CH2 and / or CH3 domains of an antibody with effector functions provided by the CH2 and CH3 domains. Other sequences in DDpp fusions that provide effector functions and are encompassed by the present invention will be apparent to those skilled in the art and can be routinely selected and designed into DDpp fusion proteins encompassed herein based on the desired effector function.

[0283] In one embodiment, the DDpp fusion contains a full-length antibody or an antibody fragment that is an antigen-binding fragment. In a further embodiment, the antibody or antibody fragment binds to a disease-associated antigen. In one embodiment, the DDpp fusion protein comprises an antibody or antibody fragment that specifically binds to a cancer antigen. In another embodiment, the DDpp fusion protein comprises an antibody or antibody fragment that specifically binds to a particular pathogen (e.g., bacterial cells (e.g., tuberculosis, smallpox, anthrax)), virus (e.g., HIV), parasite (e.g., malaria, leishmania), fungal infection, mold, mycoplasma, or prion antigen. In another embodiment, the DDpp fusion protein comprises an antibody or antibody fragment that specifically binds to a particular pathogen (e.g., bacterial cells (e.g., tuberculosis, smallpox, anthrax)), virus (e.g., HIV), parasite (e.g., malaria, leishmania), fungal infection, mold, mycoplasma, or prion antigen. In another embodiment, the DDpp fusion protein comprises an antibody or antibody fragment that specifically binds to an antigen associated with a disease or disorder of the immune system.

[0284] In preferred embodiments, DDpp fusion proteins containing antibody fragments or domains retain the activity of the parent antibody. Thus, in certain embodiments, DDpp fusion proteins containing antibody fragments or domains are capable of inducing complement-dependent cytotoxicity. In certain embodiments, DDpp fusion proteins containing antibody fragments or domains are capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC).

[0285] Thus, in some embodiments, the DDpp fusion protein comprises an antibody fragment that confers the biological or biochemical properties of an immunoglobulin to the DDpp fusion protein. In some embodiments, the antibody fragment confers a property selected from the ability to non-covalently dimerize, the ability to localize at tumor sites, and an increased serum half-life, when compared to a DDpp fusion protein in which one or more DDpps have been deleted. In certain embodiments, the DDpp fusion protein is at least as stable as the corresponding antibody without the attached DDpp. In certain embodiments, the DDpp fusion protein is more stable than the corresponding antibody without the attached DDpp. The stability of the DDpp fusion protein can be measured using established methods, including, for example, ELISA techniques. In some embodiments, the DDpp fusion protein is stable in whole blood (in vivo or ex vivo) at 37°C for at least about 10 hours, at least about 15 hours, at least about 20 hours, at least about 24 hours, at least about 25 hours, at least about 30 hours, at least about 35 hours, at least about 40 hours, at least about 45 hours, at least about 48 hours, at least about 50 hours, at least about 55 hours, at least about 60 hours, at least about 65 hours, at least about 70 hours, at least about 72 hours, at least about 75 hours, at least about 80 hours, at least about 85 hours, at least about 90 hours, at least about 95 hours, or at least about 100 hours (including any time in between those listed). In one embodiment, the DDpp fusion contains an immunoglobulin effector domain or half-life-affecting domain corresponding to an immunoglobulin domain or fragment, where at least a small portion of one or more of the constant region domains has been altered to provide a desired biological property, such as reduced or increased effector function, ability to noncovalently dimerize, increased ability to localize at tumor sites, reduced serum half-life, or increased serum half-life, when compared to an immunoglobulin fragment having a corresponding unaltered immunoglobulin sequence. These constant region domain alterations can be amino acid substitutions, insertions, or deletions.

[0286] In one embodiment, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers antibody-dependent cellular cytotoxicity (ADCC) to the DDpp fusion protein. In additional embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to increase ADCC (e.g., Bruhns, Blood 113: 3716-3725 (2009); Shields, J. Biol. Chem. 276: 6591-6604 (2001); Lazar, PNAS 103: 4005-4010 (2006); Stavenhagen, Cancer Res. 67: 8882-8890 (2007); Horton, Cancer Res. 68: 8049-8057 (2008); Zalevsky, Blood 113: 3735-3743 (2009); Bruckheimer, Neoplasia 11: 509-517 (2009); WO06 / 020114; Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and WO04 / 074455, the contents of each of which are incorporated herein by reference in their entireties.Examples of modifications to engineer immunoglobulin fragments contained in amino acid sequences in DDpp fusion proteins that increase ADCC include immunoglobulin effector domain sequences having one or more modifications corresponding to IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L, with the numbering of residues in the Fc region being as described in Kabat et al. (Sequences of proteins of The text is from the EU Index of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety.

[0287] In other embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to reduce ADCC (e.g., Idusogie et al., J. Immunol. 166: 2571-2575 (2001); Sazinsky et al., PNAS 105: 20167-20172 (2008); Davis et al., J. Rheumatol. 34: 2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23: 403-411 (1993); Alegre et al., Transplantation 57: 1537-1543 (1994); Xu et al., Cell Immunol. 200: 16-26 (2000); Cole et al., Transplantation 68: 563-571 (2001)). (1999); Hutchins et al., PNAS 92: 11980-11984 (1995); Reddy et al., J. Immunol. 164: 1925-1933 (2000); WO97 / 11971; WO07 / 106585; US2007 / 0148167A1; McEarchern et al., Blood 109: 1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Kumagai et al., J. Clin. Pharmacol. 47: 1489-1497 (2007), the contents of each of which are incorporated herein by reference in their entireties.Examples of modifications that manipulate the immunoglobulin fragment sequence contained in the amino acid sequence of the DDpp fusion protein to reduce ADCC include IgG1-K326W, E333S; IgG2-E333S; IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2-118-260; IgG IgG1-C220S, C226S, C229S, E233P, L234V, L235A; or IgG1-L234F, L235E, P331S, where the numbering of the residues is that of the EU index of Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety).

[0288] In a further embodiment, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers antibody-dependent cellular phagocytosis (ADCP) to the DDpp fusion protein. In additional embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to increase antibody-dependent cellular phagocytosis (ADCP) (e.g., Shields et al., J. Biol. Chem. 276: 6591-6604 (2001); Lazar et al., PNAS 103: 4005-4010 (2006); Stavenhagen et al., Cancer Res. 67: 8882-8890 (2007); Richards et al., Mol. Cancer Ther. 7: 2517-2527 (2008); Horton et al., Cancer Res. 68: 8049-8057 (2008); Zalevsky et al., Blood 113: 3735-3743 (2009); Bruckheimer et al., Neoplasia 11: 509-517 (2009); WO06 / 020114; Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and WO04 / 074455, the contents of each of which are incorporated herein by reference in their entireties.Examples of modifications that manipulate immunoglobulin fragments contained in the amino acid sequence of the DDpp fusion protein to increase ADCP include IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S with or without S298D or V. IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; and IgG1-G236A, S239D, I332E, where the numbering of the residues is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety).

[0289] In other embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to reduce ADCP (e.g., Sazinsky et al., PNAS 105: 20167-20172 (2008); Davis et al., J. Rheumatol. 34: 2204-2210 (2007); Bolt et al., Eur. J. Immunol. 23: 403-411 (1993); Alegre et al., Transplantation 57: 1537-1543 (1994); Xu et al., Cell Immunol. 200: 16-20 (2000); Cole et al., Transplantation 68: 563-571 (1999); Hutchins et al., PNAS 92: 11980-11984 (1995); Reddy et al., J. Immunol. 164: 1925-1933 (2000); WO97 / 11971; WO07 / 106585; US2007 / 0148167A1; McEarchern et al., Blood 109: 1185-1192 (2007); Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Kumagai et al., J. Clin. Pharmacol. 47: 1489-1497 (2007), the contents of each of which are incorporated herein by reference in their entireties.By way of example, DDpp fusion proteins may be modified with the following modifications to reduce ADCC: IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2 IgG4 - EU sequence 261-447; IgG2 - H268Q, V309L, A330S, A331S; IgG1 - C220S, C226S, C229S, p268S; IgG1 - C226S, C229S, E233P, L234V, L235A; and IgG1 - L234F, L235E, P331S, where the numbering of the residues is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety).

[0290] In additional embodiments, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers complement-dependent cytotoxicity (CDC) to the DDpp fusion protein. In additional embodiments, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain that has been modified to increase complement-dependent cytotoxicity (CDC) (see, e.g., Idusogie et al., J. Immunol. 166: 2571-2575 (2001); Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Natsume et al., Cancer Res. 68: 3863-3872 (2008), the contents of each of which are incorporated herein by reference in their entirety). By way of example, the DDpp fusion protein may contain an antibody fragment or domain containing one or more of the following modifications that increase CDC: IgG1-K326A, E333A; IgG1-K326W, E333S, IgG2-E333S, where the residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety).

[0291] In a further embodiment, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers the ability to bind to the Fc gamma RIIb receptor to the DDpp fusion protein. In a further embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain that has been modified to increase inhibitory binding to the Fc gamma RIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45: 3926-3933 (2008)). An example of a modification engineered immunoglobulin fragment contained in the amino acid sequence of the DDpp fusion protein that increases binding to the inhibitory Fc gamma RIIb receptor is IgG1-S267E, L328F.

