D domain-containing polypeptides and uses thereof

Abstract

To provide D domain (DD)-containing polypeptides based on a non-antibody structural scaffold, specifically binding to target proteins of BCMA, CD123, AFP, AFPp26, CS1, or HER2.SOLUTION: The present invention provides DDpp that bind to the target proteins having specific sequences, fusion proteins including DDpp (such as chimeric antibody receptors), nucleic acids encoding these proteins, vectors and host cells containing the nucleic acids, and methods for treatment and diagnosis of a disease, using the proteins, nucleic acids, vectors, cells, or pharmaceutical compositions.SELECTED DRAWING: None

Description

[Background technology]

[0001] Antibody-based reagents have accelerated the pace of biological research and development. Antibody compositions are among the most important and successful therapeutic and diagnostic agents utilized in the pharmaceutical industry. However, cost, time, and efficacy considerations have led to a growing momentum toward the development of alternative affinity reagents.

[0002] A variety of non-antibody conjugation formats have emerged for applications historically achieved with antibodies. While many successful cases have been reported with amorphous linear peptides, more specific results have been achieved by imposing structural constraints on the peptide sequence—usually through the introduction of disulfide bonds. This constraint provides higher affinity and specificity through more favorable thermodynamics of fixed shape complementarity and surface presentation of residues (e.g., hydrophobic amino acids) that might otherwise be buried and therefore not be anti-targets (Ladner, Trends in Biotech. 13(10):426-430 (1995)). Conversely, formats that include disulfide bonds are typically prone to improper pairing of intra- or inter-domain cysteines, which can lead to poor expression, product yield, and product quality.

[0003] Structures found in protein subdomains provided another source of structural constraints. Structures such as fibronectin type III repeats (adnectins), z-proteins (affibodies), knottins, lipocalins (anticalins), and ankyrin repeats (DARPins) have been shown to confer antibody-like affinities to a variety of different targets (Hey et al., Trends in Biotech. 23(10):514-422 (2005)). These domains typically contain two features similar to the framework and complementarity-determining regions (CDRs) found in antibody variable domains: a structural scaffold that confers high thermodynamic stability and residues or loops that form a platform for displaying library variability. Summary of the Invention

[0004] There remains a substantial unmet need for novel target-binding compositions, particularly such reagents comprising alternative binding scaffolds (e.g., non-antibody scaffolds). Reagents 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 scaffolds. In some embodiments, the D-domain polypeptides (DDpp) are characterized by high target binding affinity and a non-antibody scaffold. In some embodiments, the DDpp are target-specific binding polypeptides that can be advantageously used as targeted therapeutics (e.g., immune cells) to specific cells (e.g., diseased cells) with reduced or no off-target effects. In some embodiments, the provided DDpps are used as therapeutics that bind to cells or soluble factors involved in disease.

[0005] In some embodiments, the present disclosure provides a protein (DDpp) comprising a D domain (DD) target binding domain, wherein the DD specifically binds to a target selected from the group consisting of BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), CS1 (SEQ ID NO: 965), HER2 (SEQ ID NO: 967), AFP (SEQ ID NO: 9), AFP p26 (SEQ ID NO: 10), or a fragment thereof. 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 DDs are also provided, as are methods of making and using the fusion proteins. Nucleic acids encoding the DDpp and vectors, and host cells comprising 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.

[0006] In a further embodiment, the present disclosure provides a protein (DDpp) comprising a D domain (DD) target binding domain, wherein the DD is a member selected from the group consisting of: (a) a DD that specifically binds to BCMA (SEQ ID NO: 7) and comprises the amino acid sequence of SEQ ID NO: 11-305, or 306; (b) a DD that specifically binds to CD123 (SEQ ID NO: 8) and comprises the amino acid sequence of SEQ ID NO: 307-739, or 740; (c) a DD that specifically binds to AFP (SEQ ID NO: 9) or a fragment thereof and comprises the amino acid sequence of SEQ ID NO: 741-874, or 886-895; (d) a DD that specifically binds to AFP p26 (SEQ ID NO: 10) and comprises the amino acid sequence of SEQ ID NO: 741-874, or 886-895; (e) a DD that specifically binds to CS1 (SEQ ID NO: 965) or a fragment thereof and comprises the amino acid sequence of SEQ ID NO: 896-909, or 910; and (f) a DD that specifically binds to HER2 or a fragment thereof and comprises the amino acid sequence of SEQ ID NO: 911-949, or 950. Proteins comprising variants of (a)-(f) that retain the ability to specifically bind to their respective targets are also provided. 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 thereof. 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 antibody fragment that is an Fc. In further embodiments, the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane-binding 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 comprises a heterologous polypeptide comprising the extracellular domain or a fragment of the extracellular domain of BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), CD19 (SEQ ID NO: 3), or CS1 (SEQ ID NO: 965). In some embodiments, the protein comprises 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. In some embodiments, the protein comprises a heterologous polypeptide comprising an antigenic portion of a serum protein (e.g., AFP and AFP p26). In some embodiments, the protein comprises 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 further embodiments, the protein is conjugated to a therapeutic or cytotoxic agent. In some embodiments, the protein comprises a heterologous polypeptide that binds to one or more major histocompatibility complexes (MHC) class I or class II complexes.

[0007] In some embodiments, the DD of the DDpp specifically binds to BCMA. In some embodiments, the DD specifically binds to a BCMA protein having an amino acid sequence consisting of SEQ ID NO:7. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:11-305, and 306. In other embodiments, the BCMA-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:11-305, and 306. In some embodiments, the DDpp comprises multiple target binding domains (e.g., a dimer, a trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to BCMA. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with the same sequence. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to different epitopes of BCMA. In some embodiments, the DDpp comprises a DD that specifically binds BCMA and further comprises two, three, four, five, or more than five additional, different DDs or target-binding binding domains (e.g., scFvs) that bind BCMA or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds BCMA and further comprises one or more additional DDs or other target-binding binding domains that bind one or more antigens expressed on the surface of B cells. In some embodiments, the DDpp comprises a DD that specifically binds BCMA and further comprises one or more additional DDs or other target-binding binding domains that bind one or more cancer antigens. In some embodiments, the DDpp specifically binds to two, three, four, five, or more than five different targets. In further embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to cancer antigens expressed on the surface of two, three, four, five, or more than five different cancer cells.

[0008] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to BCMA. In some embodiments, the DDpp fusion protein comprises a DD that specifically binds to BCMA protein having the amino acid sequence of SEQ ID NO:7. In some embodiments, the DDpp is a fusion protein comprising a DD that comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:11-305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:11-305, and 306. 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 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 BCMA-binding DDpp is an Fc-fusion protein.

[0009] In some embodiments, the DDpp fusion protein comprises a BCMA-binding DD operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP or AFP p26, or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP (e.g., SEQ ID NO: 9), or a fragment thereof. In other embodiments, the DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof.

[0010] In some embodiments, the DDpp fusion protein is a soluble protein comprising one or more target-binding DDpp and a p29 protein (e.g., having the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974). Fusion proteins comprising such p29 sequences have surprisingly been discovered herein to have long serum half-lives. 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 more than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours in mice. 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.

[0011] In some embodiments, the BCMA-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: 11-305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In further embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of CD123 (e.g., SEQ ID NO: 8), or a fragment thereof. In some embodiments, the BCMA-binding DDpp fusion protein comprises the extracellular domain of a receptor, or a fragment thereof, selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, CS1, TSLPR, IL7R, and gp96.

[0012] In further embodiments, the BCMA-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: 11-305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the BCMA-binding DDpp fusion protein comprises the extracellular domain of a serum protein (e.g., HSA, AFP, and AFP p26), a receptor (e.g., BCMA, CD123, CS1, and CD19), or a fragment of an intracellular protein (e.g., a nuclear protein), and consists 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 acid residues.

[0013] In some embodiments, the DD of the DDpp specifically binds to CD123. In some embodiments, the DDpp specifically binds to a CD123 protein having an amino acid sequence consisting of SEQ ID NO:8. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:307-739, and 740. In other embodiments, the CD123-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:307-739, and 740. In some embodiments, the DDpp comprises multiple target-binding domains (e.g., a dimer, a trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to CD123. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with the same sequence. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to different epitopes of CD123. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 and further comprises two, three, four, five, or more than five additional, different DDs or target-binding binding domains (e.g., scFvs) that bind BCMA or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 and further comprises one or more additional DDs or other target-binding binding domains that bind one or more antigens expressed on the surface of B cells. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 and further comprises one or more additional DDs or other target-binding binding domains that bind one or more cancer antigens. In some embodiments, the DDpp specifically binds to two, three, four, five, or more than five different targets. In further embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to cancer antigens expressed on the surface of two, three, four, five, or more than five different cancer cells.

[0014] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to CD123. In some embodiments, DD specifically binds to a CD123 protein having the amino acid sequence of SEQ ID NO: 8. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. 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 comprises a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN8828). In other embodiments, the CD123-binding DDpp is an Fc-fusion protein.

[0015] In some embodiments, the DDpp fusion protein comprises a CD123-binding DD operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP or AFP p26, or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP (e.g., SEQ ID NO: 9), or a fragment thereof. In other embodiments, the DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof.

[0016] In some embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In further embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or a fragment thereof. In further embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7), or CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965), or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain or a fragment thereof of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, CS1, TSLPR, IL7R, and gp96.

[0017] In further embodiments, the CD123-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740.

[0018] In some embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of a serum protein (e.g., HSA, AFP, and AFP 26), a receptor (e.g., BCMA, CS1, CD123, and CD19), or a fragment of an intracellular protein (e.g., a nuclear protein), and consists 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.

[0019] In some embodiments, the DD of the DDpp specifically binds to CS1. In some embodiments, the DDpp specifically binds to a CS1 protein having an amino acid sequence consisting of SEQ ID NO: 965. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In other embodiments, the CS1-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In some embodiments, the DDpp comprises multiple target-binding domains (e.g., a dimer, a trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to CS1. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with the same sequence. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to different epitopes of CS1. 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 target-binding binding domains (e.g., scFvs) that bind BCMA or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 and further comprises one or more additional DDs or other target-binding binding domains that bind one or more antigens expressed on the surface of B cells. In some embodiments, the DDpp comprises a DD that specifically binds to CS1 and further comprises one or more additional DDs or other target-binding binding domains that bind one or more cancer antigens. In some embodiments, the DDpp specifically binds to two, three, four, five, or more than five different targets. In further embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to cancer antigens expressed on the surface of two, three, four, five, or more than five different cancer cells.

[0020] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to CS1. In some embodiments, DD specifically binds to a CS1 protein having an amino acid sequence consisting of SEQ ID NO: 965. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. 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: 896-909, and 910. 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 comprises a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN8828). In another embodiment, the CS1-binding DDpp is an Fc fusion protein.

[0021] In some embodiments, the DDpp fusion protein comprises a CS1-binding DD operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. 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: 896-909, and 910. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP or AFP p26, or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP (e.g., SEQ ID NO: 9), or a fragment thereof. In other embodiments, the DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof.

[0022] In some embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. 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: 896-909, and 910. In further embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965), 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, CS-1, TSLPR, IL7R, and gp96, or a fragment thereof.

[0023] In further embodiments, the CS1-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. 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: 896-909, and 910.

[0024] In some embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of a serum protein (e.g., HSA, AFP, and AFP 26), a receptor (e.g., BCMA, CS1, CD123, and CD19), or a fragment of an intracellular protein (e.g., a nuclear protein), and consists 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.

[0025] In some embodiments, the DD of the DDpp specifically binds to AFP or a fragment thereof. In some embodiments, the DDpp specifically binds to an AFP protein having an amino acid sequence consisting of SEQ ID NO:9 or a fragment thereof. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:741-874 and 886-895. In other embodiments, the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:741-874 and 886-895. In some embodiments, the DDpp comprises multiple target binding domains (e.g., a dimer, a trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to AFP or fragments thereof. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs having the same sequence. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to different epitopes of AFP or fragments thereof. In some embodiments, the DDpp comprises a DD that specifically binds to AFP or a fragment thereof, and further comprises two, three, four, five, or more than five additional, different DDs or target-binding binding domains (e.g., scFvs) that bind to AFP, an AFP fragment, or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to AFP or a fragment thereof, 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 cells. In some embodiments, the DDpp comprises a DD that specifically binds to AFP, 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 specifically binds to two, three, four, five, or more than five different targets. In further embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp 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 specifically binds to cancer antigens expressed on the surface of two, three, four, five, or more than five different cancer cells.

[0026] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to AFP, or a fragment thereof. In some embodiments, the DD specifically binds to an AFP protein having the amino acid sequence of SEQ ID NO:9, or a fragment thereof. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:741-874 and 886-895. In other embodiments, the AFP-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:741-874 and 886-895. In some embodiments, the fusion protein comprises an AFP-binding DD operably linked to 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 comprises a commercially approved therapeutic antibody (e.g., rituximab, ofatumumab, ocrelizumab, veltuzumab, MEDI-551, epratuzumab, belimumab, tabalumab, AMG-557, MEDI-570, and NN882). In some embodiments, the AFP-binding DDpp is an Fc-fusion protein.

[0027] In some embodiments, the DDpp fusion protein comprises a DD that specifically binds to AFP or an AFP fragment operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof.

[0028] In some embodiments, the DDpp fusion protein comprises a DD that specifically binds to AFP or an AFP fragment and further comprises an extracellular domain of a receptor or a fragment thereof. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or a fragment thereof. In some embodiments, the DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7), or CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965), or a fragment thereof. In some embodiments, the 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, and gp96, or a fragment thereof.

[0029] In some embodiments, the DDpp fusion protein comprises a DD that specifically binds to AFP or an AFP fragment and further comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895.

[0030] In some embodiments, the AFP-binding DDpp fusion protein comprises a fragment of a serum protein (e.g., HSA), the extracellular domain of a receptor (e.g., BCMA, CS1, CD123, and CD19), or an intracellular protein (e.g., a nuclear protein), and consists 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.

[0031] In some embodiments, the DD of the DDpp specifically binds to AFP p26. In some embodiments, the DDpp specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO: 10 or a fragment thereof. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the DD specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO: 10 but does not specifically bind to AFP having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the DDpp comprises multiple target-binding domains (e.g., a dimer, a trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to AFP p26. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs having the same sequence. In some embodiments, the DDpp comprises a DD that binds to two, three, four, five, or more than five different epitopes of AFP p26. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 and further comprises two, three, four, five, or more than five additional, different DDs or target-binding binding domains (e.g., scFvs) that bind to AFP p26 or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 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 cells. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 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 specifically binds to two, three, four, five, or more than five different targets. In further embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens.In some embodiments, DDpp specifically binds to 2, 3, 4, 5, or more than 5 different cancer antigens expressed on the surface of cancer cells. In some embodiments, DDpp specifically binds to 2, 3, 4, 5, or more than 5 different cancer antigens expressed on the surface of cancer cells.

[0032] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to AFP p26. In some embodiments, the DD specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP p26-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the DD of the DDpp fusion protein specifically binds to AFP p26 but does not specifically bind to AFP having the amino acid sequence of SEQ ID NO: 9. In further embodiments, the DDpp is a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the DDpp comprises a variant of the amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP p26-binding DDpp is an Fc fusion protein.

[0033] In some embodiments, the DDpp fusion protein comprises a DD that specifically binds AFP p26. In other embodiments, the DDpp fusion protein comprises an AFP p26-binding DD operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof.

[0034] In some embodiments, the DDpp fusion protein comprises an AFP p26-binding DD and further comprises the extracellular domain of a receptor or a fragment thereof. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or a fragment thereof. In some embodiments, the DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965), or a fragment thereof. In some embodiments, the 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, and gp96, or a fragment thereof.

[0035] In further embodiments, the DDpp fusion protein comprises an AFP p26-binding DD and further comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the AFP p26-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In other embodiments, the AFP p26-binding DDpp fusion protein comprises a fragment of a serum protein (e.g., HSA), the extracellular domain of a receptor (e.g., BCMA, CS1, CD123, and CD19), or an intracellular protein (e.g., a nuclear protein), and consists 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.

[0036] In some embodiments, the DD of the DDpp specifically binds to HER2. In some embodiments, the DDpp specifically binds to a HER2 protein having an amino acid sequence consisting of SEQ ID NO: 967. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, the HER2-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the DDpp comprises multiple target-binding domains (e.g., a dimer, a trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that bind to HER2. 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 bind to different epitopes of HER2. In some embodiments, the DDpp comprises a DD that specifically binds to HER2 and further comprises two, three, four, five, or more than five additional, different DDs or target-binding binding domains (e.g., scFvs) that bind to BCMA or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to HER2 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 cancer cells. In some embodiments, the DDpp comprises a DD that specifically binds to HER2 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 specifically binds to two, three, four, five, or more than five different targets. In further embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to two, three, four, five, or more than five different cancer antigens. In some embodiments, the DDpp specifically binds to cancer antigens expressed on the surface of two, three, four, five, or more than five different cancer cells.

[0037] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to HER2. In some embodiments, the DD specifically binds to a HER2 protein having an amino acid sequence consisting of SEQ ID NO: 967. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, the HER2-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. 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 comprises a commercially approved therapeutic antibody. In other embodiments, the HER2-binding DDpp is an Fc-fusion protein.

[0038] In some embodiments, the DDpp fusion protein comprises a HER2-binding DD operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, the HER2-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP or AFP p26, or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP (e.g., SEQ ID NO: 9), or a fragment thereof. In other embodiments, the DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof.

[0039] In some embodiments, the HER2-binding DDpp fusion protein comprises the extracellular domain of a receptor or a fragment thereof. In some embodiments, the HER2-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, the HER2-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In further embodiments, the HER2-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965), or a fragment thereof. In some embodiments, the HER2-binding DDpp fusion protein comprises the extracellular domain of a receptor selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, CS-1, TSLPR, IL7R, and gp96, or a fragment thereof.

[0040] In further embodiments, the HER2-binding DDpp fusion protein comprises an intracellular protein (e.g., a nuclear protein) or a fragment thereof. In some embodiments, the HER2-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, the HER2-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950.

[0041] In some embodiments, the HER2-binding DDpp fusion protein comprises the extracellular domain of a serum protein (e.g., HSA, AFP, and AFP 26), a receptor (e.g., BCMA, CS1, CD123, and CD19), or a fragment of an intracellular protein (e.g., a nuclear protein), and consists 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.

[0042] In some embodiments, the DDpp fusion protein comprises a full-length antibody. In further embodiments, the DDpp is a fusion protein comprising a full-length antibody that specifically binds to a cancer antigen. In further embodiments, the DDpp is a fusion protein comprising a full-length antibody, wherein the antibody specifically binds to a cancer antigen believed to be expressed by the cancer of the subject to which the DDpp fusion protein is administered.

[0043] 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, an enzyme label, a fluorescent label, a luminescent label, or a bioluminescent label. 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.

[0044] In other embodiments, the DDpp fusion protein is bound to a solid support, hi some embodiments, the solid support is selected from the group consisting of beads, glass slides, chips, gelatin, and agarose.

[0045] In some embodiments, the DDpp (e.g., a DDpp fusion protein) is bound to the liposome. In some embodiments, the DDpp is bound to the liposome by a covalent bond. In some embodiments, the DDpp is a fusion protein. In further embodiments, the DDpp is a CAR. In further embodiments, the DDpp is bound to the liposome by an ionic bond rather than a covalent bond.

[0046] In some embodiments, the target-binding DDpp is conjugated to a therapeutic or cytotoxic agent (eg, a chemotherapeutic or radiotherapeutic agent).

[0047] In further embodiments, the present disclosure provides a chimeric antigen receptor (CAR) comprising a target binding domain comprising a DD disclosed herein (e.g., a DD comprising the amino acid sequence of SEQ ID NO: 11-949, or 950). In some embodiments, the DD binds to BCMA and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the DD binds to CD123 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the DD binds to AFP and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the DD binds to AFP p26 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. 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 41BB or CD28 transmembrane domain. In some embodiments, the CAR comprises an intracellular signaling domain 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. In some embodiments, the CAR 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 (LFAl), 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 is expressed in an immune cell. In some embodiments, the immune effector cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell). In some embodiments, the CAR is conjugated to a liposome.

[0048] In some embodiments, the CAR comprises two, three, four, five, or more than five DD and / or other binding domains (e.g., scFvs) that specifically bind to a target (e.g., BCMA or CD123) expressed on the surface of a cancer cell. In further embodiments, the CAR comprises two, three, four, five, or more than five DD or other binding domains (e.g., scFvs) that specifically bind to a second, different target expressed on the surface of a cancer cell. In further embodiments, the administered CAR further comprises two, three, four, five, or more than five DD or other binding domains (e.g., scFvs) that specifically bind to a second, different target expressed by a second, different cancer cell or vascular endothelial cell.

[0049] Nucleic acids encoding the disclosed DDpp (e.g., nucleic acid DDpp fusion proteins) are also provided. Additionally, vectors (e.g., plasmids, viral vectors, and non-viral vectors) comprising nucleic acids encoding DDpp (e.g., DDpp fusion proteins) and host cells comprising the nucleic acids and vectors are also 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 further embodiments, the vector comprises one or more additional standard components for expression of a protein-encoding nucleic acid (e.g., a promoter, packaging components, etc.). In some embodiments, the vector is a lentiviral vector.

[0050] The present disclosure also provides host cells comprising a nucleic acid molecule encoding a target-binding DDpp disclosed herein. In some embodiments, the host cell (e.g., a cell line of cells) is genetically modified to express a protein comprising a DD disclosed herein (e.g., a DD having an amino acid sequence of SEQ ID NO: 11-949, or 950). In some embodiments, expression of the DDpp by the host cell allows for production and isolation of the DDpp. In some embodiments, expression results in the DDpp being expressed on the surface and / or integrated into the membrane of the host cell. In some embodiments, the host cell is a viral particle, or 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 human immune cell displays the DDpp on its cell surface.

[0051] The present disclosure further provides host cells 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 comprising a DD comprising an amino acid sequence selected from SEQ ID NOs: 11-305, and 306; SEQ ID NOs: 307-739, and 740; SEQ ID NOs: 741-874 and 886-895; SEQ ID NOs: 896-909 and 910; or SEQ ID NOs: 911-949, and 950, and a transmembrane domain. 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 host cell is an immune 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 (a different epitope of the same target, or a second target expressed by a cancer cell).

[0052] Also provided are mammalian cells that produce membrane-bound virus-like particles (VLPs), wherein the mammalian cells are genetically engineered to express a fusion protein comprising a D-domain polypeptide (DDpp) fused to a chimeric antigen receptor (CAR), and the fusion protein is expressed (e.g., as a transmembrane protein) on the produced VLPs. Depending on the embodiment, the VLPs produced by the mammalian cells are suitable for use as immunogens for antibody production.

[0053] Pharmaceutical compositions comprising the DD-containing proteins disclosed herein, nucleic acids encoding the proteins, vectors comprising the nucleic acids, and host cells comprising the nucleic acids and / or vectors are also provided. Kits (e.g., therapeutic kits, diagnostic kits, kits for research use, etc.) comprising one or more of the disclosed target-binding DDpp (e.g., DDpp fusion proteins such as DD-Fc and DD-CAR), nucleic acid molecules, vectors, and host cells are also provided.

[0054] The DDpps provided herein have the ability to specifically bind to targets of interest (e.g., therapeutic and / or diagnostic targets such as BCMA, CD123, CS1, HER2, AFP, and AFP p26, peptide tags, and serum proteins such as alphafetoprotein, human serum albumin (HSA) or immunoglobulins) in vitro or in vivo, and the ability to serve as reactive sites for linking or conjugating proteins, such as DDpp fusion proteins, to one or more additional moieties (e.g., solid supports) and / or for other modifications. The DDpps provided herein may also have additional desirable properties and / or functions useful for manufacturing, formulation, and biological, diagnostic, and therapeutic applications.

[0055] In some embodiments, the DDpp is used to bind to, detect, and / or quantify a target (e.g., BCMA, CD123, CS1, HER2, AFP, or AFP p26) in a sample containing the target. In one embodiment, the present disclosure provides a method for detecting a target (e.g., BCMA, CD123, CS1, HER2, AFP, or AFP p26) in a sample, the method comprising: (a) contacting the sample with a DDpp comprising a DD that specifically binds to the target and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949 and 950, under conditions favorable for specific binding of the DDpp to the target to form a target / DDpp complex; and (b) detecting the presence of the complex and / or captured target. In some embodiments, the DDpp is immobilized on a solid support. In some embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305 or 306, and the DDpp is used to bind to, detect, and / or quantify BCMA or a fusion protein comprising BCMA in the sample. In some embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739 or 740, and the DDpp is used to bind to, detect, and / or quantify CD123 or a fusion protein comprising CD123 in a sample. In some embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 or 886-895, and the DDpp is used to bind to, detect, and / or quantify AFP p26 or a fusion protein comprising AFP p26 in a sample. In some embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909 or 910, and the DDpp is used to bind to, detect, and / or quantify CS1 or a fusion protein comprising CS1 in a sample. In some embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949 or 950, and the DDpp is used to bind to, detect, and / or quantify HER2 or a fusion protein comprising HER2 in a sample.