[0292] In other embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to reduce CDC (e.g., WO97 / 11971; WO07 / 106585; US2007 / 0148167A1; McEarchern et al., Blood 109: 1185-1192 (2007); Hayden-Ledbetter et al., Clin. Cancer 15: 2739-2746 (2009); Lazar et al., PNAS 103: 4005-4010 (2006); Bruckheimer et al., Neoplasia 11: 509-517 (2009); Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Sazinsky et al., PNAS 105: 20167-20172 (2008), the contents of each of which are incorporated herein by reference in their entireties. By way of example, the DDpp fusion protein may contain antibody fragments or domains containing one or more of the following modifications that reduce CDC: IgG1-S239D, A330L, I332E; IgG2-118-260; IgG4-261-447; IgG2-H268Q, V309L, A330S, A331S; IgG1-C226S, C229S, E233P, L234V, L235A; IgG1-L234F, L235E, P331S; and IgG1-C226S, p260S, where the residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety).

[0293] The half-life of IgG is mediated by its pH-dependent binding to the fetal receptor FcRn. In certain embodiments, the DDpp fusion protein comprises an amino acid sequence of an immunoglobulin effector domain or a derivative of an immunoglobulin effector domain that confers to the DDpp fusion the ability to bind to the fetal receptor FcRn. In certain embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin FcRn-binding domain that has been modified to enhance binding to FcRn (e.g., Petkova et al., Int. Immunol. 18: 1759-1769 (2006); Dall'Acqua et al., J. Immunol. 169: 5171-5180 (2002); Oganesyan et al., Mol. Immunol. 46: 1750-1755 (2009); Dall'Acqua et al., J. Biol. Chem. 281: 23514-23524 (2006); Hinton et al., J. Immunol. 176: 346-356 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35: 86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282: 1709-1717 (2007); WO06 / 130834; Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Yeung et al., J. Immunol. 182: 7663-7671 (2009), the contents of each of which are incorporated herein by reference in their entireties.

[0294] In a further embodiment, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to have selective affinity for FcRn at pH 6.0 but not at pH 7.4. By way of example, the DDpp fusion protein may contain an antibody fragment or domain containing one or more of the following half-life-increasing modifications: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L; IgG1-H433K, N434Y; IgG1-N434A; and IgG1-T307A, E380A, N434A, where the residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 1991 Fifth Edition, the contents of which are incorporated herein by reference in their entirety).

[0295] In other embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to reduce binding to FcRn (see, e.g., Petkova et al., Int. Immunol. 18: 1759-1769 (2006); Datta-Mannan et al., Drug Metab. Dispos. 35: 86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282: 1709-1717 (2007); Strohl, Curr. Op. Biotechnol. 20: 685-691 (2009); and Vaccaro et al., Nat. Biotechnol. 23: 1283-1288 (2005), the contents of each of which are incorporated herein by reference in their entirety). By way of example, the DDpp fusion protein may contain an antibody fragment or domain containing one or more of the following half-life-reducing modifications: IgG1-M252Y, S254T, T256E; H433K, N434F, 436H; IgG1-I253A; and IgG1-P257I, N434H and D376V, N434H, where the residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety).

[0296] According to another embodiment, the DDpp fusion protein is selected from the group consisting of 238, 239, 246, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 332, 333, 334 , 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439, wherein the numbering of residues in the Fc region is according to the EU numbering system of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 Fifth edition, the contents of which are incorporated herein by reference in their entirety). In a specific embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain derivative in which at least one residue corresponding to position 434 is a residue selected from the group consisting of A, W, Y, F, and H. According to another embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector fragment derivative having the following respective substitutions S298A / E333A / K334A. In a further embodiment, the DDpp fusion protein comprises an immunoglobulin effector domain derivative having a substitution corresponding to K322A. In another embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain derivative having one or any combination of the following substitutions K246H, H268D, E283L, S324G, S239D and I332E. According to yet another embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain derivative having substitutions corresponding to D265A / N297A.

[0297] In certain embodiments, the DDpp fusion protein comprises an immunoglobulin effector domain sequence that has been glycoengineered or mutated to increase effector function using techniques known in the art. For example, inactivation (by point mutation or other means) of the constant region domain sequence contained in DDpp may reduce Fc receptor binding of circulating DDpp fusion proteins, thereby increasing tumor localization. In other cases, constant region modifications consistent with certain provided embodiments may alleviate complement binding, thus reducing serum half-life and nonspecific association of conjugated cytotoxins. Still other modifications of the constant region may be used to modify disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. The resulting physiological profile, bioavailability, and other biochemical effects of the modifications, such as tumor localization, biodistribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0298] In some embodiments, immune effector cells comprise cell surface receptors for immunoglobulins or other peptide-binding molecules, e.g., receptors for immunoglobulin constant regions, including the class of receptors commonly referred to as "Fc receptors" ("FcRs"). Several FcRs have been structurally and / or functionally characterized and are known in the art, including FcRs that have a specific ability to interact with a restricted subset of immunoglobulin heavy chain isotypes, or with Fc domains that have different affinities and / or that may be expressed on a restricted subset of immune effector cells under certain conditions (e.g., Kijimoto-Ochichai et al., Cell Mol. Life. Sci. 59: 648 (2002); Davis et al., Curr. Top. Microbiol. Immunol. 266: 85 (2002); Pawankar, Curr. Opin. Allerg. Clin. Immunol. 1: 3 (2001); Radaev et al., Mol. Immunol. 38: 1073 (2002); Wurzburg et al., Mol. Immunol. 38: 1063 (2002); Sulica et al., Int. Rev. Immunol. 20: 371 (2001);Underhill et al., Ann. Rev. Immunol. 20: 825 (2002);Coggeshall, Curr. Dir. Autoimm. 5: 1 (2002);Mimura et al., Adv. Exp. Med. Biol. 495: 49 (2001);Baumann et al. al., Adv. Exp. Med. Biol. 495: 219 (2001);Santoso et al., Ital. Heart J. 2: 811 (2001);Novak et al., Curr. Opin. Immunol. 13: 721 (2001);Fossati et al., Eur. J. Clin. Invest. 31: 821 (2001)), the contents of each of which are incorporated herein by reference in their entirety.

[0299] Cells capable of mediating ADCC are examples of immune effector cells. Other immune effector cells include natural killer cells, tumor-infiltrating T lymphocytes (TILs), cytotoxic T lymphocytes, and granulocytes, such as cells involved in allergic response mechanisms. Thus, immune effector cells include, but are not limited to, cells of hematopoietic origin, such as T lymphocytes, B lymphocytes, NK cells, monocytes, macrophages, dendritic cells, neutrophils, basophils, eosinophils, mast cells, platelets, erythrocytes, and precursors, progenitors (e.g., hematopoietic stem cells), as well as resting, activated, and mature forms of such cells, including cells at various stages of differentiation within the myeloid and lymphoid lineages and may (but need not) express one or more types of functional cell surface FcR. Other immune effector cells may include cells of non-hematopoietic origin capable of mediating immune functions, such as endothelial cells, keratinocytes, fibroblasts, osteoclasts, epithelial cells, and other cells. Immune effector cells can also include cells that mediate cytotoxic or cytostatic events, or endocytic, phagocytic or pinocytic events, or result in the induction of apoptosis, or result in microbial immunity or neutralization of microbial infection, or cells that mediate allergic, inflammatory, hypersensitivity and / or autoimmune responses. F. DDpp fusion proteins with increased half-lives

[0300] The disclosed DDpp can be fused or conjugated to a second peptide domain to increase the half-life or stability of the DDpp.

[0301] In one embodiment, DDpp further comprises one or more amino acids that facilitate the synthesis, handling, or use of the peptide, including, but not limited to, one or two lysines at the N- and / or C-terminus to increase the solubility of the polypeptide. Suitable fusion proteins include, but are not limited to, one or more polypeptides, polypeptide fragments, or proteins comprising DDpp linked to an amino acid not generally recognized as part of a protein sequence. In one embodiment, the fusion peptide comprises the entire amino acid sequence of two or more peptides, or alternatively, portions (fragments) of two or more peptides. In some embodiments, the peptide (e.g., a Protein S binding peptide) is operably linked to, for example, one or more of the following: a marker protein, a peptide that facilitates purification, a peptide sequence that promotes the formation of a multimeric protein, or a fragment of any of the foregoing. Suitable fusion partners include, but are not limited to, His tags, FLAG tags, strep tags, and myc tags.

[0302] In some embodiments, DDpp is fused to one or more moieties that enhance the half-life of the polypeptide. Half-life can be increased, for example, by increasing the molecular weight of DDpp to avoid renal clearance and / or by incorporating a binding domain for the FcRn-mediated recycling pathway. In one embodiment, DDpp is fused to or chemically conjugated with an albumin polypeptide or a fragment thereof (e.g., human serum albumin (HSA)). In certain embodiments, the fused or chemically conjugated albumin fragment comprises 10%, 25%, 50%, or 75% of the full-length albumin protein. In further or alternative embodiments, DDpp is fused to or complexed with an albumin-binding domain or a fatty acid that binds to albumin when administered in vivo. An example of an albumin binding domain is the "albu-tag," a moiety derived from 4-(p-iodophenyl)-butanoic acid (Dumelin et al., Angew Chem. Int. Ed Engl. 47: 3196-3201 (2008)).