[0056] Also provided is a method for quantifying a target of interest (e.g., BCMA, CD123, CS1, HER2, AFP, or AFP p26) in a sample containing the target, the method comprising: (a) contacting the sample with a DDpp comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, immobilized on a solid support, and comprising a DD that specifically binds to the target, under conditions favoring specific binding of the DDpp to the target to form a target / DDpp complex; and (b) detecting the presence of the target / DDpp complex and / or the captured target, wherein quantitative detection of the product is indicative of, or can otherwise correlate to, the amount of the target or the amount of a fusion protein comprising the target in the sample.

[0057] In further embodiments, the provided DDpp is used in protein analysis. In some embodiments, the DDpp is conjugated to a detectable agent and / or tag. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306; SEQ ID NOs: 307-739, and 740; SEQ ID NOs: 741-874, 886-895; SEQ ID NOs: 896-909, and 910; or SEQ ID NOs: 911-949, and 950. In other embodiments, the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306; SEQ ID NOs: 307-739, and 740; or SEQ ID NOs: 741-874, 886-895; SEQ ID NOs: 896-909, and 910; or SEQ ID NOs: 911-949, and 950. In some embodiments, the DDpp is conjugated to a detectable agent. In one embodiment, the detectable agent comprises a chromogen. In another embodiment, the detectable agent comprises a fluorescent dye. In further embodiments, the detectable agent comprises a radionuclide. In some embodiments, DDpp is covalently bound to the detectable agent. In some embodiments, DDpp is a fusion protein. In further embodiments, DDpp is a multimer. In further embodiments, DDpp is bound to a tag. In some embodiments, the tag is a member selected from the group consisting of a polyhistidine tag, a nucleotide tag, and a FLAG tag. In further embodiments, DDpp is bound to a combination of tags (e.g., a polyhistidine tag and a FLAG tag). In some embodiments, DDpp is covalently bound to the tag. In some embodiments, DDpp is a fusion protein. In some embodiments, DDpp is a multimer.

[0058] In further embodiments, the DDpp is bound to a solid support or tag, hi some embodiments, the solid support is a chromatography bead, a resin, a glass slide, a chip, gelatin, or agarose.

[0059] 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, the method comprising administering to a subject in need thereof a therapeutically effective amount of a DDpp (e.g., a DDpp fusion protein) that specifically binds to a therapeutic target of interest (e.g., BCMA, CD123, CS1, HER2, AFP, or AFP p26). In some embodiments, the disease or disorder is cancer, a B-cell malignancy, a disease or disorder of the immune system, or an infectious disease. Also provided are methods of treating a disease or disorder comprising co-administering the disclosed DDpp with an additional therapeutic agent. In some embodiments, the disease or disorder is multiple myeloma. In some embodiments, the disease or disorder is breast cancer or ovarian cancer.

[0060] The target-binding DDpp disclosed herein have uses, including diagnostic and therapeutic uses. In some embodiments, the DDpp are useful in therapeutic settings, for example, for the treatment and / or diagnosis of diseases such as cancer (e.g., solid or hematologic tumors).

[0061] In some embodiments, the present disclosure provides methods of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of: a protein (i.e., DDpp) comprising a DD disclosed herein (e.g., a DD disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NO: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid, or a host cell comprising the nucleic acid or vector. In some embodiments, the DDpp comprises a DD amino acid sequence that specifically binds to BCMA. In some embodiments, the DD of the DDpp specifically binds to BCMA having the amino acid sequence of SEQ ID NO: 7. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the DDpp comprises a DD amino acid sequence that specifically binds to CD123. In some embodiments, the DD of the DDpp specifically binds to CD123 having the amino acid sequence of SEQ ID NO: 8. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the DDpp comprises a DD amino acid sequence that specifically binds to CS1. In some embodiments, DD of DDpp specifically binds to CS1 having the amino acid sequence of SEQ ID NO: 965. In further embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In some embodiments, DDpp comprises a DD amino acid sequence that specifically binds to HER2. In some embodiments, DD of DDpp specifically binds to HER2 having the amino acid sequence of SEQ ID NO: 967. In further embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, DDpp comprises a DD amino acid sequence that specifically binds to AFP. In some embodiments, DD of DDpp specifically binds to AFP having the amino acid sequence of SEQ ID NO: 9. In further embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895.In some embodiments, DDpp specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO: 10. In further embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, DD of DDpp specifically binds to AFP p26 but does not specifically bind to AFP having the amino acid sequence of SEQ ID NO: 9. In further embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895.

[0062] In some embodiments, the present disclosure provides a method of treating a subject with a B-cell malignancy, the method comprising administering to the subject an effective amount of: a protein comprising a DD disclosed herein (e.g., a DD disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NOs: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid, or a host cell comprising the nucleic acid or vector. In some embodiments, the B-cell malignancy is selected from the group consisting of lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and myeloma. In some embodiments, the B-cell malignancy is selected from the group consisting of acute lymphocytic leukemia, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma, plasmacytoma, and multiple myeloma.

[0063] In some embodiments, the disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject immune cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a target binding domain comprising a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, and the polypeptide specifically binds to the target and transmembrane domain. In further embodiments, the target is expressed by a cancer cell. In some embodiments, the DD specifically binds to BCMA having an amino acid sequence consisting of SEQ ID NO: 7. In further embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the DD specifically binds to CD123 having an amino acid sequence consisting of SEQ ID NO: 8. In further embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the DD specifically binds to CS1 having an amino acid sequence consisting of SEQ ID NO: 965. In further embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In some embodiments, the DD specifically binds to HER2 having the amino acid sequence of SEQ ID NO: 967. In further embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the DD specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO: 10. In further embodiments, the DD comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the CAR comprises an intracellular domain. In further embodiments, the intracellular domain comprises a signaling domain, and upon administration to a subject with cancer, the target binding domain specifically binds to a target expressed by the cancer cell (e.g., BCMA and CD123), and target binding induces the immune cell to generate a cytotoxic signal that exerts a cytotoxic effect on the cancer cell. In some embodiments, the immune cell is a T cell. In other embodiments, the immune cell is a NK cell. In some embodiments, the administered CAR immune cell is not a T cell or an NK cell.In further embodiments, a combination of different CAR immune cell types (e.g., NK cells and T cells) is administered to a subject. In some embodiments, the immune cells are administered intravenously. In some embodiments, a combination of different CAR immune cell types is administered intravenously to a subject. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is breast cancer or ovarian cancer.

[0064] In some embodiments, the disclosure provides a method of treating a subject with a B-cell malignancy, the method comprising administering to the subject immune cells comprising a chimeric antigen receptor (CAR), wherein the CAR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, and comprises a target binding domain comprising a polypeptide that specifically binds to a target expressed by a cancer cell; a transmembrane domain; and an intracellular domain. In some embodiments, the target binding domain specifically binds to BCMA having the amino acid sequence of SEQ ID NO: 7. In further embodiments, the target binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the target binding domain specifically binds to CD123 having the amino acid sequence of SEQ ID NO: 8. In further embodiments, the target binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the target binding domain specifically binds to CS1 having the amino acid sequence of SEQ ID NO: 965. In further embodiments, the target binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In some embodiments, the CAR comprises an intracellular domain. In further embodiments, the intracellular domain comprises a signaling domain, and upon administration to a subject with cancer, the target binding domain specifically binds to a target expressed by the malignant B cell (e.g., BCMA, CS1, and CD123), and target binding induces the immune cell to generate a cytotoxic signal that confers a cytotoxic effect on the malignant B cancer cell. In some embodiments, the B cell malignancy is selected from the group consisting of lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and myeloma. In some embodiments, the B cell malignancy is selected from the group consisting of acute lymphocytic leukemia, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, plasmacytoma, and multiple myeloma. In some embodiments, the B cell malignancy is multiple myeloma. In some embodiments, the immune cell is a T cell. In other embodiments, the immune cell is a NK cell.In some embodiments, the administered CAR immune cells are not T cells or NK cells. In further embodiments, a combination of different CAR immune cell types (e.g., NK cells and T cells) is administered to a subject. In some embodiments, the immune cells are administered intravenously. In some embodiments, a combination of different CAR immune cell types is administered intravenously to a subject.

[0065] In some embodiments, the present disclosure provides a method of treating a subject with a B cell-associated disorder, the method comprising administering to the subject an effective amount of: a protein comprising a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NO: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid, or a host cell comprising the nucleic acid or vector.

[0066] In some embodiments, the present disclosure provides a method of treating a subject with an immune system disorder, the method comprising administering to the subject an effective amount of: a protein comprising a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NOs: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid, or a host cell comprising the nucleic acid or vector. In some embodiments, the immune system disorder is an autoimmune disease such as rheumatoid arthritis.

[0067] In some embodiments, the present disclosure provides use of a composition for treating cancer, the composition comprising a protein comprising a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NOs: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid, or a host cell comprising the nucleic acid or vector. In some embodiments, the cancer is a B-cell malignancy. In some embodiments, the B-cell malignancy is selected from the group consisting of lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and myeloma. In some embodiments, the B-cell malignancy is selected from the group consisting of acute lymphocytic leukemia, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, plasmacytoma, and multiple myeloma. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is breast cancer or ovarian cancer.

[0068] In some embodiments, the present disclosure provides use of a composition for treating a B-cell malignancy, the composition comprising a protein comprising a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NOs: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid; or a host cell comprising the nucleic acid or vector. In some embodiments, the B-cell malignancy is selected from the group consisting of lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and myeloma. In some embodiments, the B-cell malignancy is selected from the group consisting of acute lymphocytic leukemia, chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, plasmacytoma, and multiple myeloma. In some embodiments, the B-cell malignancy is multiple myeloma.

[0069] In some embodiments, the present disclosure provides for the use of a composition for treating a B cell-related disorder, the composition comprising a protein comprising a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NO: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid; or a host cell comprising the nucleic acid or vector.

[0070] In some embodiments, the present disclosure provides use of a composition for treating an immune system disorder, the composition comprising a protein comprising a DD disclosed herein (e.g., a DD having an amino acid sequence disclosed in Table 1, or a DD having an amino acid sequence of SEQ ID NOs: 11-949, or 950); a nucleic acid encoding the protein; a vector comprising the nucleic acid; or a host cell comprising the nucleic acid or vector. In some embodiments, the immune system disorder is an autoimmune disease such as rheumatoid arthritis.

[0071] In further embodiments, the present disclosure provides use of immune cells comprising a chimeric antigen receptor (CAR) for the treatment of cancer, the CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949 and 950, comprising a target binding domain comprising a polypeptide that specifically binds to a target expressed by a cancer cell; a transmembrane domain; and an intracellular domain, wherein the intracellular domain comprises a signaling domain, and upon administration to a subject with cancer, the target binding domain specifically binds to a target expressed by the cancer cell, and target binding induces the immune cell to generate a cytotoxic signal that produces a cytotoxic effect against the cancer cell. In some embodiments, the immune cell is a T cell or a natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is not a T cell or an NK cell. In further embodiments, a combination of different CAR immune cell types (e.g., a T cell and an NK cell) is used. In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is breast cancer or ovarian cancer.

[0072] In further embodiments, the present disclosure provides use of immune cells comprising a chimeric antigen receptor (CAR) for the treatment of B cell malignancies, wherein the CAR comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, and specifically binds to a target (e.g., a target expressed by malignant B cells, such as BCMA, CS1, or CD123), and comprises a target binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-306, 307-740, and 896-910; a transmembrane domain; and an intracellular domain, wherein the intracellular domain comprises a signaling domain, and upon administration to a subject with cancer, the target binding domain specifically binds to the target expressed by the malignant B cells, and target binding induces the immune cell to generate a cytotoxic signal that produces a cytotoxic effect against the malignant B cells. In some embodiments, the immune cell is a T cell or a natural killer (NK) cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is not a T cell or an NK cell. In further embodiments, a combination of different CAR immune cell types (e.g., T cells and NK cells) is used. In some embodiments, the B cell malignancy is multiple myeloma.

[0073] In further embodiments, the present disclosure provides use of immune cells comprising a chimeric antigen receptor (CAR) for the treatment of an immune system disorder, the CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, comprising a target binding domain comprising a polypeptide that specifically binds to a target expressed by the targeted immune cell; a transmembrane domain; and an intracellular domain, wherein the intracellular domain comprises a signaling domain, and upon administration to a subject with an immune system disorder, the target binding domain specifically binds to a target expressed by the targeted immune cell, and target binding induces the recombinant immune cell to generate a cytotoxic signal that produces a cytotoxic effect against the targeted immune cell. In some embodiments, the immune cell is a T cell or a natural killer (NK) cell. In some embodiments, the immune cell is not a T cell or an NK cell. In some embodiments, the immune cell is a natural killer (NK) cell. In some embodiments, the immune cell is not a T cell or an NK cell. In further embodiments, a combination of different CAR immune cell types (e.g., a T cell and an NK cell) is used.

[0074] The methods summarized above and / or described herein describe specific acts performed by a practitioner; however, it is understood that the methods can also include teachings of those acts by another party. Thus, an act such as "administering T cells comprising a target-specific binding polypeptide-CAR" includes "teachings of administering T cells comprising a target-specific binding polypeptide-CAR."

[0075] In some embodiments, the present disclosure provides: [1] A protein comprising a D domain (DD) target binding domain, the D domain being a member selected from the group consisting of: (a) a DD that specifically binds to BCMA and comprises the amino acid sequence of SEQ ID NO: 11 to 305, or 306; (b) a DD that specifically binds to CD123 and comprises the amino acid sequence of SEQ ID NO: 307 to 739 or 740; (c) a DD that specifically binds to AFP and includes the amino acid sequence of SEQ ID NOs: 741 to 874 or 886 to 895; (d) a DD that specifically binds to AFP p26 and comprises the amino acid sequence of SEQ ID NOs: 741 to 874 or 886 to 895; (e) a DD that specifically binds to CS1 and includes the amino acid sequence of SEQ ID NO: 896 to 909 or 910; (f) A DD that specifically binds to HER2 and comprises the amino acid sequence of SEQ ID NO: 911 to 949 or 950. [2] The protein according to [1], wherein the DD is fused to a heterologous polypeptide. [3] The protein of [2], wherein the heterologous polypeptide comprises a full-length antibody or an antibody fragment. [4] The protein of [2], wherein the heterologous polypeptide comprises a member selected from the group consisting of: (a) transmembrane domain, (b) membrane-binding 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 the receptor or a fragment thereof. [5] The protein of [3], wherein the DD is fused to the amino terminus of the full-length antibody heavy chain; the amino terminus of the full-length antibody light chain; the carboxyl terminus of the full-length antibody heavy chain; or the carboxyl terminus of the full-length antibody light chain. [6] The protein according to [3], wherein the heterologous polypeptide is Fc. [7]. The protein according to [2], wherein the heterologous polypeptide comprises an extracellular domain, or a fragment of an extracellular domain, of a receptor selected from the group consisting of BCMA, CD123, CS1, and CD19. [8]. A labeled protein according to any one of [1] to [7]. [9]. The protein of [9], 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.

[10] . The protein according to any one of [1] to

[10] , which is conjugated to a therapeutic agent or a cytotoxic agent.

[11] . A chimeric antigen receptor (CAR) comprising a target binding domain comprising a protein according to any one of [1] to [5].

[12] . The CAR described in

[11] , comprising a target binding domain, a transmembrane domain, and an intracellular signaling domain.

[13] . The CAR according to

[11] or

[12] , wherein the transmembrane domain comprises a 41BB or CD28 transmembrane domain.

[14] . A CAR described in any one of

[11] to

[13] , 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.

[15] . A CAR according to any one of

[11] to

[14] , 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 (LFA1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.

[16] . The protein according to any one of [1] to

[15] , further comprising a second target binding domain having the same or a different target as the DD target binding domain.

[17] . An isolated nucleic acid encoding the protein according to any one of [1] to

[16] .

[18] . A vector comprising the nucleic acid according to

[17] .

[19] . The vector of

[18] , wherein the nucleic acid is operably linked to a nucleotide sequence that regulates expression of the protein encoded by the nucleic acid.

[20] . The vector according to

[19] , which is a lentiviral vector.

[21] A host cell comprising the nucleic acid according to

[17] or the vector according to any one of

[18] to

[21] .

[22] . A cell genetically modified to express a protein according to any one of [1] to

[16] .

[23] The cell according to

[21] or

[22] , which is a T cell or a natural killer (NK) cell.

[24] . A pharmaceutical composition comprising a protein according to any one of [1] to

[16] , a nucleic acid according to

[17] , a vector according to

[18] ,

[19] or

[20] , or a cell according to any one of

[21] to

[23] .

[25] A kit comprising the protein according to any one of [1] to

[16] .

[26] . A method of treating a subject for cancer, comprising: A method comprising administering to a subject an effective amount of a protein according to any one of [1] to

[16] , a nucleic acid according to

[17] , a vector according to

[18] ,

[19] or

[20] , a cell according to any one of

[21] to

[23] , or a pharmaceutical composition according to

[24] .

[26] . The method of

[26] , wherein the cancer is a B-cell malignancy selected from the group consisting of lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, plasmacytoma, and myeloma.

[28] . The method of

[27] , wherein the B-cell malignant tumor is selected from the group consisting of chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma, plasmacytoma, and multiple myeloma.

[27] . The method of

[26] , wherein the cancer is a myeloid malignancy selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, leukemia, plasmacytoma, and myeloma.

[29] A method for treating a subject with a B-cell-related disorder (e.g., monoclonal gammopathy of undetermined significance (MGUS)), comprising administering to the subject an effective amount of the protein described in any one of [1] to

[16] , the nucleic acid described in

[17] , the vector described in

[18] ,

[19] , or

[20] , the cell described in any one of

[21] to

[23] , or the pharmaceutical composition described in

[24] .

[30] . A method for treating a subject with a disorder of the immune system, comprising administering to the subject an effective amount of a protein according to any one of [1] to

[16] , a nucleic acid according to

[17] , a vector according to

[18] ,

[19] or

[20] , a cell according to any one of

[21] to

[23] , or a pharmaceutical composition according to

[24] .

[31] The method according to

[30] , wherein the immune system disorder is an autoimmune disease such as rheumatoid arthritis.

[32] A method of treating a subject for cancer, comprising administering to the subject immune cells comprising a chimeric antigen receptor (CAR), the CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, and comprising a target binding domain comprising a DD, optionally capable of specifically binding to a target expressed by a cancer cell; a transmembrane domain; and an intracellular domain, the intracellular domain comprising a signaling domain, wherein upon administration to the subject, the target binding domain specifically binds to a target expressed by the cancer cell, and target binding induces the immune cell to generate a cytotoxic signal that produces a cytotoxic effect against the cancer cell.

[33] The method according to

[32] , wherein the immune cells are T cells.

[34] The method according to

[32] , wherein the immune cells are NK cells.

[35] . The method of

[32] , wherein the administration is intravenous.

[36] . Use of a composition for treating cancer, wherein the composition comprises a protein according to any one of [1] to

[16] , a nucleic acid according to

[17] , a vector according to

[18] ,

[19] or

[20] , or a cell according to any one of

[21] to

[23] .

[37] . The use according to

[36] , wherein the cancer is a B-cell malignancy.

[38] . The use of

[37] , wherein the B-cell malignancy is selected from the group consisting of lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma (NHL)), leukemia, and myeloma.

[39] . The use according to

[38] , wherein the B-cell malignancy is selected from the group consisting of chronic lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma.

[40] Use of a composition for treating a B-cell-related disorder, the composition comprising a protein according to any one of [1] to

[16] , a nucleic acid according to

[17] , a vector according to

[18] ,

[19] or

[20] , a cell according to any one of

[21] to

[23] , or a pharmaceutical composition according to

[24] .

[41] . Use of a composition for treating an immune system disorder, wherein the composition comprises a protein according to any one of [1] to

[16] , a nucleic acid according to

[17] , a vector according to

[18] ,

[19] or

[20] , a cell according to any one of

[21] to

[23] , or a pharmaceutical composition according to

[24] .

[42] The use according to

[41] , wherein the immune system disorder is an autoimmune disease such as rheumatoid arthritis.

[43] . Use of immune cells comprising a chimeric antigen receptor (CAR) for the treatment of cancer, wherein the CAR comprises a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, and optionally comprises a target binding domain that may specifically bind to a target expressed by a cancer cell; a transmembrane domain; and an intracellular domain, wherein the intracellular domain comprises a signaling domain, wherein upon administration to a subject with cancer, the target binding domain specifically binds to a target expressed by the cancer cell, and target binding induces the immune cell to generate a cytotoxic signal that produces a cytotoxic effect against the cancer cell.

[44] . The use according to

[43] , wherein the immune cells are T cells or natural killer (NK) cells. [Brief explanation of the drawings]

[0076] [Figure 1](Figures 1A-1B) DDpp, as part of a fusion protein (e.g., an antibody-DDpp fusion protein), confers a new binding specificity to another molecule (e.g., a full-length antibody). DDpp-antibody fusions were created using an RSV-specific antibody (SYN) and a non-targeting peptide (DD) of SEQ ID NO: 1 or the CD137-specific DDpp (bb10) (SEQ ID NO: 876). DDpp is fused to the N-terminus (bb10-SYN and DD-SYN) or C-terminus (SYN-bb10 and SYN-DD). All four antibody fusions bind to RSV (Figure 1A). However, fusion of bb10 to the N-terminus (bb10-SYN) or C-terminus (SYN-bb10) of the antibody heavy chain confers a new CD137-binding specificity to a normally monospecific antibody (Figure 1B). [Figure 2-1] (Figures 2A-2F) CD123-DDpp-CAR T cells produce cytokines in response to target binding. Figures 2A and 2B show data related to interferon gamma (IFNγ) production by CD123-DDpp-CAR-expressing T cells when co-cultured with the CD123-negative tumor K562 and the CD123-positive tumor BDCM, respectively. Figures 2C and 2D show similar data measuring interleukin-2 (IL2) production by CD123-DDpp-CAR T cells when co-cultured with K562 and BDCM, respectively. Figures 2E and 2F show similar target-driven cytokines (IFNγ, IL2) by PD-L1-DDpp CAR T cells when co-cultured with PDL1-expressing tumor cells (SUDHL-1). [Figure 2-2] See description of Figure 2-1. [Figure 3](Figures 3A-3B) DDpp-CAR-expressing T cells do not undergo over-depletion to a greater extent than scFv. Figure 3A shows the expression of three exhaustion markers (LAG3, PD1, and TIM3) on T cells expressing various DDpp-CARs at levels similar to the expression of these markers on scFv-CAR 32716 (3278 (Du X1, Ho M, Pastan I. 2007. New immunotoxins targeting CD123, a stem cell antigen on acute myeloid leukemia cells. J Immunother. 30(6):607-13). Figure 3B shows flow cytometry data demonstrating similar exhaustion marker expression on DDpp-CAR T cells (expressing CD123-targeting cg06 DDpp) compared to CAR T cells expressing a CD123-specific scFv (32716). [Figure 4] (Figure 4A-4C) When co-cultured with target-expressing tumor cells (BDCM), DDpp-CAR-expressing T cells (CD123-targeted) undergo degranulation. Figures 4A-4C show results obtained by culturing T cells alone (Figure 4A) or in the presence of K562 cells (Figure 4B) or BDCM cells (Figure 4C). [Figure 5-1] (Figures 5A-5D) DDpp-CAR-expressing T cells mediate target-specific tumor cytotoxicity. Figure 5A shows data related to % killing of CD123-negative K562 tumor cells by CD123-DDpp-CAR T cells. Figure 5B shows % killing when CD123-targeted DDpp-CAR T cells were co-cultured with CD123-positive BDCM cells. Data from Figures 5A and 5B were generated using T cells from a first donor blood sample. Figures 5C and 5D show similar data from collected T cells from a second donor. [Figure 5-2] See description of Figure 5-1. [Figure 6](Figures 6A-6E) Bispecific DDpp-CAR T cells. Figure 6A shows the % of T cells expressing CD123-targeted DDpp-CAR (cg06). Figure 6B shows the % of T cells expressing PDL1-targeted DDpp-CAR (pb04). Figure 6C shows the % of T cells expressing bispecific CD123-PDL1-targeted DDpp-CAR (expressed with cg06 DDpp distal to the T cell membrane compared to pb04 DDpp). Figure 6D shows the % of T cells expressing bispecific PDL1-CD123-targeted DDpp-CAR (expressed with pb04 DDpp distal to the T cell membrane compared to cg06 DDpp). Figure 6E shows data related to the increased intracellular signaling of bispecific DDpp. [Figure 7] (Figures 7A-7B) Dual-binding domain adaptor proteins promote enhanced signaling by CAR-expressing Jurkat NFAT-luciferase reporter cells compared with single-binding domain adaptor proteins. In Figure 7A, 50,000 reporter cells previously transduced with AFP (p26 domain)-linked CAR (af03) were cultured for 5 hours in the presence of the CD123-specific Cg06 adaptor (Cg06-p26) or Cg06-dual adaptor protein (Cg06-p26-Cg06) in the presence of 50,000 CD123+ MOLM13 or CD123-deficient MOLM13 cells, and then luciferase activity was assessed. CD123-deficient cells were generated using CRISPR / Cas9 genetic engineering technology. In Figure 7B, 50,000 reporter cells previously transduced with AFP (p26 domain)-conjugated CAR (af03) were cultured for 5 hours in the presence or absence of 50,000 BCMA+ U266 cells in the presence of the BCMA-specific Bc40-adapter (Bc40-p26) or Bc40-dual adaptor protein (Bc40-p26-Bc40), and luciferase activity was then assessed. [Figure 8] A BCMA-targeted DDpp-CAR containing bc40 DDpp (sequence number 164) eradicates the BCMA-expressing tumor, U226, in a mouse model of B-cell cancer. DETAILED DESCRIPTION OF THE INVENTION