[0303] In one embodiment, DDpp is fused or chemically conjugated to a transferrin polypeptide or a fragment thereof (e.g., human transferrin). In certain embodiments, the fused or chemically conjugated transferrin fragment comprises 10%, 25%, 50%, or 75% of the full-length transferrin protein. In further or alternative embodiments, DDpp is fused to or complexed with a transferrin-binding domain that binds to transferrin when administered in vivo.

[0304] In some embodiments, DDpp is fused or chemically conjugated to a proline-alanine-serine multimer (PASylation; XL-Protein GmbH), an imprecise repeat peptide sequence (XTenylation, rPEG), a homopolymer of glycine residues (HAPylation), an elastin-like repeat sequence (ELPylation; see, e.g., U.S. Patent Application No. 61 / 442,106, the contents of which are incorporated herein by reference in their entirety), an artificial GLK (GLK fusion; Huang et al., Eur. J. Pharm. Biopharm. 72: 435-41 (2010)), or a CTP peptide derived from the human CG beta subunit (CTP fusion). G. Additional DDpp Fusion Proteins

[0305] In some embodiments, the DDpp fusion protein specifically binds to CS1 and further binds to a disease-associated antigen. The disease-associated antigen can be an antigen characteristic of cancer, and / or a particular cell type (e.g., a hyperproliferative cell), and / or a pathogen (e.g., a bacterial cell (e.g., tuberculosis, smallpox, and anthrax), a virus (e.g., HIV), a parasite (e.g., malaria and leishmania), a fungal infection, a mold, a mycoplasma, a prion antigen, or an antigen associated with a disorder of the immune system. In further embodiments, the DDpp fusion protein is conjugated to a therapeutic or cytotoxic agent.

[0306] In additional embodiments, the DDpp fusion protein is linked to one or more chemical moieties (e.g., labels) that facilitate detection, multimerization, binding to interaction partners, or characterization of DDpp activity. An exemplary chemical moiety is biotin. Other moieties suitable for conjugation to DDpp include, but are not limited to, photosensitizers, dyes, fluorescent dyes, radionuclides, radionuclide-containing complexes, enzymes, toxins, and cytotoxic agents. Photosensitizers include, for example, Photofrin®, Visudyne®, Levulan®, Foscan®, Metvix®, Hexvix®, Cysview™, Laserphyrin®, Antrin®, Photochlor®, Photosens®, Photrex™, LumaCAN®, Cevira®, Visonac®, BF-200 ALA, and Amphiminex®. In additional embodiments, a His tag, a FLAG tag, a strep tag, or a myc tag is conjugated to the DDpp.

[0307] In another embodiment, the DDpp fusion protein comprises a DD that binds to CS1 or a fragment thereof and further binds to a peptide tag present on a target of interest. Such a peptide tag provides a useful means of detecting and / or attaching to a target of interest that contains the peptide tag. In one embodiment, the DDpp fusion protein specifically binds to a peptide tag selected from the group of a hexahistidyl (His6) tag, a myc tag, or a FLAG tag. Other peptide tags are described herein or otherwise known in the art. H. DDpp fusion protein with epitope tag

[0308] In some embodiments, the DDpp fusion protein comprises a peptide epitope tag. In some embodiments, the peptide tag is selected from the group consisting of a hexahistidyl (His6) tag, a myc tag, and a FLAG tag. In additional embodiments, peptide tags include, but are not limited to, avitag (which allows for biotinylation of the tag and isolation with streptavidin), calmodulin, E-tag, hemagglutinin (HA), S-tag, SBP-tag, softag1, streptavidin, tetra- or poly-cysteine, V5, VSV, and Xpress tags. Additionally, polyhistidyl tags (other than 6 residues) can be used. In additional embodiments, covalent peptide tags, protein tags, and the like can be used. Covalent peptide tags include, but are not limited to, isopeptag (covalently linked to pilinC protein), Spytag (covalently linked to SpyCatcher protein), and Snooptag (covalently linked to SnoopCatcher protein). In still further embodiments, protein tags, including but not limited to biotin carboxyl carrier protein (BCCP), glutathione-S-transferase, green fluorescent protein (or other fluorophores), Halo tag, Nus tag, thioredoxin, and Fc tag, may be used as needed. In still further embodiments, multiple types of tags may be used. In still further embodiments, no tags are used. In still further embodiments, the DDpp fusion protein includes a removable tag. Any combination of extracellular, transmembrane, and intracellular domains disclosed herein may be used, depending on the embodiment. I. DDpp as a Chemical Conjugate

[0309] DDpp fusion proteins (e.g., adapters) that facilitate specific binding to a target of interest can be chemically conjugated to a variety of compounds, such as fluorescent dyes, radioisotopes, chromatographic compositions (e.g., beads, resins, gels, etc.), and chemotherapeutic agents. DDpp fusion protein conjugates have uses including, but not limited to, diagnostic, analytical, manufacturing, and therapeutic applications.

[0310] The natural lack of a cysteine ​​in the DD sequence provides an opportunity for the introduction of a unique cysteine ​​for the purpose of site-specific conjugation.

[0311] In some embodiments, the DDpp fusion protein (e.g., adapter) contains at least one reactive residue. The reactive residue is useful, for example, as a site for attachment of a conjugate, e.g., a chemotherapeutic agent. The reactive residue can be, for example, a cysteine, a lysine, or another reactive residue. Thus, a cysteine ​​can be added to a DDpp either at the N- or C-terminus or within the DDpp sequence. A cysteine ​​can substitute for another amino acid in the DDpp sequence. Additionally, a lysine can be added to a DDpp either at the terminus or within the DDpp sequence and / or a lysine can substitute for another amino acid in the DDpp sequence. In one embodiment, the reactive residue (e.g., a cysteine, a lysine, etc.) is located in the loop sequence of a DDpp (e.g., amino acid residues corresponding to residues 22-24 and 46-49 of SEQ ID NO: 5). In one embodiment, the reactive residue is located in a linker located between components of a DDpp fusion, for example, between a DDpp and another component of a DDpp fusion protein. Reactive residues (e.g., cysteine, lysine, etc.) can also be located within the sequence of DDpp or other components of a DDpp fusion protein. In one embodiment, a DDpp or DDpp fusion protein comprises at least one, at least two, or at least three reactive residues. In one embodiment, a DDpp, e.g., a DDpp fusion protein, comprises at least one, at least two, or at least three cysteine ​​residues.

[0312] Expressions such as "binding affinity for a target," "binding to a target," etc. refer to properties of a polypeptide that can be directly measured by determining affinity constants, such as the amount of DDpp associated and dissociated at a given antigen concentration. Different methods can be used to characterize molecular interactions, including but not limited to competitive analysis, equilibrium analysis, and microcalorimetry, as well as real-time interaction analysis based on surface plasmon resonance interaction (for example, using a Biacore® instrument). These methods are known to those skilled in the art and are described, for example, in Neri et al., Tibtech 14: 465-470 (1996) and Jansson et al., J Biol Chem 272: 8189-8197 (1997).

[0313] The affinity requirements for a given DDpp binding event depend on a variety of factors, including, but not limited to, the composition and complexity of the binding matrix, the valency and density of both the DDpp and the target molecule, and the functional application of the DDpp. In one embodiment, the DDpp is 5×10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 -5 M or 10 -5 In a further embodiment, DDpp binds to a target of interest (e.g., CS1) with a dissociation constant (KD) less than or equal to M. In a further embodiment, DDpp binds to a target of interest (e.g., CS1) with a dissociation constant (KD) less than or equal to 5×10 -6 M, 10 -6 M, 5 x 10 -7 M, 10 -7 M, 5 x 10 -8 M, or 10 -8 In a further embodiment, the DDpp binds to the target of interest with a KD of less than or equal to 5×10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M, or 10 -15 In some embodiments, the DDpp provided binds to the target of interest with a KD of less than or equal to 10 -4 M~10 -5 M, 10 -5 M~10 -6 M, 10 -6 M~10 -7 M, 10 -7 M~10 -8 M, 10 -8 M~10 -9 M, 10 -9 M~10 -10 M, 10 -10 M~10 -11 M and 10 -11 M~10 -12 M has a dissociation constant selected from the group consisting of:

[0314] In some embodiments, DDpp binds to a target of interest (e.g., CS1) in its active form. In one embodiment, DDpp reversibly binds to a target of interest in its active form and releases the bound target in its active form. In some embodiments, DDpp binds to a target of interest in its native form. In a specific embodiment, DDpp binds to a target of interest in its native form. -10 seconds -1 , 5×10 -9 seconds -1 , 10 -9 seconds -1 , 5×10 -8 seconds -1 , 10 -8 seconds -1 , 5×10 -7 seconds -1 , 10 -7 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -5 seconds -1 , 10 -5 seconds -1 , 5×10 -4 seconds-1 and 10 -4 seconds -1 and 5×10 -3 seconds -1 and 10 -3 seconds -1 and 5×10 -2 seconds -1 and 10 -2 seconds -1 and 5×10 -1 seconds -1 or 10 -1 seconds -1 at an off-rate or K greater than or equal to off and binds to the target of interest.