[0077] Any section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0078] Definition of Terms Whenever an embodiment is described herein with the term "comprising," other similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of." However, when used as transitional phrases in the claims, each should be construed separately and in the appropriate legal and factual context (e.g., "comprising" is considered the more open-ended phrase, "consisting of" is considered more exclusive, and "consisting essentially of" is considered intermediate).

[0079] As used herein, the singular forms "a," "an," and "the" include plural references unless specifically stated otherwise.

[0080] The term "and / or" as used in phrases such as "A and / or B" is intended herein to include both A and B; A or B; A alone; and B alone. Similarly, the term "and / or" as used in phrases such as "A, B and / or C" encompasses 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.

[0081] The terms "protein" and "polypeptide" are used interchangeably herein to refer to biological polymers comprising units derived from amino acids joined via peptide bonds; a protein may be composed of two or more polypeptide chains.

[0082] As used interchangeably herein, the terms "antibody" or "immunoglobulin" include full-length antibodies and antibody fragments containing any functional domain of an antibody, such as an antigen-binding fragment or single chain thereof, an effector domain, a salvage receptor-binding epitope, or a portion thereof. A typical antibody contains at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region consists of one domain, Cl. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FW). Each VH and VL consists of three CDRs and four FWs, arranged from the amino terminus to the carboxy 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, and combinations thereof, for example, DDpp is covalently bound (e.g., via a peptide bond or via a chemical linker) to the N-terminus of the heavy and / or light chains of a typical whole (full-length) antibody, or intercalated into the H and / or L chains of a full-length antibody.

[0083] 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, a salvage receptor-binding epitope, or a 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. As used herein, an "antibody fragment" comprises an antigen-binding site or an epitope-binding site. In one embodiment, the DDpp fusion protein comprises an effector domain or a portion thereof. In one embodiment, the DDpp fusion protein comprises a salvage receptor-binding epitope or a portion thereof.

[0084] 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 can 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 class of immunoglobulin from which the heavy chain constant region is derived is IgG (Igγ) (γ subclasses 1, 2, 3, or 4). Other classes of immunoglobulins, such as IgA (Igα), IgD (Igδ), IgE (Igε), and IgM (Igμ), may also be used. Although the boundaries of the Fc region vary, the human IgG heavy chain Fc region is usually defined to include residues C226 or p260 at its carboxyl terminus, with numbering 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 can refer to this region alone, or in the context of a full-length antibody, an antibody fragment, or an Fc fusion protein.Polymorphisms have been observed at a number of different Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358 according to the EU index numbering system; therefore, slight differences may exist between the presented sequence and prior art sequences. 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 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 of either IgA, IgD, IgE, or IgM. Additionally, amino acid substitutions or deletions within the immunoglobulin heavy chain constant region may be useful in practicing the methods and compositions disclosed herein. One example would be to introduce amino acid substitutions in the upstream CH2 region to generate Fc variants with reduced affinity for Fc receptors (Cole, J. Immunol. 159:3613 (1997)).

[0085] "Antibody-dependent cellular 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 lyse (or cause other cytotoxic effects on) the target cells. To assess 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, ADCC activity of a molecule of interest can be assessed in vivo using an animal model such as that disclosed in Clynes et al., PNAS 95:652-656 (1998).

[0086] As used herein, a "single-chain variable fragment," or "scFv" antibody, refers to a form of an antibody (e.g., an antibody fragment) that contains only the variable regions of the heavy and light chains joined by a linker peptide. In one embodiment, a DDpp fusion protein comprises DDpp and an scFv.

[0087] The term "linker" refers to a peptide or other chemical linker positioned between a DDpp and another polypeptide of a DDpp fusion protein. Suitable linkers for joining two or more linked DDpps will be apparent to those skilled in the art, and non-limiting examples are described herein.

[0088] As used herein, the term "operably linked" refers to the association of two molecules such that each retains at least some level of functional activity that each molecule had alone (each molecule retains the functional activity). In some embodiments where one molecule does not have a functional activity, it is operably linked to another molecule if the other molecule has at least some level of that functional activity. Operably linked can also refer to the linkage of two non-functional molecules. Two molecules can be "operably linked" whether they are directly or indirectly (e.g., via a linker).

[0089] The terms "specifically bind" or "having selective affinity for," "bind," or "binding" are used interchangeably and mean that a binding agent, such as DDpp, reacts with or binds to an epitope, protein, or target molecule more frequently, more rapidly, for a longer period of time, with greater affinity, or some combination of the above, than with another agent, including an unrelated protein, for the target epitope, protein, or target molecule. Due to sequence identity between homologous proteins in different species, specific binding, in some embodiments, includes a binding agent that recognizes proteins or targets in more than one species. Similarly, due to homology within sequence regions of particular polypeptides of different proteins, specific binding can include a binding agent that recognizes more than one protein or target. 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 (although it can include) exclusive binding, e.g., binding to a single target. 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 the binding agent.

[0090] By "target" is meant any molecule or combination of molecules that can be bound by a DDpp, such as a DDpp fusion protein, or other component of a DDpp fusion protein, such as an antibody or antibody variable domain fragment.

[0091] The terms "epitope" and "antigenic determinant" are used interchangeably herein and refer to that portion of any molecule (e.g., a target such as BCMA, CD123, AFP, or AFP p26) that can be recognized and specifically bound by a specific binding agent (e.g., DDpp or an antibody). When the recognized molecule is a polypeptide, an epitope is formed from contiguous and non-contiguous amino acids and / or chemically active surface groups of other molecules (e.g., carbohydrates) juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturation, whereas epitopes formed by tertiary folding are typically lost upon protein denaturation. An epitope typically comprises at least three amino acids, more usually at least five or eight to ten amino acids, in a unique spatial conformation.

[0092] As used herein, "peptide tag" refers to a peptide sequence that is part of or attached (e.g., by genetic engineering) to another protein to confer function to the resulting fusion. Peptide tags are typically short relative to the protein to which they are fused; for example, peptide tags in some embodiments are 4 or more amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more amino acids in length. In some embodiments, DDpp is a fusion protein comprising the peptide tag. In other embodiments, DDpp specifically binds to the peptide tag. Many peptide tags having the uses provided herein are known in the art. Examples of peptide tags that may be components of a DDpp fusion protein or target bound by a DDpp (e.g., a DDpp fusion protein) 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® (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 of cells, ELISA assays, and immunostaining.

[0093] The term "natural" when used in reference to biological materials such as nucleic acid molecules, polypeptides, and host cells means that which is found in nature and has not been modified by humans. Conversely, the terms "non-natural" or "synthetic" when used in reference to biological materials means that which is not found in nature and has been modified by humans.

[0094] As used herein, "modifications" to the sequence of a reference sequence include substitutions, deletions, insertions, and / or additions to the sequence at corresponding amino acid positions in the reference sequence (e.g., a DD disclosed herein).

[0095] A "substitution" relative to a sequence of a reference sequence refers to the substitution of a particular amino acid residue with a different amino acid residue at the corresponding amino acid position in the reference sequence.

[0096] 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, including 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), and cysteine ​​(D). Conservative substitutions include: alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W)), nonpolar 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 a DDpp result in altered or unchanged specific binding of the DDpp containing the substitution to the target to which it binds (e.g., BCMA, CD123, AFP, or AFP p26). In one embodiment, conservative substitutions in the sequence of a DDpp do not abrogate binding of the DDpp containing the substitution to the target to which it binds. Methods for identifying nucleotide and amino acid conservative and non-conservative 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)).

[0097] A "non-conservative" amino acid substitution is one in which one amino acid residue is replaced with another amino acid residue having a heterologous side chain. In one embodiment, the non-conservative substitution in the sequence of DDpp results in specific binding of the DDpp containing the substitution to its target (e.g., BCMA, CD123, AFP, or AFP p26). In one embodiment, the non-conservative substitution in the sequence of DDpp does not abrogate binding of the DDpp containing the substitution to its target.

[0098] 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 with the DDpp provided herein are known in the art. In one embodiment, the unnatural amino acid is 4-hydroxyproline, which can be substituted for proline, 5-hydroxylysine, which can be substituted for lysine, 3-methylhistidine, which can be substituted for histidine, homoserine, which can be substituted for serine, and ornithine, which can be substituted for lysine. Additional examples of unnatural amino acids that can be substituted with the DDpp disclosed herein include, but are not limited to, molecules such as D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-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, β-alanine, lanthionine, dehydroalanine, γ-aminobutyric acid, selenocysteine, and pyrrolidine fluoroamino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, and Nα-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 acid or amino acid analog can include one or more amino acid deletions from the sequence.

[0099] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric forms of nucleotides of any length, ribbon nucleotides or deoxynucleotides, including, but 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.

[0100] As used herein, the term "naked DNA" refers to DNA (e.g., histone-free DNA) encoding a protein such as a DDpp (e.g., a CAR) disclosed herein that is cloned into a suitable expression vector (e.g., a plasmid) in the proper orientation for expression. Viral vectors that can be used to carry and / or express DNA encoding DDpp include, but are not limited to, SIN lentiviral vectors, retroviral vectors, foamy virus vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, hybrid vectors, and / or plasmid transposons (e.g., the Sleeping Beauty transposon system), or integrase-based vector systems. Other vectors that can be used in connection with the production and use of DDpp are described herein or are known in the art.

[0101] As used herein, the terms "vector," "cloning vector," and "expression vector" refer to a vehicle by which a nucleic acid sequence (e.g., a disclosed DDpp coding sequence) can be maintained or propagated 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.

[0102] A "host cell" includes an individual cell or cell culture that can be or has been a recipient of a nucleic acid encoding a disclosed DDpp. Host cells include, but are not limited to, virus particles, phagemids, bacteria, yeast, plant, animal, and mammalian cells. A host cell includes the progeny of a single host cell, which progeny need not be exactly identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations and / or changes. A host cell includes cells transfected or infected with a nucleic acid encoding a disclosed DDpp in vivo, in vitro, or ex vivo. In some examples, a host cell can express and display a disclosed DDpp on its surface, for example, in phage display or CAR T cells. "Expression" includes transcription and / or translation.

[0103] 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 filter membrane) to which DDpp, antibodies, or other proteins can be bound (e.g., coupled, linked, or attached) directly or indirectly (e.g., via other antibodies or other binding partner intermediates such as protein A), or into which DDpp or antibodies can be embedded (e.g., via receptors or channels). Reagents and techniques for binding 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, chromatographic resins or matrices (e.g., Sepharose-4FF 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 immobilized on support materials non-covalently or covalently using reagents and techniques known in the art. In one embodiment, DDpp is attached to the chromatographic material using a linker.

[0104] As used herein, the terms "pharmaceutically acceptable" or "physiologically acceptable" and grammatical variations thereof, when referring to compositions, carriers, diluents, and reagents, are used interchangeably to indicate that the substance can be administered to a human without producing therapeutically prohibited undesirable physiological effects, such as nausea, dizziness, acute gastric peristalsis, etc.