[0315] Binding experiments to determine KD and off-rate can be routinely performed under several conditions, including, but not limited to, [pH 6.0, 0.01% Tween® 2], [pH 6.0, 0.1% gelatin], [pH 5.0, 0.01% Tween® 2], [pH 9.0, 0.1% Tween® 2], [pH 6.0, 15% ethylene glycol, 0.01% Tween® 2], [pH 5.0, 15% ethylene glycol, 0.01% Tween® 2], and [pH 9.0, 15% ethylene glycol, 0.01% Tween® 2]. The buffers for making these solutions can be routinely determined by those skilled in the art and mainly depend on the desired pH of the final solution. Low pH solutions (<pH 5.5) can be made, for example, in citrate buffer, glycine-HCl buffer, or succinate buffer. High pH solutions can be made, for example, in Tris-HCl, phosphate buffer, or sodium bicarbonate buffer. For the purpose of determining, for example, the optimal pH and / or salt concentration, several conditions are routinely used by those skilled in the art to determine KD and off-rate.

[0316] In one embodiment, DDpp is 0.1 to 10 -7 seconds -1 and 10 -2 to 1​​​​-2 ~10 -7 seconds -1 K in the range Off In a specific embodiment, the DDpp (e.g., DDpp fusion protein) specifically binds to a target of interest (e.g., CS1) at a concentration of 5×10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , or 10 -3 seconds -1 Off-rate (K Off ) and binds to the target of interest. In a further embodiment, the DDpp is 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 , or 10 -7 seconds -1 Off-rate (K Off ) and binds to the desired target.

[0317] In one embodiment, DDpp is 10 3 ~10 7 M -1 seconds -1 , 10 3 ~10 6 M -1 seconds -1 , or 10 3 ~10 5 M -1 seconds -1 K in the range On In a specific embodiment, the DDpp (e.g., DDpp fusion protein) specifically binds to a target of interest (e.g., CS1) at 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 104 M -1 seconds -1 , or 5 × 10 4 M -1 seconds -1 On-rate (K On ) and binds to the target of interest. In a further embodiment, the DDpp is 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5 × 10 6 M -1 seconds -1 , or 10 7 M -1 seconds -1 K is larger than On and binds to the desired target.

[0318] The disclosed nucleic acid molecules encoding DDpp are encompassed herein, as are vectors containing these nucleic acids, host cells containing these nucleic acid vectors, and methods for making DDpp-albumin fusion proteins and using these nucleic acids, vectors, and / or host cells. The invention also encompasses pharmaceutical formulations comprising a DDpp-albumin fusion protein and a pharmaceutically acceptable diluent or carrier. Such formulations can be used in methods for treating, preventing, ameliorating, or diagnosing a disease or disease symptom in a patient, preferably a mammal, most preferably a human, comprising administering the pharmaceutical formulation to the patient. DDpp drug conjugates

[0319] In further embodiments, the DDpp fusion protein can be linked to other organic or inorganic molecules or substrates by chemical conjugation. In one embodiment, the DDpp-drug conjugate is intended to facilitate the local delivery of cytotoxic agents through the specific targeting of DDpp. This combination of targeting specificity and cytotoxic agent allows targeted delivery of the drug to tumors and their intracellular accumulation, where systemic administration of these unconjugated drug agents can result in unacceptable levels of toxicity not only to the tumor cells being eliminated, but also to normal cells (Baldwin et al., Lancet pages 603-605 (1986); Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al., (ed.s), pp. 475-506 (1985)).

[0320] Cytotoxic agents include chemotherapeutic agents, growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), radioactive isotopes (i.e., radioconjugates), and the like. Chemotherapeutic agents useful in producing such immunoconjugates include, for example, methotrexate, adriamycin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents. Chemotherapeutic agents useful in producing such immunoconjugates also include antitubulin agents, such as auristatins, including monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). Enzymatically active toxins and fragments thereof that can be used in accordance with the disclosed methods include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0321] In one embodiment, the DDpp (e.g., DDpp fusion protein) is conjugated to a radioisotope. In a further embodiment, the DDpp is conjugated to a radioisotope using any one of several known chelators or direct labeling. 90 Y, 125 I, 131 I, 123 I, 111 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188In other embodiments, the DDpp is coupled to a drug, prodrug, or lymphokine such as interferon. Conjugates of DDpp and cytotoxins can be routinely prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyidithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In a specific embodiment, the toxin is conjugated to the DDpp fusion protein through an enzyme-cleavable linker system (such as that present in SGN-35). Conjugates of DDpp with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothenes, and CC1065, and derivatives of these toxins that have toxin activity, can also be used.

[0322] In some embodiments, the cytotoxic agent is covalently attached to the DDpp by a linker. In some embodiments, the linker attaching the DDpp and the cytotoxic agent is cleavable by a protease. Affinity maturation and deimmunization of DD and DDpp

[0323] Affinity maturation strategies can be used to create high-affinity DDs and DDpps that can be used in the DDpp fusion proteins described herein. Improved DDs and DDpps that specifically bind to desired targets (e.g., CS1) can also be prepared based on previous DDpp reference sequences. For example, at least one, two, three, four, five, or more amino acid mutations (e.g., conservative or non-conservative substitutions), deletions, or insertions can be introduced into the DD sequences (i.e., reference sequences) disclosed in Table 1, and the resulting DDpps can be screened for binding to their respective targets and biological activity, for example, for their ability to antagonize or agonize the biological activity of their respective targets.

[0324] The disclosed DDpp, particularly those administered to a subject, are preferably not antigenic to the subject (e.g., human). In some embodiments, the sequence of the DDpp does not contain a human HLA-DR binding motif or a cleavage site for the proteasome and immune-proteasome. In certain embodiments, the DDpp sequence does not contain an antigenic sequence determined by a computer prediction model version existing as of the filing date of this application. In certain embodiments, the DDpp sequence is selected from the group consisting of ProPred (see, e.g., Singh, Bioinformatics 17(12): 1236-1237 (2001)), ProPred1 (Singh, Bioinformatics 19(8): 1009-14 (2003)), SYFPEITHI (see, e.g., Schuler, Immunoinf. Meth. in Mol. Biol. 409(1): 75-93 (2007)), SMM-align (see, e.g., Nielsen, BMC Bioinformatics 8: 238 (2007)), RANKPEP (see, e.g., Reche, Hum Immunol 63: 701-709. (2004)), or TEPITOPE (Sturniolo, Nat Biotechnol 17: 555-561 (2004)). (1999)), where the version of the algorithm and the applied database are those existing as of the filing date of this application. In some embodiments, the DDpp does not contain sequences that share characteristics with high affinity (less than 6% binding threshold) T cell epitopes (Singh, Bioinformatics 17: 1236-1237 (2001)). In some embodiments, the DDpp does not contain sequences that share characteristics with promiscuous (present in more than 50% of relevant alleles) T cell epitopes (Singh, Bioinformatics 17: 1236-1237 (2001)). In some embodiments, the DDpp does not contain sequences that share characteristics with high affinity or promiscuous T cell epitopes.In certain embodiments, the DDpp does not contain the sequence LAAIKTRLQ (SEQ ID NO: 6). Techniques for generating, screening, and identifying affinity-matured DDpp variants and target-binding DDpp variants containing sequence mutations that remove predicted MHC (class I or class II) binding site sequences are known in the art. Polynucleotides

[0325] Also provided is a polynucleotide comprising a nucleotide sequence encoding a DDpp (e.g., adapter or CAR). Such a polynucleotide may further comprise one or more expression control elements, if necessary. For example, the polynucleotide may comprise one or more promoters or transcription enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals as expression control elements. The polynucleotide may be inserted into any suitable vector, which may be contained in any suitable host cell for expression.

[0326] In some embodiments, a polynucleotide disclosed herein encodes a polypeptide comprising a D domain that binds to CS1. In some embodiments, the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60.

[0327] In some embodiments, the polynucleotides disclosed herein encode a chimeric antigen receptor (CAR), wherein the CAR comprises a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain. In some embodiments, the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO: 108.

[0328] In some embodiments, a polynucleotide disclosed herein encodes an adaptor comprising (a) a D domain (DD) that binds to CS1 and (b) an antigenic determinant (AD). In some embodiments, the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the adaptor is a monovalent adaptor comprising a single D domain that binds to CS1. In some embodiments, the adaptor is a bivalent adaptor comprising two D domains that bind to CS1. In some embodiments, the two D domains that bind to CS1 are the same. In some embodiments, the two D domains that bind to CS1 are different. In some embodiments, the adaptor is a bivalent adaptor comprising a first D domain that binds to CS1 and a second D domain that binds to a second AD. In some embodiments, the second AD is BCMA. In some embodiments, the monovalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the monovalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the bivalent adaptor comprises a DD comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the bivalent adaptor comprises a D domain comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the bivalent adaptor comprises two identical D domains comprising the amino acid sequence of SEQ ID NO: 46. In some embodiments, the bivalent adaptor comprises two identical D domains comprising the amino acid sequence of SEQ ID NO: 60. In some embodiments, the adaptor comprises an AFP p26 antigenic determinant (AD). In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14 and 15. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the monovalent adaptor comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the monovalent adaptor comprises the amino acid sequence of SEQ ID NO: 110.In some embodiments, the bivalent adaptor comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the monovalent adaptor comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the adaptor comprises one or more linkers.

[0329] In some embodiments, the polynucleotide disclosed herein encodes an adapter comprising (a) a D domain that binds to CS1 and (b) an antigenic determinant binding domain (ADBD) that binds to AFP p26 AD. In some embodiments, the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14, and 15. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 10. In some embodiments, the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105, and 106. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 46. In some embodiments, the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO: 60. In some embodiments, the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD. In some embodiments, the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO: 21 or 22. In some embodiments, the adapter comprises one or more linkers.

[0330] In some embodiments, the polynucleotide is DNA.

[0331] In some embodiments, the polynucleotide is RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, the RNA, e.g., mRNA, comprises modified ribonucleotides.

[0332] In some embodiments, the mRNA disclosed herein comprises a coding region encoding a polypeptide disclosed herein, and additionally comprises one or more of a 5' untranslated region, a 3' untranslated region, a 5' cap, and a polyadenylation signal. In some embodiments, the mRNA disclosed herein comprises a coding region encoding a polypeptide disclosed herein, a 5' untranslated region, a 3' untranslated region, a 5' cap, and a polyadenylation signal. In some embodiments, the mRNA disclosed herein comprises a modified ribonucleotide. In some embodiments, the mRNA comprises N1-methylpseudouridine or N1-ethylpseudouridine. In some embodiments, the 5' end cap is 7mG(5')ppp(5')N1mpNp. See, e.g., US20200261572, US20190351040, and US20190211065, each of which is incorporated by reference in its entirety.

[0333] In some embodiments, the polynucleotide is a vector comprising the polynucleotide described herein. In some embodiments, the vector is a transfer vector suitable for use in producing recombinant lentivirus. In some embodiments, the vector is a lentiviral vector encoding a polypeptide (e.g., a CAR) disclosed herein. In some embodiments, the lentiviral vector is suitable for transducing immune cells (e.g., T cells or NK cells) to produce cells that express the polypeptide (e.g., a CAR).

[0334] In some embodiments, the polynucleotide is a recombinant virus comprising the polynucleotide described herein.In some embodiments, the polynucleotide is a recombinant lentivirus comprising the polynucleotide encoding the polypeptide (e.g., CAR) described herein.In some embodiments, the polynucleotide is a recombinant adenovirus vector or adeno-associated virus (AAV). DDpp production

[0335] The disclosed DDpp (eg, and adapters) can be made routinely using commercially available reagents and techniques known in the art. In one embodiment, DDpp is prepared using methods known in the art, e.g., Merrifield, J. Am. Chem. Soc. 85: 2149 (1963); Davis et al., Biochem. Intl. 10: 394-414 (1985); Larsen et al., J. Am. Chem. Soc. 115: 6247 (1993); Smith et al., J. Peptide Protein Res. 44: 183 (1994); O'Donnell et al., J. Am. Chem. Soc. 118: 6070 (1996); Stewart and Young, Solid Phase Peptide Synthesis, Freeman (1969); Finn et al., The Proteins, 3rd ed., 2: 105-253 (1976); and Erickson et al., The Proteins, 3rd ed., 2: 257-527 (1976). The present disclosure contemplates synthetic peptides. Alternatively, peptides are recombinantly expressed by introducing nucleic acid encoding the disclosed DDpp into host cells and culturing them to express the peptide. Such peptides are purified from the culture medium or cell pellets.

[0336] Production of DDpp useful in practicing the provided methods can be carried out using a variety of standard techniques for chemical synthesis, semi-synthesis, and recombinant DNA methodology known in the art. Methods for producing DDpp as a soluble agent and cell-associated protein, individually or as part of a multidomain fusion protein, are also provided.

[0337] If desired, the reference sequence and / or the modified polypeptide (e.g., DDpp) can be deimmunized. For example, potentially immunogenic residues or motifs can be identified and modified to reduce or eliminate a potential immune response to DDpp. Additional details regarding various embodiments of the production, selection, and isolation of DDpp are provided in more detail below. A. Recombinant expression of DDpp

[0338] In some embodiments, a DDpp, e.g., a DDpp fusion protein (e.g., an adapter), is "recombinantly produced" (i.e., produced using recombinant DNA technology). Exemplary recombinant methods available for synthesizing a DDpp fusion protein include, but are not limited to, polymerase chain reaction (PCR)-based synthesis, concatemerization, seamless cloning, and recursive directional ligation (RDL) (see, e.g., Meyer et al., Biomacromolecules 3: 357-367 (2002); Kurihara et al., Biotechnol. Lett. 27: 665-670 (2005); Haider et al., Mol. Pharm. 2: 139-150 (2005); and McMillan et al., 32: 3643-3646 (1999), the contents of each of which are incorporated herein by reference in their entirety).

[0339] Nucleic acids comprising polynucleotide sequences encoding DDpp (e.g., adapters or CARs) are also provided. Such polynucleotides may further comprise one or more expression control elements, if necessary. For example, the polynucleotide may comprise one or more promoters or transcription enhancers, ribosome binding sites, transcription termination signals, and polyadenylation signals as expression control elements. The polynucleotide may be inserted into any suitable vector, which may be contained in any suitable host cell for expression.

[0340] Expression of a nucleic acid encoding a DDpp (e.g., an adapter or CAR) is typically achieved by operably linking the nucleic acid encoding the DDpp to a promoter in an expression vector. Typical expression vectors contain transcription and translation terminators, initiation sequences, and a promoter useful for regulating expression of the desired nucleic acid sequence. Expression vectors containing a nucleic acid sequence encoding a DDpp together with appropriate transcriptional / translational control signals can be routinely constructed using methods known in the art. These methods include, but are not limited to, in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. Expression of polynucleotides can be carried out in any suitable expression host known in the art, including, but not limited to, bacterial cells, yeast cells, insect cells, plant cells, or mammalian cells. In one embodiment, the nucleic acid sequence encoding a DDpp is operably linked to a suitable promoter sequence such that the nucleic acid sequence is transcribed and / or translated into a DDpp in the host.

[0341] In one embodiment, a vector containing a nucleic acid encoding DDpp (e.g., an adapter) is introduced into a host cell for expression of DDpp. The vector can remain episomal or be chromosomally integrated, as long as the insert encoding the therapeutic agent can be transcribed. Vectors can be constructed by standard recombinant DNA techniques. Vectors can be plasmids, phages, cosmids, phagemids, viruses, or any other available type used for replication and expression in prokaryotic or eukaryotic cells. Those skilled in the art will recognize that a wide variety of components (such as expression control elements) can be included in such vectors, including a wide variety of transcription signals, e.g., promoters, and other sequences that regulate RNA polymerase binding to the promoter. Any promoter known or demonstrated to be effective in the cells in which the vector is expressed can be used to initiate expression of DDpp. Suitable promoters can be inducible (e.g., regulatable) or constitutive. Non-limiting examples of suitable promoters include the SV40 early promoter region, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the HSV-1 (herpes simplex virus-1) thymidine kinase promoter, the regulatory sequence of the metallothionein gene, and the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: the elastase I gene control region, which is active in pancreatic acinar cells; the insulin gene control region, which is active in pancreatic beta cells; the mouse mammary tumor virus control region, which is active in testicular, breast, lymphoid, and mast cells; the albumin gene control region, which is active in the liver; the alpha-fetoprotein gene control region, which is active in the liver; the alpha 1-antitrypsin gene control region, which is active in the liver; the beta-globin gene control region, which is active in red blood cells; the myelin basic protein gene control region, which is active in oligodendrocyte cells in the brain; the myosin light chain-2 gene control region, which is active in skeletal muscle; and the gonadotropin-releasing hormone gene control region, which is active in the hypothalamus.In certain embodiments, the promoter is an immunoglobulin gene regulatory region that is active in lymphoid cells.

[0342] In one embodiment, one or several nucleic acids encoding DDpp (e.g., adapters) are expressed under the control of a constitutive promoter, or alternatively, a regulated expression system. Suitable regulated expression systems include, but are not limited to, a tetracycline-regulated expression system, an ecdysone-inducible expression system, a lac switch expression system, a glucocorticoid-inducible expression system, a temperature-inducible promoter system, and a metallothionein metal-inducible expression system. When several nucleic acids encoding different DDpps are contained within a host cell system, some of the nucleic acids may be expressed under the control of a constitutive promoter, while others may be expressed under the control of a regulated promoter. Expression levels may be determined by methods known in the art, including Western blot analysis and Northern blot analysis.

[0343] A variety of host-expression vector systems can be utilized to express nucleic acids encoding DDpp (e.g., adapters). Vectors containing nucleic acids encoding DDpp (e.g., individual DD subunits or DDpp fusions), or portions or fragments thereof, include plasmid vectors, single-stranded and double-stranded phage vectors, and single-stranded and double-stranded RNA or DNA viral vectors. Phage and viral vectors can also be introduced into host cells in the form of packaged or enclosed viruses using known techniques for infection and transduction. Viral vectors can also be replication-competent or, alternatively, replication-defective. Alternatively, cell-free translation systems can be used to produce proteins using RNA derived from DNA expression constructs (see, e.g., WO86 / 05807 and WO89 / 01036; and U.S. Pat. No. 5,122,464, the contents of each of which are incorporated herein by reference in their entireties).

[0344] Generally, any type of cell or cultured cell line can be used to express the DDpp (e.g., adapter) provided herein. In some embodiments, the background cell line used to generate the engineered host cell is a bacterial cell, a yeast cell, or a mammalian cell. A variety of host-expression vector systems may be used to express the coding sequence of the DDpp fusion protein. Mammalian cells can be used as host cell lines transfected with recombinant plasmid DNA or cosmid DNA expression vectors containing the coding sequence of the target of interest and the coding sequence of the fusion polypeptide.

[0345] The cells may be primary isolates from organisms (including humans), cultures, or cell lines of transformed or transgenic nature. In some embodiments, the host cells are human cells. In some embodiments, the host cells are human T cells. In some embodiments, the host cells are derived from a human patient.