[0105] "Modulate" refers to the regulation or control of magnitude, frequency, extent, or activity. In another related aspect, such modulation can be positive (e.g., an increase in frequency, extent, or activity) or negative (e.g., a decrease in frequency, extent, or activity). In some embodiments, the positive or negative modulation is relative to the function of the cell, tissue, or organ prior to administration of the therapeutic agent. In further embodiments, the positive or negative modulation is relative to the function of a normal, healthy cell, tissue, or organ.

[0106] An "effective amount" of a DDpp, such as a DDpp fusion protein provided herein, is an amount sufficient to achieve a specifically stated purpose, such as causing an observable change in the level of one or more biological activities associated with a target to which the DDpp (e.g., DDpp fusion protein) binds. In certain embodiments, the change increases the level of target activity. In other embodiments, the change decreases the level of target activity. An "effective amount" can be determined empirically and routinely with respect to the stated purpose. The term "therapeutically effective amount" refers to an amount of a DDpp, such as a DDpp fusion protein, or other therapeutic agent effective to "treat" (e.g., reduce the symptoms of a disease or disorder) a disease or disorder in a subject (mammal). The term "therapeutically effective amount" also refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result.

[0107] "Patient," "subject," "animal," and "mammal" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, mice, and other animals. Animals include all vertebrates, e.g., mammals, such as chickens, amphibians, and reptiles, and non-mammals. As used herein, "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cows, sheep, pigs, goats, and horses; domestic animals, such as dogs and cats; and laboratory animals, including rodents, such as mice, rats, and guinea pigs. In certain embodiments, the patient is a human. The term does not denote a particular age or sex. Thus, adult and neonatal / pup subjects, as well as fetuses / pups, regardless of male or female, are intended to be included within the scope of this term.

[0108] As used herein, "treat," "treatment," and "treating" refer to both therapeutic and prophylactic or preventative treatment, where the objective is to prevent or delay (reduce or slow) the symptoms, complications, or biochemical manifestations of a disease, condition, or disorder, or to alleviate the disease, condition, or disorder or to arrest or inhibit its further progression. "Treatment" can target a pathological condition; prophylactic (preventing or delaying the onset of the disease or preventing the onset of clinical or asymptomatic symptoms) or therapeutic suppression or alleviation of symptoms after the onset of a disease, condition, or disorder; prevent a pathological condition; pursue or achieve a beneficial outcome; or reduce the likelihood of the development of a particular condition, even if the treatment is ultimately unsuccessful. Subjects in need of treatment include those already with the condition, as well as those prone to the condition or those in whom the condition is to be prevented. Treatment can involve the use of DDpp fusion proteins alone or in combination with additional therapeutic agents.

[0109] "Cancer," "tumor," or "malignant tumor" are used interchangeably and refer to any of a number of diseases characterized by uncontrolled, abnormal proliferation of cells, spread of infected cells locally or to other parts of the body via the bloodstream and lymphatic system (metastasis), and numerous distinctive structural and / or molecular features. As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. A "cancerous tumor," or "malignant cell," is understood to be a cell that has specific structural characteristics, lacks differentiation, and is capable of invasion and metastasis. Cancers that can be treated using the DDpp fusion proteins provided herein include hematological tumors, such as leukemia and lymphoma, or solid tumors. In certain embodiments, the cancer being treated is a leukemia or lymphoma. Cancer types that can be treated with DDBpp include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Cancer and tumor types that can be treated with DDpp include, but are not limited to, breast cancer, lung cancer, brain cancer, bone cancer, liver cancer, kidney cancer, colon cancer, head and neck cancer, ovarian cancer, hematopoietic cancers (e.g., leukemia), and prostate cancer. In some embodiments, cancers and tumors that can be treated with DDpp include breast cancer and ovarian cancer. Other types of cancers and tumors that can be treated with DDpp-containing antibodies are described herein or are known in the art.

[0110] The terms "tumor antigen" and "cancer antigen" are used interchangeably herein. Tumor and cancer antigens can be 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 do not occur in other cells in the body. TAAs are antigens found in both tumors and some normal cells. Due to the dynamic nature of tumors, in some cases, tumor cells may express antigens unique to a particular stage, and in other cases, express antigens that are also expressed on non-tumor cells. Therefore, the inclusion of a particular marker as a TAA does not exclude it from being considered a TSA.

[0111] As used herein, "target cell" refers to a cell involved in a disease and capable of being targeted by a DDpp-containing composition. Other target cells include any cell in a subject (e.g., a human or animal) that can be targeted by the disclosed DDpp. A target cell can be a cell that expresses or overexpresses the target specifically bound by the DDpp fusion protein.

[0112] As used herein, the term "effector cell" refers to a leukocyte that expresses one or more FcRs and performs effector function. Preferably, the cell expresses at least FcRIII 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 natural sources, such as blood or PBMCs, as described herein or known in the art. In certain embodiments, the effector cells are human effector cells.

[0113] The term "effector function" refers to the differentiated function of a differentiated cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0114] As used herein, the term "immune cell" refers to a cell of the 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.

[0115] 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.

[0116] As used herein, the term "immune response" means immunity, including, but not limited to, innate immunity, humoral immunity, cell-mediated immunity, immunity, inflammatory response, acquired (acquired) immunity, autoimmunity, and / or hyperactive immunity.

[0117] As used herein, the term "transduction" refers to the introduction of foreign nucleic acid into a cell using a viral vector. As used herein, the term "gene transfer" 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, where the host cell expresses the introduced nucleic acid to produce a substance, such as a protein or enzyme, encoded by the desired 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 also include nonfunctional 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.

[0118] "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 a metastatic cell. As used herein, "cell surface receptor" also encompasses molecules expressed on the cell surface, including DDpp, that can bind to a target (e.g., BCMA, CD123, CS1, HER2, AFP, or AFP p26). The term "receptor" refers to a cell-associated protein that binds to or interacts with a molecule (e.g., a ligand) and mediates the action 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, which includes a ligand-binding domain, a transmembrane domain, and an intracellular effector domain that is typically involved in signal transduction.

[0119] As used herein, the term "CS1" refers to an NK cell receptor that regulates immune function and is expressed on B cells, T cells, dendritic cells, NK-T cells, and monocytes alike. CS1 is overexpressed in multiple myeloma and has been successfully targeted for immunotherapy of multiple myeloma. Malaer & Mathew, Am J Cancer Res. 7(8):1637-1641 (2017). CS1 is also known as SLAM7, protein 19A, CRACC, and CD319. The term "CS1" includes variants, isoforms, homologs, orthologs, and paralogs. CS1 is a transmembrane protein with various alternatively spliced ​​isoforms. In some embodiments, the amino acid sequence of human CS1 comprising a 22 amino acid residue N-terminal signal sequence (MAGSPTCLTLIYILWQLTGSAA, SEQ ID NO: 964) and an extracellular domain comprising 226 N-terminal residues (SEQ ID NO: 965) has GenBank Accession Number NP_067004 (SEQ ID NO: 966). In some embodiments, the amino acid sequence of human CS1 has GenBank Accession Number NP_001269517, NP_001269518, NP_001269519, NP_001269520, NP_001269521, NP_001269522, NP_001269523, NP_001269524, or NP_001269525.

[0120] As used herein, "chimeric antigen receptor" or "CAR" or "CARs" refers to a genetically engineered receptor that transfers antigen or target specificity into a cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell; a NK cell; a NKT cell, or a combination thereof). CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors.

[0121] D domain polypeptide (DDpp) Unless otherwise indicated, the practice of the disclosed compositions and methods employs standard techniques of molecular biology (including recombinant techniques, tissue culture, and cell transformation), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are typically performed according to manufacturer's specifications or as commonly practiced, using known procedures, or routine modifications thereof, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)); PCR Technology: Principles and Applications for DNA Amplification (ed. H.A. Erlich, Freeman Press, NY, 1992); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Handbook of Experimental Immunology (Weir et al., eds.); Gene Transfer Vectors for Mammalian Cells (Miller, ed., 1987); Current Protocols in Molecular Biology (Ausubel, ed., 1987); PCR Protocols: A Guide to Methods and Applications (Innis, ed., Academic Press, San Diego, Calif., 1990); Mattila, et al., Nucleic Acids Res. 19:967 (1991); Eckert, et al., PCR Methods and Applications 1:17 (1991); PCR (McPherson, ed., IRL Press, Oxford); PCR: The Polymerase Chain Reaction, (Mullis, ed.,1994); Harlow, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988) and Kontermann, ed., “The Antibody Engineering Lab Manual” (Springer Verlag, Heidelberg / New York, 2000); Current Protocols in Immunology (Coligan, ed., 1991); The Immunoassay Handbook (Wild, ed., Stockton Press NY, 1994); and Methods of Immunological Analysis (Masseyeff., ed., Weinheim: VCH Verlags Gesellschaft mbH, 1993); and Gennaro, et al. 2000, Remington: the Science and Practice of Pharmacy, 20th Ed. Lipincott Williams and Wilkins: Baltimore, Md. Unless specific definitions are provided, the nomenclature used in connection with, and the laboratory methods and techniques for, analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those known and used in the art. Moreover, standard techniques can be used for chemical synthesis, chemical analysis, recombinant production, purification, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0122] In various embodiments, the present disclosure provides a DDpp that specifically binds to a target selected from the group consisting of BCMA, CD123, CS1, HER2, AFP, and AFP p26. In some embodiments, the DD of the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950. In some embodiments, the DDpp comprises a D domain (DD) selected from the group consisting of: (a) a DD that specifically binds to BCMA and comprises the amino acid sequence of SEQ ID NO: 11-305, or 306; (b) a DD that specifically binds to CD123 and comprises the amino acid sequence of SEQ ID NO: 307-739, or 740; (c) a DD that specifically binds to AFP or a fragment thereof and comprises the amino acid sequence of SEQ ID NO: 741-874, or 886-895; (d) a DD that specifically binds to AFP p26 and comprises the amino acid sequence of SEQ ID NO: 741-874, or 886-895; (e) a DD that specifically binds to CS1 (SEQ ID NO: 965) or a fragment thereof and comprises the amino acid sequence of SEQ ID NO: 896-909, or 910; or (f) a DD that specifically binds to HER2 or a fragment thereof and comprises the amino acid sequence of SEQ ID NO: 911-949, or 950. Proteins comprising variants of (a)-(f) that retain the ability to specifically bind to their respective targets are also provided.

[0123] In further embodiments, the DD of the DDpp is a variant of a DD reference sequence selected from the group consisting of SEQ ID NOs: 11-949, and 950, which retains the ability to specifically bind to the target of the reference DD. 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: 11-949, and 950, wherein the variant DD retains the ability to specifically bind to the target of the reference DD sequence.

[0124] In some embodiments, the DD of the DDpp is a variant of a BCMA-binding DD reference sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306, and retains the ability to specifically bind to BCMA. 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: 11-305, and 306, and wherein the variant DD retains the ability to specifically bind to BCMA.

[0125] In some embodiments, DD of DDpp is a variant of a CD123-binding DD reference sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740, and retains the ability to specifically bind to CD123. 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: 307-739, and 740, and wherein the variant DD retains the ability to specifically bind to CD123.

[0126] 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: 896-909, and 910, and 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: 896-909, and 910, and wherein the variant DD retains the ability to specifically bind to CS1.

[0127] In some embodiments, the DD of the DDpp is a variant of a HER2-binding DD reference sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950, and retains the ability to specifically bind to HER2. 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: 911-949, and 950, and wherein the variant DD retains the ability to specifically bind to HER2.

[0128] In some embodiments, the DD of the DDpp is a variant of an AFP-binding DD reference sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895, and retains the ability to specifically bind to AFP. 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: 741-874 and 886-895, and the variant DD retains the ability to specifically bind to AFP.

[0129] In some embodiments, the DD of the DDpp is a variant of an AFP p26-binding DD reference sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895, and retains the ability to specifically bind to AFP p26. 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: 741-874 or 886-895, and the variant DD retains the ability to specifically bind to AFP p26.