[0346] Useful host cells include, but are not limited to, microorganisms such as bacteria (e.g., E. coli, B. subtilis, P. fluorescens) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the DDpp (e.g., adapter) coding sequence; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the DDpp coding sequence; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the DDpp coding sequence; and plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) containing the DDpp coding sequence or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the DDpp coding sequence. In certain embodiments, mammalian cell systems are used to produce DDpp. Mammalian cell systems typically utilize recombinant expression constructs containing promoters derived from the genomes of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[0347] Prokaryotes useful as host cells for producing DDpp, e.g., DDpp fusion proteins (e.g., adapters), include gram-negative or gram-positive organisms, such as E. coli and B. subtilis. Expression vectors for use in prokaryotic host cells generally contain one or more phenotypic selection marker genes (e.g., genes encoding proteins that confer antibiotic resistance or supply autotrophic requirements). Examples of useful prokaryotic host expression vectors include pKK223-3 (Pharmacia, Uppsala, Sweden), pGEM1 (Promega, Wis., USA), pET (Novagen, Wis., USA), and pRSET (Invitrogen, Calif., USA) series vectors (see, e.g., Studier, J. Mol. Biol. 219: 37 (1991) and Schoepfer, Gene 124: 83 (1993)). Exemplary promoter sequences frequently used in prokaryotic host cell expression vectors include T7 (Rosenberg et al., Gene 56: 125-135 (1987)), beta-lactamase (penicillinase), lactose promoter systems (Chang et al., Nature 275: 615 (1978)); and Goeddel et al., Nature 281: 544 (1979)), tryptophan (trp) promoter systems (Goeddel et al., Nucl. Acids Res. 8: 4057 (1980)), and the tac promoter (Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0348] In one embodiment, eukaryotic host cell systems are used, including yeast cells transformed with a recombinant yeast expression vector containing the coding sequence for DDpp (e.g., an adapter), such as the expression systems taught in U.S. Application No. 60 / 344,169 and WO 03 / 056914 (Methods for Producing Human-Like Glycoproteins in Non-Human Eukaryotic Host Cells), the contents of each of which are incorporated herein by reference in their entireties. Exemplary yeast that can be used to produce provided compositions, such as DD, include yeast from the genera Saccharomyces, Pichia, Actinomycetes, and Kluyveromyces. Yeast vectors typically contain an origin of replication sequence from the 2mu yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Examples of promoter sequences for yeast expression constructs include promoters from metallothionein, 3-phosphoglycerate kinase (Hitzeman, J. Biol. Chem. 255: 2073 (1980)), and other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Additional suitable vectors and promoters for use in yeast expression and yeast transformation protocols are known in the art. See, for example, Fleer, Gene 107: 285-195 (1991) and Hinnen, PNAS 75: 1929 (1978).

[0349] Insect and plant host cell culture systems are also useful for producing the compositions encompassed by the present disclosure.Such host cell systems include, for example, the insect cell system that is infected with the recombinant virus expression vector (for example, baculovirus) that contains the coding sequence of DD; the plant cell system that is infected with the recombinant virus expression vector (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) that contains the coding sequence of DD or that is transformed with the recombinant plasmid expression vector (for example, Ti plasmid) that contains the coding sequence of DD, including but not limited to the expression systems taught in U.S. Patent No. 6,815,184; U.S. Publication No. 60 / 365,769 and U.S. Publication No. 60 / 368,047; and WO04 / 057002, WO04 / 024927 and WO03 / 078614, the contents of each of which are incorporated herein by reference in their entirety.

[0350] In additional embodiments, host cell lines may be used, including animal cell lines infected with recombinant viral expression vectors (e.g., adenovirus, retrovirus, adeno-associated virus, herpesvirus, lentivirus), including cell lines engineered to contain multiple copies of DNA encoding DDpp, either stably amplified (CHO / dhfr) or unstably amplified in double minute chromosomes (e.g., murine cell lines). In one embodiment, the vector containing the polynucleotide encoding DDpp is polycistronic. Exemplary mammalian cells useful for producing these compositions include 293 cells (e.g., 293T and 293F), CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 (Crucell, Netherlands) cells VERY, Hela cells, COS cells, MDCK cells, 3T3 cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, CRL7O30 cells, HsS78Bst cells, hybridoma cells, and other mammalian cells. Additional exemplary mammalian host cells useful in practicing the provided embodiments include, but are not limited to, T cells. Some examples of expression systems and selection methods are described in the following references and references cited therein: Borth et al., Biotechnol. Bioen. 71(4): 266-73 (2000), ...

Claims

1. A protein that specifically binds to CS1 and comprises a D domain target binding domain comprising the amino acid sequence of SEQ ID NO: 41-105 or 106.

2. The protein of claim 1, wherein the D domain comprises the amino acid sequence of SEQ ID NO:

46.

3. The protein of claim 1, wherein the D domain comprises the amino acid sequence of SEQ ID NO:

60.

4. The protein of any one of claims 1 to 3, wherein the D domain is fused to a heterologous polypeptide.

5. The protein of claim 4 , wherein the heterologous polypeptide comprises a full-length antibody or an antibody fragment.

6. The protein of claim 4 , wherein the D domain is fused to the amino terminus of a full-length antibody heavy chain; the amino terminus of a full-length antibody light chain; the carboxyl terminus of a full-length antibody heavy chain; or the carboxyl terminus of a full-length antibody light chain.

7. The protein of claim 4 , wherein the heterologous polypeptide is an Fc domain.

8. the heterologous polypeptide is (a) a transmembrane domain; (b) membrane-associated domain; (c) human serum albumin or a fragment thereof; (d) AFP or a fragment thereof; (e) AFP p26 or a fragment thereof; and (f) the extracellular domain of a receptor or a fragment thereof 5. The protein of claim 4, comprising a member selected from the group consisting of:

9. The protein of claim 4 , wherein the heterologous polypeptide comprises the extracellular domain or a fragment of the extracellular domain of a receptor selected from the group consisting of BCMA and CS1.

10. The protein of any one of claims 2 to 9, further comprising a peptide linker.

11. The protein according to any one of claims 1 to 10, which is labeled.

12. 12. The protein of claim 11, wherein the label is selected from the group consisting of an enzyme label, a fluorescent label, a luminescent label, a bioluminescent label, and a biotin moiety.

13. The protein of any one of claims 1 to 12, conjugated to a therapeutic or cytotoxic agent.

14. A chimeric antigen receptor (CAR) comprising a target binding domain comprising the protein of any one of claims 1 to 3.

15. The CAR of claim 14, comprising a target binding domain, a transmembrane domain, and an intracellular signaling domain.

16. The CAR of claim 14 or 15, wherein the transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain.

17. The CAR according to any one of claims 14 to 16, wherein the intracellular signaling domain is selected from the group consisting of a human T-cell receptor alpha, beta, or zeta chain domain; a human 41BB domain; a human CD28 domain; and any combination thereof.

18. The CAR according to any one of claims 14 to 17, wherein the intracellular signaling domain comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.

19. The CAR according to any one of claims 14 to 18, further comprising a peptide linker.

20. The CAR of claim 14, comprising the amino acid sequence of SEQ ID NO:

107.

21. The CAR of claim 14, comprising the amino acid sequence of SEQ ID NO:

108.

22. The protein of any one of claims 1 to 13, or the CAR of any one of claims 14 to 21, further comprising a second target binding domain having the same or a different target as the D domain target binding domain.

23. (a) a D domain target binding domain that specifically binds to CS1 and comprises the amino acid sequence of SEQ ID NO: 41-105 or 106, and (b) an adapter comprising an antigenic determinant (AD).

24. 24. The adapter of claim 23, wherein the D domain comprises the amino acid sequence of SEQ ID NO:

46.

25. 24. The adapter of claim 23, wherein the D domain comprises the amino acid sequence of SEQ ID NO:

60.

26. The adapter of any one of claims 23 to 25, wherein the AD comprises AFP p26 or a fragment thereof.

27. 27. The adapter of claim 26, wherein AFP p26 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14 and 15.

28. 27. The adapter of claim 26, wherein AFP p26 comprises the amino acid sequence of SEQ ID NO:

8.

29. 27. The adapter of claim 26, wherein AFP p26 comprises the amino acid sequence of SEQ ID NO:

10.

30. The adapter of any one of claims 23 to 25, wherein the AD comprises BCMA (SEQ ID NO: 2) or a fragment thereof.

31. The adapter of any one of claims 23 to 30, further comprising a peptide linker.

32. 32. The adapter of any one of claims 23 to 31, comprising a single D domain that specifically binds to CS1.

33. 33. The adapter of claim 32, comprising the amino acid sequence of SEQ ID NO:

109.

34. 33. The adapter of claim 32, comprising the amino acid sequence of SEQ ID NO:

110.

35. 32. The adapter of any one of claims 23 to 31, comprising two D domains that specifically bind to CS1.

36. 36. The adapter of claim 35, comprising the amino acid sequence of SEQ ID NO:

111.

37. 36. The adapter of claim 35, comprising the amino acid sequence of SEQ ID NO:

112.

38. An isolated polynucleotide encoding a protein according to any one of claims 1 to 10 or 22, or an adapter according to any one of claims 23 to 37.

39. A vector comprising the polynucleotide of claim 38.

40. 40. The vector of claim 39, wherein the polynucleotide is operably linked to a nucleotide sequence that regulates expression of the protein encoded by the polynucleotide.

41. 41. A host cell comprising a polynucleotide according to claim 38, or a vector according to claim 39 or claim 40.

42. 42. A method for producing a protein according to any one of claims 1 to 10 or 22, or an adaptor according to any one of claims 23 to 37, the method comprising culturing a host cell according to claim 41 under conditions suitable for producing said protein or adaptor.

43. An isolated polynucleotide encoding the CAR of any one of claims 14 to 22.

44. A vector comprising the polynucleotide of claim 43.

45. 45. The vector of claim 44, wherein the polynucleotide is operably linked to a nucleotide sequence that regulates expression of the protein encoded by the polynucleotide.

46. 46. ​​The vector of claim 45, which is a lentiviral vector.

47. A host cell comprising the polynucleotide of claim 43 or the vector of any one of claims 44 to 46.

48. A cell engineered to express the CAR according to any one of claims 14 to 22.

49. 49. The cell of claim 47 or claim 48, which is a T cell or a natural killer (NK) cell.

50. A pharmaceutical composition comprising the protein of any one of claims 1 to 13 or 22, and a pharmaceutically acceptable excipient.

51. 45. A pharmaceutical composition comprising the vector of claim 44 and a pharmaceutically acceptable excipient.

52. 52. The pharmaceutical composition of claim 51, wherein the vector is a lentiviral vector.

53. A pharmaceutical composition comprising a cell expressing the CAR according to any one of claims 14 to 22, and a pharmaceutically acceptable excipient.

54. 54. The pharmaceutical composition of claim 53, wherein the cell is a T cell or a natural killer (NK) cell.

55. A kit comprising an adapter according to any one of claims 23 to 37.

56. 45. A kit comprising the vector of claim 44.

57. A kit comprising a cell expressing the CAR according to any one of claims 14 to 22.

58. 58. The kit of claim 57, wherein the cells are T cells or natural killer (NK) cells.

59. 1. A method of producing an immune response against one or more target cells, the method comprising contacting a composition comprising the target cells with a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

60. 1. A method of killing a target cell, comprising contacting a composition comprising the target cell with a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

61. 61. The method of claim 59 or claim 60, wherein the target cells express CS1.

62. 62. The method of any one of claims 59 to 61, wherein the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106.

63. 62. The method of any one of claims 59 to 61, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

46.

64. 62. The method of any one of claims 59 to 61, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

60.

65. 65. The method of any one of claims 59 to 64, wherein the transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain.

66. 66. The method of any one of claims 59-65, wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T-cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof.

67. 67. The method of any one of claims 59-66, wherein the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

68. The method of any one of claims 59 to 61, wherein the CAR comprising a D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

107.

69. The method of any one of claims 59 to 61, wherein the CAR comprising a D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

108.

70. The method of any one of claims 59 to 69, wherein the cell expressing the CAR is an immune cell.

71. 71. The method of claim 70, wherein the immune cells are autologous cells.

72. 71. The method of claim 70, wherein the immune cells are allogeneic cells.

73. 73. The method of any one of claims 70-72, wherein the cell expressing the CAR is an immune effector cell.

74. The method of any one of claims 70 to 72, wherein the cell expressing the CAR is a T cell.

75. The method of any one of claims 70 to 72, wherein the cell expressing the CAR is a natural killer (NK) cell.

76. 76. The method of any one of claims 59 to 75, wherein the target cell is a cancer cell.

77. 77. The method of claim 76, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

78. 78. The method of any one of claims 59-77, wherein said contacting occurs in a human patient.

79. 1. A method for producing an immune response against a target cell, the method comprising contacting a composition comprising the target cell with an adaptor, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprises (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

80. A method for producing an immune response against a target cell, the method comprising contacting a composition comprising the target cell with an adaptor, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, the CAR comprising (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprises (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).

81. A method for generating an immune response against a target cell, the method comprising contacting a composition comprising the target cell with an adaptor, wherein (a) the composition comprising the target cell further comprises a cell expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprising (i) a D domain that binds to CS1 and (ii) the AD to which the CAR binds.

82. A method for producing an immune response against a target cell, the method comprising contacting a composition comprising the target cell with a cell expressing a CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

83. A method for generating an immune response against a target cell, the method comprising contacting a composition comprising the target cell with a cell expressing a CAR, wherein (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).

84. A method for generating an immune response against a target cell, the method comprising contacting a composition comprising the target cell with a cell expressing a CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cell further comprises an adaptor comprising (i) a D domain that binds to CS1 and (ii) the AD to which the CAR binds.

85. A method for killing target cells, comprising contacting a composition comprising the target cells with an adaptor, wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprises (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

86. A method for killing target cells, comprising contacting a composition comprising the target cells with an adaptor, wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, the CAR comprising (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprises (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).

87. A method for killing target cells, comprising contacting a composition comprising the target cells with an adaptor, wherein (a) the composition comprising the target cells further comprises cells expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the adaptor comprising (i) a D domain that binds to CS1 and (ii) the AD to which the CAR binds.

88. A method for killing target cells, comprising contacting a composition comprising the target cells with cells expressing a CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cells further comprises an adaptor comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

89. A method for killing target cells, comprising contacting a composition comprising the target cells with cells expressing a CAR, wherein (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cells further comprises an adaptor comprising (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to the AFP p26 antigenic determinant (AD).

90. A method for killing target cells, comprising contacting a composition comprising the target cells with cells expressing a CAR, wherein (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain; and (b) the composition comprising the target cells further comprises an adaptor comprising (i) a D domain that binds to CS1 and (ii) the AD to which the CAR binds.

91. 91. The method of any one of claims 79 to 90, wherein the target cells express CS1.

92. 92. The method of any one of claims 79 to 91, wherein the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106.

93. 92. The method of any one of claims 79 to 91, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

46.

94. 92. The method of any one of claims 79 to 91, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

60.

95. 95. The method of any one of claims 79 to 94, wherein the transmembrane domain comprises a CD8a, 41BB, or CD28 transmembrane domain.

96. 96. The method of any one of claims 79-95, wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T-cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof.

97. 97. The method of any one of claims 79-96, wherein the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

98. The method of any one of claims 79 to 91, wherein the CAR comprising a D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

107.

99. The method of any one of claims 79 to 91, wherein the CAR comprising a D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

108.

100. 100. The method of any one of claims 79-99, wherein the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14 and 15.

101. 101. The method of any one of claims 79 to 100, wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

8.

102. 101. The method of any one of claims 79 to 100, wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

10.

103. The method of any one of claims 79 to 102, wherein the ADBD that binds to the AFP p26 AD comprises an scFv that binds to the AFP p26 AD.

104. The method of any one of claims 79 to 102, wherein the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD.

105. 105. The method of claim 104, wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

21.

106. 105. The method of claim 104, wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

22.

107. The method of claim 104, wherein the CAR comprising a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

23.

108. The method of claim 104, wherein the CAR comprising a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

24.

109. The method of any one of claims 79 to 108, wherein the CAR comprises an ADBD that binds to an AD other than CS1, and the ADBD binds to a tumor antigen.

110. 110. The method of claim 109, wherein the tumor antigen is selected from the group of BCMA, CD19 and CD22.

111. 110. The method of claim 109, wherein the tumor antigen is BCMA.

112. 110. The method of claim 109, wherein the tumor antigen is CD19.

113. 113. The method of any one of claims 79 to 112, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

109.

114. 113. The method of any one of claims 79 to 112, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

110.

115. 113. The method of any one of claims 79 to 112, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

111.

116. 113. The method of any one of claims 79 to 112, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

112.

117. The method of any one of claims 79 to 116, wherein the cell expressing the CAR is an immune cell.

118. 118. The method of claim 117, wherein the immune cells are autologous cells.

119. 118. The method of claim 117, wherein the immune cells are allogeneic cells.

120. 120. The method of any one of claims 117-119, wherein the cell expressing the CAR is an immune effector cell.

121. The method of any one of claims 117 to 119, wherein the cell expressing the CAR is a T cell.

122. The method of any one of claims 117 to 119, wherein the cell expressing the CAR is a natural killer (NK) cell.

123. 123. The method of any one of claims 79 to 122, wherein the target cells are cancer cells.

124. 124. The method of claim 123, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

125. 125. The method of any one of claims 79 to 124, wherein said contacting occurs in a human patient.

126. 126. The method of Claim 125, comprising administering to the human patient the cells expressing the CAR and the adaptor in any order.

127. 126. The method of Claim 125, comprising administering said adaptor to said human patient, wherein said human patient has been administered said cells expressing said CAR.

128. 126. The method of Claim 125, comprising administering said adaptor to said human patient, wherein said human patient comprises said cells expressing said CAR.

129. 1. A method of producing an immune response against target cells in a patient, the method comprising administering to the patient cells expressing a chimeric antigen receptor (CAR) comprising: (i) a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

130. 1. A method of killing target cells in a patient in need thereof, comprising administering to the patient cells expressing a chimeric antigen receptor (CAR) comprising: (i) a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

131. The method of claim 129 or claim 130, wherein the target cells express CS1.

132. 132. The method of any one of claims 129 to 131, wherein the target cells are cancer cells.

133. 133. The method of claim 132, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

134. 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient cells expressing a chimeric antigen receptor (CAR) comprising: (i) a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

135. 135. The method of claim 134, wherein the lymphocytes express CS1.