[0130] In certain embodiments, the identity between a variant DD (query) sequence and a reference DD sequence, also referred to as a 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). 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 subject amino acid sequence. In this embodiment, if the reference DD sequence is shorter than the variant DD query sequence due to N- or C-terminal deletions rather than because of internal deletions, a manual correction is made to the results to account for the fact that the FASTDB program does not take into account the N- and C-terminal truncations of the reference DD sequence when calculating the percent global identity. For reference sequences that are N- and C-terminally truncated relative to the query sequence, the % identity is corrected by calculating the number of residues in the query sequence that are N- and C-terminal to the reference sequence, as a percentage of the total bases in the query sequence, that do not match / align with the corresponding subject residue. The determination of whether a residue matches / aligns is determined by the results of a FASTDB sequence alignment. This % is then subtracted from the % identity and calculated by the FASTDB program above using the specified parameters to arrive at a final % identity score. This final % identity score is what is used for the purposes of this embodiment.

[0131] In some embodiments, DDpp includes variant DDs that contain amino acid sequences that differ in two or more sequence modification categories (i.e., substitutions, deletions, insertions, and additions) from the corresponding reference DDs of SEQ ID NOs: 11-949, or 950, and the variant DDs retain the ability to bind to the respective reference DD targets (e.g., BCMA, CD123, CS1, HER2, AFP, and AFP p26). For example, the variant DD sequence can contain a combination of amino acid deletions, insertions, and substitutions compared to the reference DD sequence. In some embodiments, the variant DD sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid substitutions compared to the reference DD sequence of SEQ ID NOs: 11-949, or 950. In some embodiments, the variant DD sequence contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 conservative amino acid substitutions compared to the reference DD sequence of SEQ ID NOs: 11-949, or 950. In some embodiments, the sequence of the variant DD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 non-conservative amino acid substitutions relative to the reference DD sequence of SEQ ID NOs: 11-949, or 950. In some embodiments, the sequence of the variant DD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 conservative and non-conservative amino acid substitutions relative to the reference DD sequence of SEQ ID NOs: 11-949, or 950. In some embodiments, the sequence of the variant DD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid deletions relative to the reference DD sequence of SEQ ID NOs: 11-949, or 950. In some embodiments, the sequence of the variant DD comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid insertions into the reference DD sequence of SEQ ID NOs: 11-949, or 950, wherein the variant DD retains the ability to bind to the target of the reference DD. Additionally, DDpp are provided that comprise variant DDs having amino acid residues deleted from the amino terminus, carboxy terminus, or both the amino and carboxy termini of the corresponding reference DDs of SEQ ID NOs: 866 and 867.In some embodiments, the sequence of the variant DD comprises a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues have been deleted from the amino terminus of a reference DD sequence of SEQ ID NOs: 11-949, or 950, wherein the variant DD retains the ability to bind to the target of the reference DD. In some embodiments, the sequence of the variant DD comprises a sequence in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues have been deleted from the carboxy terminus of a reference DD sequence of SEQ ID NOs: 11-949, or 950, wherein the variant DD retains the ability to bind to the target of the reference DD. In some embodiments, the sequence of the variant DD comprises 1 to 5, 1 to 10, or 1 to 15 amino acid residues deleted from the amino terminus of a reference DD sequence of SEQ ID NO: 11-949, or 950, and the variant DD sequence has 1 to 5, 1 to 10, or 1 to 15 amino acid residues deleted from the carboxy terminus of the reference DD sequence, and the variant DD retains the ability to bind to the target of the reference DD.

[0132] DDpp fusion protein "Fusion proteins," "chimeric polypeptides," "chimeric proteins," "chimeric antigens," and DDpps consisting of / comprising heterologous polypeptides consist of at least two polypeptides and, optionally, a linker that operably links the two polypeptides into one contiguous polypeptide, e.g., produced by a recombinant process. The two polypeptides can be operably linked directly or indirectly.

[0133] A "DDpp fusion protein" provided herein comprises at least one DDpp disclosed herein that specifically binds to a target (e.g., BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), CS1 (SEQ ID NO: 965), HER2 (SEQ ID NO: 967), AFP (SEQ ID NO: 9), AFP p26 (SEQ ID NO: 10), or a fragment thereof). In one embodiment, the DDpp fusion protein comprises one DDpp.

[0134] 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 the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974). Fusion proteins comprising such p26 sequences have surprisingly been discovered herein to have long serum half-lives. 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 more than 65 hours, or 1-10 hours, 2-10 hours, 4-10 hours, 6-10 hours, or 6-9 hours in mice. 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.

[0135] In some embodiments, the present disclosure provides methods for altering the in vivo half-life (e.g., in mice or humans) of a soluble fusion protein comprising a p26 protein (e.g., having the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974). 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 extended or shortened 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 another embodiment, the p26 sequence of the soluble fusion protein is modified by making 1, 2, 3, 5, 5, 10, or 1-20, 1-10, 3-10, or 3-5 amino acid substitutions (conservative and / or non-conservative substitutions), deletions, and / or insertions 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: 10 of 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 deleting 1-150, 1-100, 1-50, 1-25, or 1-10 amino acid residues to increase or decrease the in vivo half-life of the soluble fusion protein. In further embodiments, the p26 sequence of the soluble fusion protein is modified by making 1, 2, 3, 5, 5, 10, or 1-20, 1-10, 3-10, or 3-5 amino acid substitutions (conservative and / or non-conservative substitutions), deletions, and / or insertions to increase or decrease the interaction of the soluble fusion protein with FeRn.

[0136] Multimeric DDpp fusion protein 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 a further embodiment, 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 2, 3, 4, or 5, or more than 5, DDpps. In some embodiments, a DDpp fusion protein comprises 1 to 3, 1 to 4, 1 to 5, or more than 5 different DDpps. In some embodiments, a DDpp fusion protein comprises at least 2, 3, 4, or 5, or more than 5 different DDpps. Thus, a DDpp fusion protein can be a monomeric DDpp (i.e., comprising one DDpp) or a multimeric DDpp (i.e., comprising two or more DDpps operably linked in tandem, optionally via a linker). In some embodiments, the use of a multimeric DDpp results in enhanced (e.g., synergistic) target binding. In a further embodiment, multimeric DDpps allow targeting of two or more targets using a single DDpp construct (e.g., bispecific, trispecific, etc.). The combination of two or more identical DDpps results in multivalent molecules that offer distinct advantages over monovalent compositions (e.g., increased avidity, target clustering, and receptor activation). The combination of two or more different DDpps results in multivalent and multispecific molecules that have the potential to bind two or more target antigens, separately or simultaneously.

[0137] Multimeric DDpp fusion proteins can be DDpp homomultimers (i.e., containing two or more identical DDpps in tandem, optionally joined 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 in a multimeric composition can vary depending on the embodiment and can be determined, at least in part, by the expression system in which the DDpp is produced. However, in some embodiments, the fusion protein can include multimers 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 intermediate numbers and endpoints between the recited numbers). Furthermore, the multiple tandem components of a DDpp fusion protein can include the same or different DDpps. In some DDpp fusions, DDpp exists as a monomer or in homomultimers or heteromers, eg, homodimers or heterodimers, homotrimers or heterotrimers, homotetramers or heterotetramers.

[0138] DDpp fusion proteins can be "monospecific" or "multispecific." A "multispecific" (e.g., bispecific, trispecific, or even more multispecific) DDpp fusion protein recognizes and binds to two or more different epitopes present on one or more different molecules (e.g., proteins, solid support structures, etc.).

[0139] In some embodiments, two or more DDs are fused together as a multivalent DDpp. The DDs of a multivalent DDpp may 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 DDs are different), which contain any of the DDs described herein and may be optionally linked by one or more linkers.

[0140] In some embodiments, two or more DDs are linked by a multimerization domain or by chemical linkage to generate a multivalent DD complex. The DDs in a multivalent DD complex may be the same or different. Thus, 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 DDs are different), which include any of the DDs described herein and may be optionally linked by one or more linkers.

[0141] In one embodiment, the multispecific DDpp fusion protein comprises at least two DDpps that bind to at least two different epitopes on a single target (e.g., BCMA, CD123, CS1, HER2, AFP, or AFP p26). 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, the bispecific DDpp fusion protein specifically binds to a target on a cancer cell and a target on an immune effector cell. In one embodiment, the bispecific DDpp fusion protein specifically binds to a target expressed on a cancer cell (e.g., BCMA, CS1, CD123, and CD19) and a target expressed on the surface of a T lymphocyte (e.g., CD3). In one embodiment, the bispecific DDpp fusion protein specifically binds to BCMA and CS1.

[0142] In a further embodiment, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to one epitope on a target and at least one domain or sequence (e.g., an antibody fragment or domain such as an scFv) that confers the function of specifically binding to a different epitope on the same target. In one embodiment, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope on a target 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. In one embodiment, the multispecific DDpp fusion protein comprises at least one DDpp that specifically binds to an epitope on a target 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 that confers a function, e.g., an antibody fragment or a domain that specifically binds to a solid support.

[0143] In further embodiments, multimeric DDpp fusions comprising two or more DDpps may be further fused to other heterologous proteins (or subdomains thereof), thereby conferring multivalency and multispecificity 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) α chains, T cell receptor β 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 and their respective positions within the fusion protein may vary. For example, DDpps may be located at one or all termini of the fusion partner and / or may be interspersed within heterologous subunits within the DDpp fusion partner.

[0144] In further embodiments, the DDpp fusion protein comprises a polypeptide sequence comprising 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 a portion thereof. In further embodiments, the Fc domain is a variable human Fc domain.

[0145] 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 thereof. 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 antibody fragment that is an Fc. In further embodiments, the heterologous polypeptide comprises a member selected from the group consisting of: (i) a transmembrane domain; (ii) a membrane-binding 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 of a cell surface receptor or a fragment of the extracellular domain.

[0146] 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, antibody fragment, serum protein (e.g., human serum albumin) or serum protein fragment, or cell surface receptor, T cell receptor (TCR) alpha chain, T cell receptor beta chain, cytokine, growth factor, hormone, or enzyme, or fragment thereof. Incorporation of the DD into multidomain and / or multifunctional complexes 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 further include other optional components, such as linkers and other components described herein.

[0147] DDpp fusion protein as CAR In addition to incorporating DDs into soluble multidomain proteins, the present invention provides a means to generate cell-bound DDpps consisting of at least one DDpp designed to confer binding specificity to a membrane-bound fusion protein. The DDpp receptor can be expressed by any cell type.

[0148] In one embodiment, the DDpp receptor fusion protein comprises a chimeric antigen receptor (CAR), or DDpp-CAR, which comprises an extracellular targeting domain and a transmembrane domain. In another embodiment, the DDpp-CAR is composed of an extracellular targeting domain, a transmembrane domain, and a cytoplasmic domain, with the cytoplasmic domain comprising a signaling domain. In a further embodiment, the DDpp-CAR extracellular domain comprises one or more DDpps, each comprising a specific binding domain with the same or different specificity. In some embodiments, the target-specific domain is directed to one or more cancer or tumor antigens disclosed herein, such as, by way of non-limiting example, BCMA, CD123, CS1, HER2, AFP, and AFP p26. In one embodiment, the intracellular domain (e.g., cytoplasmic domain) of the DDpp-CAR comprises the intracellular domain of the CD3 zeta chain. In another embodiment, the intracellular signaling domain of the DDpp is composed of a portion of the intracellular domain of the CD3 zeta chain. In a further embodiment, the intracellular domain of the DDpp-CAR comprises the intracellular domain of the CD3 zeta chain and a costimulatory signaling region. The costimulatory signaling region refers to a portion of a DDpp-CAR that includes all or part of 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 efficiently respond to antigens. Costimulatory molecules and some of these molecules that can confer costimulatory properties to AR are known in the art and can be incorporated into DDpp-CARs in a conventional manner. In addition, truncations or mutations to these intracellular signaling and costimulatory domains can be incorporated to further enhance or reduce receptor signaling. In a preferred embodiment, T cells are genetically modified to stably express DDpp-CAR. In such embodiments, the cytoplasmic domain of the DDpp-CAR can include the CD28 and / or 41BB signaling domains alone, or can be combined with any other desired cytoplasmic domain useful in the context of the disclosed embodiments. In one embodiment, the cytoplasmic domain of the DDpp-CAR can be designed to further include the signaling domain of the CD3 zeta chain.In one embodiment, the DDpp-CAR comprises an extracellular targeting domain, an extracellular protein linker having a transmembrane domain that spans the cell membrane (as found in T cells or NK cells), and a cytoplasmic domain, and may optionally comprise multiple signaling modules. In some embodiments, the DDpp-CAR may also comprise an epitope tag. In some embodiments, the cytoplasmic domain of the DDpp-CAR may include, but is not limited to, CD3 zeta, 41BB, and CD28 signaling modules, and combinations thereof.