136. 1. A method of treating cancer, comprising administering to a patient in need of cancer treatment a cell expressing a chimeric antigen receptor (CAR) comprising (i) a D domain that binds to CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

137. 137. The method of claim 136, wherein the cancer is a blood cancer.

138. 138. The method of claim 137, wherein the hematological cancer is myeloma, optionally multiple myeloma.

139. 139. The method of any one of claims 129 to 138, wherein the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106.

140. The method of any one of claims 129 to 138, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

46.

141. The method of any one of claims 129 to 138, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

60.

142. 142. The method of any one of claims 129 to 141, wherein the transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain.

143. 143. The method of any one of claims 129-142, wherein the intracellular signaling domain is selected from the group consisting of a domain of the human T-cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof.

144. 144. The method of any one of claims 129-143, wherein the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

145. The method of any one of claims 129 to 138, wherein the CAR comprising a D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

107.

146. The method of any one of claims 129 to 138, wherein the CAR comprising a D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

108.

147. The method of any one of claims 129 to 146, wherein the cell expressing the CAR is an immune cell.

148. 148. The method of claim 147, wherein the immune cells are autologous cells.

149. 148. The method of claim 147, wherein the immune cells are allogeneic cells.

150. 150. The method of any one of claims 147-149, wherein the cell expressing the CAR is an immune effector cell.

151. The method of any one of claims 147 to 149, wherein the cell expressing the CAR is a T cell.

152. The method of any one of claims 147 to 149, wherein the cell expressing the CAR is a natural killer (NK) cell.

153. The method of any one of claims 129-152, wherein administering the cells expressing the CAR comprises administering a pharmaceutical composition comprising the cells expressing the CAR.

154. 1. A method of producing an immune response against a target cell in a patient, the method comprising administering to the patient an adaptor comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

155. 1. A method of killing target cells in a patient in need thereof, comprising administering to said patient an adaptor comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

156. The method of claim 154 or claim 155, wherein the target cells express CS1.

157. 157. The method of any one of claims 154 to 156, wherein the target cell is a cancer cell.

158. 158. The method of claim 157, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

159. 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to said patient an adaptor comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

160. 160. The method of claim 159, wherein the lymphocytes express CS1.

161. A method of treating cancer, comprising administering to a patient in need of cancer treatment an adaptor comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD.

162. 162. The method of claim 161, wherein the cancer is a blood cancer.

163. 163. The method of claim 162, wherein the hematological cancer is myeloma, optionally multiple myeloma.

164. The method of any one of claims 154 to 163, wherein the patient has been administered a cell expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.

165. The method of any one of claims 154-163, wherein the patient comprises cells expressing a CAR, the CAR comprising (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.

166. The method of any one of claims 154 to 163, further comprising administering a cell expressing a CAR, wherein the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.

167. 1. A method of generating an immune response against target cells in a patient, the method comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).

168. A method of killing target cells in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).

169. The method of claim 167 or claim 168, wherein the target cells express CS1.

170. 170. The method of any one of claims 167 to 169, wherein the target cell is a cancer cell.

171. 171. The method of claim 170, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

172. 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).

173. 173. The method of claim 172, wherein the lymphocytes express CS1.

174. 1. A method of treating cancer, comprising administering to a patient in need of cancer treatment an adapter comprising (i) a D domain that binds to CS1 and (ii) an antigenic determinant binding domain (ADBD) that binds to an AFP p26 antigenic determinant (AD).

175. 175. The method of claim 174, wherein the cancer is a blood cancer.

176. 176. The method of claim 175, wherein the hematological cancer is myeloma, optionally multiple myeloma.

177. The method of any one of claims 167-176, wherein the patient has been administered a cell expressing a CAR, and (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.

178. 177. The method of any one of claims 167-176, wherein the patient comprises cells expressing a CAR, and (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.

179. The method of any one of claims 167-176, further comprising administering a cell expressing a CAR, wherein (a) the CAR comprises (i) an AFP p26 antigenic determinant (AD), (ii) a transmembrane domain, and (iii) an intracellular domain.

180. 1. A method for generating an immune response against a target cell in a patient, the method comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient has been administered cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

181. A method for killing target cells in a patient in need thereof, comprising administering to the patient an adaptor comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient has been administered cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain; and the patient has been administered cells expressing a CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

182. The method of claim 180 or claim 181, wherein the target cells express CS1.

183. The method of any one of claims 180 to 182, wherein the target cells are cancer cells.

184. 184. The method of claim 183, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

185. 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient has been administered cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

186. 186. The method of claim 185, wherein the lymphocytes express CS1.

187. A method of treating cancer, comprising administering to a patient in need of cancer treatment an adapter comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient has been administered cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

188. 188. The method of claim 187, wherein the cancer is a blood cancer.

189. 189. The method of claim 188, wherein the hematological cancer is myeloma, optionally multiple myeloma.

190. 1. A method for generating an immune response against a target cell in a patient, the method comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient comprises cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

191. 1. A method of killing target cells in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient comprises cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

192. The method of claim 190 or claim 191, wherein the target cells express CS1.

193. 193. The method of any one of claims 190 to 192, wherein the target cells are cancer cells.

194. 194. The method of claim 193, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

195. 1. A method of lymphodepleting lymphocytes in a patient in need thereof, comprising administering to the patient an adapter comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient comprises cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

196. 196. The method of claim 195, wherein the lymphocytes express CS1.

197. A method for treating cancer, comprising administering to a patient in need of cancer treatment an adapter comprising (i) a D domain that binds to CS1 and (ii) an AD to which a CAR binds, wherein the patient comprises cells expressing the CAR, and (a) the CAR comprises (i) an antigenic determinant binding domain (ADBD) that binds to an antigenic determinant (AD) other than CS1, (ii) a transmembrane domain, and (iii) an intracellular domain.

198. 198. The method of claim 197, wherein the cancer is a blood cancer.

199. 199. The method of claim 198, wherein the hematological cancer is myeloma, optionally multiple myeloma.

200. 200. The method of any one of claims 154 to 199, wherein the D domain that binds to CS1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 41-105 and 106.

201. The method of any one of claims 154 to 200, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

46.

202. The method of any one of claims 154 to 200, wherein the D domain that binds to CS1 comprises the amino acid sequence of SEQ ID NO:

60.

203. 203. The method of any one of claims 154 to 202, wherein the transmembrane domain comprises a CD8a, 41BB or CD28 transmembrane domain.

204. 204. The method of any one of claims 154-203, wherein the intracellular signaling domain is selected from the group consisting of: a domain of the human T-cell receptor alpha, beta, or zeta chain; a human 41BB domain; a human CD28 domain; and any combination thereof.

205. 205. The method of any one of claims 154-204, wherein said intracellular signaling domain comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, and any combination thereof.

206. The method of any one of claims 154-205, wherein the AFP p26 AD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8-14 and 15.

207. 207. The method of any one of claims 154 to 206, wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

8.

208. 207. The method of any one of claims 154 to 206, wherein the AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

10.

209. The method of any one of claims 154 to 208, wherein the ADBD that binds to the AFP p26 AD comprises an scFv that binds to the AFP p26 AD.

210. The method of any one of claims 154 to 208, wherein the ADBD that binds to AFP p26 AD comprises a D domain that binds to AFP p26 AD.

211. 211. The method of claim 210, wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

21.

212. 211. The method of claim 210, wherein the D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

22.

213. The method of claim 210, wherein the CAR comprising a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

23.

214. The method of claim 210, wherein the CAR comprising a D domain that binds to AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

24.

215. The method of any one of claims 154 to 214, wherein the CAR comprises an ADBD that binds to an AD other than CS1, and the ADBD binds to a tumor antigen.

216. 216. The method of claim 215, wherein the tumor antigen is selected from the group of BCMA, CD19 and CD22.

217. 217. The method of any one of claims 154 to 216, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

109.

218. 217. The method of any one of claims 154 to 216, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

110.

219. 217. The method of any one of claims 154 to 216, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

111.

220. 217. The method of any one of claims 154 to 216, wherein the adapter comprising (i) a D domain that binds to CS1 and (ii) an AFP p26 AD comprises the amino acid sequence of SEQ ID NO:

112.

221. The method of any one of claims 154 to 220, wherein the cell expressing the CAR is an immune cell.

222. 222. The method of claim 221, wherein the immune cells are autologous cells.

223. 222. The method of claim 221, wherein the immune cells are allogeneic cells.

224. The method of any one of claims 221-223, wherein the cell expressing the CAR is an immune effector cell.

225. The method of any one of claims 221 to 223, wherein the cell expressing the CAR is a T cell.

226. The method of any one of claims 221 to 223, wherein the cell expressing the CAR is a natural killer (NK) cell.

227. The method of any one of claims 154 to 226, wherein the target cell is a cancer cell.

228. 228. The method of claim 227, wherein the target cell is a myeloma cell, optionally a multiple myeloma cell.

229. The method of any one of claims 154-228, wherein the cells expressing the CAR and the adaptor are administered separately, in any order.

230. 230. The method of any one of claims 154-229, wherein administering the adapter comprises administering a pharmaceutical composition comprising the adapter.

231. The method of any one of claims 154-229, wherein administering the adaptor and cells expressing the CAR comprises administering a pharmaceutical composition comprising the adaptor and a pharmaceutical composition comprising the cells expressing the CAR.

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