[0149] In further embodiments, the present disclosure provides a chimeric antigen receptor (CAR), wherein the CAR comprises a DDpp disclosed herein, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain is selected from the group consisting of a human CD3 zeta domain, a 41BB domain, a CD28 domain, and / or any combination thereof. Depending on the embodiment, the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of, for example, CD27, CD28, 41BB, OX40, CD30, CD40, PD1, lymphocyte function-associated antigen 1 (LFAl), CD2, CD7, LIGHT, NKG2C, B7H3, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the targeting domain of the CAR comprises multiple binding domains (e.g., a DD or one or more DDs and an scFv) that comprise additional target-binding polypeptides. Also provided are isolated nucleic acids encoding CARs that comprise a target-binding polypeptide as part (or all) of the targeting region.

[0150] The present disclosure also provides cells comprising a nucleic acid sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain composed, at least in part, of the disclosed DDpp that binds to a target (e.g., BCMA, CD123, CS1, HER2, AFP, and AFP p26), a transmembrane domain, and a signaling domain. In some embodiments, the CAR specifically binds to a tumor antigen (and thus functions to deliver the CAR-expressing cell to the tumor). In some embodiments, the tumor antigen is associated with a hematologic tumor. In some embodiments, the tumor antigen is BCMA. In some embodiments, the tumor antigen is CD123. In some embodiments, the tumor antigen is CS1. In further embodiments, the tumor antigen is associated with a solid tumor. In some embodiments, the tumor antigen is HER2. In some embodiments, both solid and hematologic tumors are targeted. In some embodiments, the CAR-expressing cell is a T cell, natural killer (NK) cell, or other immune cell type. In some embodiments, the CAR-expressing cell (whether a T cell, NK cell, or other cell type) exhibits anti-tumor immunity upon binding of the polypeptide to its corresponding tumor.

[0151] Extracellular domain Depending on the desired antigen to be targeted, the DDpp-CAR can be genetically modified to include an antigen-binding DDpp that is specific for the desired antigen target. For example, if BCMA is the desired antigen to be targeted, one or more BCMA-binding DDpps are incorporated into the target-specific binding domain of the DDpp-CAR. Furthermore, the DDpp-CAR can include two or more DDpps to confer multispecificity or multivalency to the DDpp-CAR. In some embodiments, the DDpp-CAR includes a BCMA-binding DDpp. In some embodiments, the DDpp-CAR includes a CS1-binding DDpp. In some embodiments, the DDpp-CAR includes a BCMA-binding DDpp and a CS1-binding DDpp.

[0152] The choice of DDpp to be incorporated into the extracellular domain of a DDpp receptor (e.g., DDpp-CAR) depends on the unique properties of the cell to be targeted. For example, the DDpp-CAR may specifically bind to a cell surface protein, such as a receptor on the same cell or another cell. In other embodiments, the DDpp-CAR specifically binds to a soluble molecule, such as an immunoglobulin. In other embodiments, targets of interest bound by the DDpp-CAR include those associated with viral, bacterial, and parasitic infections, and diseases and disorders of the immune system (e.g., autoimmune diseases).

[0153] In other embodiments, the DDpp-CAR can be selected to recognize a ligand that functions as a cell surface marker on target cells associated with cancer. In some embodiments, the DDpp-CAR targets and binds to a tumor antigen (e.g., a TAA or other tumor antigen described herein or known in the art). Accordingly, provided herein are methods for making DDpp-CARs, their use in generating chimeric cells, such as human T cells and natural killer cells, and the use of these chimeric T cells in adoptive immunotherapy.

[0154] In the context provided herein, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder, such as cancer. Disclosed herein is a tumor antigen specifically bound by a DDpp in a DDpp-CAR. In one embodiment, the DDpp in a DDpp-CAR specifically binds to a tumor-specific antigen (TSA) or tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur in other cells in the body. A TAA-associated antigen is not unique to tumor cells, but instead is expressed on normal cells under conditions that cannot induce a state of immune tolerance to the antigen. Expression of an antigen on a tumor can occur under conditions that allow the immune system to respond to the antigen. A TAA can be an antigen expressed on normal cells during fetal development when the immune system is immature and unable to respond, or it can be an antigen that is usually present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0155] In some embodiments, the DDpp in the antigen-binding portion of the DDpp-CAR specifically binds to BCMA, CS1, HER2, or CD123. In some embodiments, the DDpp specifically binds to a BCMA protein having an amino acid sequence consisting of SEQ ID NO:7. In further embodiments, the DDpp specifically binds to BCMA and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:11-305, and 306. In some embodiments, the DDpp specifically binds to a CD123 protein having an amino acid sequence consisting of SEQ ID NO:8. In further embodiments, the DDpp specifically binds to CD123 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:307-739, and 740. In some embodiments, the DDpp specifically binds to a CS1 protein having an amino acid sequence consisting of SEQ ID NO:965. In further embodiments, the DDpp specifically binds to CS1 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:896-909, and 910. In some embodiments, the DDpp specifically binds to CS1 and BCMA. In some embodiments, the DDpp specifically binds to a HER2 protein having an amino acid sequence consisting of SEQ ID NO:967. In a further embodiment, the DDpp specifically binds to HER2 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In a further embodiment, the antigen-binding portion of the DDpp-CAR further binds to a target selected from HVEM, BTLA, DR3, CD19, CD20, and CD22.

[0156] In one embodiment, the DDpp in the DDpp-CAR specifically binds to a tumor antigen associated with a malignant tumor. In one embodiment, the DDpp in the DDpp-CAR binds to an antigen selected from B-cell lymphoma-specific idiotypic immunoglobulins; B-cell differentiation antigens such as CD19, CD20, and CD37; TSLPR and IL7R on myeloid cells, and heat shock protein gp96 on multiple myeloma cells.

[0157] In some embodiments, the DDpp in the antigen-binding portion of the DDpp-CAR specifically binds to AFP, AFP p26, or a fragment thereof. In some embodiments, the DDpp specifically binds to an AFP protein having an amino acid sequence consisting of SEQ ID NO: 9, or a fragment thereof. In some embodiments, the DDpp specifically binds to an AFP p26 protein having an amino acid sequence consisting of SEQ ID NO: 10, or a fragment thereof. In further embodiments, the DDpp specifically comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the antigen-binding portion of the DDpp-CAR further binds to a tumor antigen. In further embodiments, the antigen-binding portion of the DDpp-CAR further binds to a target selected from BCMA, CD123, CS1, HER2, HVEM, BTLA, DR3, CD19, CD20, and CD22.

[0158] Transmembrane domain As used herein, the term "transmembrane domain" (TMD) refers to a region of a cell surface-expressed DDpp fusion protein, such as DDpp-CAR, that spans the cell membrane. In some embodiments, the transmembrane domain of DDpp-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 provided herein.

[0159] A DDpp receptor (e.g., DDpp-CAR) can be designed to include a transmembrane domain fused to the extracellular domain of the DDpp receptor. As described above, fusion of the extracellular domain and the transmembrane domain 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 DDpp-CAR is used. In a specific embodiment, the transmembrane domain in the DDpp-CAR is a CD8 transmembrane domain. In some cases, the transmembrane domain of the DDpp-CAR comprises a CD8 hinge domain. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to promote or inhibit binding to other surface membrane proteins.

[0160] The transmembrane domain can be derived from natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. For purposes herein, a transmembrane region of particular use can be derived (i.e., comprise at least the transmembrane region) from a member selected from the group: the α, β, or ζ chain of the T cell receptor; CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, the transmembrane domain can be synthetic, in which case the DDpp-CAR transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In a further embodiment, the transmembrane domain comprises a triplet of phenylalanine, tryptophan, and valine at each end of the synthetic transmembrane domain.

[0161] As used herein, "extracellular spacer domain" (ESD) refers to a hydrophilic region between the antigen-specific targeting region and the transmembrane domain. In some embodiments, the DDpp-CAR comprises an extracellular spacer domain. In other embodiments, the DDpp-CAR does not comprise an extracellular spacer domain. Extracellular spacer domains include, but are not limited to, the Fc fragment of an antibody or a fragment or derivative thereof, the hinge region of an antibody or a fragment or derivative thereof, the CH2 region of an antibody, the 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, the CD8a hinge and an artificial spacer made from a polypeptide that may be as small as Gly3, or the CHI and CH3 domains of 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 region and CH2 of IgG4, (iv) the hinge region of CD8a, (v) the hinge region, CH2, and CH3 regions of IgG1, (vi) the hinge region of IgG1, or (vi) the hinge region and CH2 regions of IgG1. Other extracellular spacer domains will be apparent to those of skill in the art and may be used in connection with alternative embodiments provided herein.

[0162] In some embodiments, short oligo- or polypeptide linkers, approximately 1-100 amino acids in length, are used to link either domain of the DDpp-CAR. The linker can 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 each other. The amino acid sequence composition of the linker can be selected to minimize the potential immunogenicity of the DDpp-CAR or DDpp fusion protein. Longer linkers can be used if it is desirable to ensure that two adjacent domains do not sterically interfere with each other. In some embodiments, a linker of 2-10 amino acids in length is preferred to form the bond between the transmembrane domain and the cytoplasmic signaling domain of the DDpp-CAR. In further embodiments, the linker is 10-15 amino acids in length, or 15-20 amino acids in length, or 20-30 amino acids in length, or 30-60 amino acids in length, or 60-100 amino acids in length (or any range between the recited lengths). In further embodiments, the linker is a glycine-serine doublet sequence. Further embodiments use a fragment of the hinge region from the human T-cell surface glycoprotein CD8 alpha chain (e.g., spanning amino acid positions 138-182 of the CD8 alpha chain of SwissProt Accession Number P01732). Further embodiments use a fragment of the CD8 hinge region that has been further modified by amino acid substitutions to improve expression function or immunogenicity. Further embodiments use a fragment of the extracellular region from human CD28. Further embodiments use a fragment of the CD28 extracellular region that has been further modified by amino acid substitutions to improve expression function or immunogenicity.

[0163] Intracellular domain As used herein, the term "intracellular signaling domain" (ISD) or "cytoplasmic domain" refers to a portion of a DDpp-CAR that transmits an effector function signal and instructs a cell to perform its differentiated function. The cytoplasmic domain (i.e., the intracellular signaling domain) of a DDpp-CAR is involved in activating at least one normal effector function of an immune cell genetically modified to express the DDpp-CAR. The term "effector function" refers to a differentiated function of a cell. The effector function of a T cell includes, for example, helper activity, including cytolytic activity and cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a DDpp-CAR protein that transmits an effector function signal and instructs a cell to perform its differentiated function. Typically, the entire intracellular signaling domain corresponding to the native 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 can be used, such a truncated portion can be used in place of the complete chain, as long as it transmits the effector function signal. The term intracellular signaling domain is therefore intended to include any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal. In one embodiment, the intracellular signaling domain in a DDpp-CAR also includes the cytoplasmic sequence of a T cell receptor (TCR) and a co-receptor sequence that functions in concert to initiate signal transduction after antigen receptor binding, or any derivative or variant of these sequences that has functional capability. Examples of domains that transmit effector function signals include, but are not limited to, the zeta chain of the T cell receptor complex or any of its homologs (e.g., the eta chain, FcsRly and β chain, MB1 (Iga) chain, B29 (Ig) chain, etc.), human CD3 zeta chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (e.g., Syk, ZAP70, 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.

[0164] It is known that signals generated by the TCR alone are insufficient for full activation of T cells; secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences) and those that function to provide antigen-independent secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0165] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs, known as immunoreceptor tyrosine-based activation motifs (ITAMs).

[0166] In the provided embodiments, examples of ITAMs comprising primary cytoplasmic signaling sequences of particular use include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling sequence from CD3 zeta.

[0167] As used herein, a "costimulatory domain" (CSD) refers to a portion of a CAR or DDpp-CAR that enhances the proliferation, survival, and / or development of memory cells. A DDpp-CAR may contain one or more costimulatory domains. Each costimulatory domain comprises, for example, one or more costimulatory domains of a member of the TNFR superfamily selected from CD28, CD137 (41BB), CD134 (OX40), Dap10, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), Lck, TNFR-I, TNFR-II, Fas, CD30, and CD40, or a combination 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 encompassed by the present disclosure.

[0168] In a preferred embodiment, the cytoplasmic domain of DDpp-CAR comprises a CD3 zeta signaling domain alone or in combination with any other desired cytoplasmic domain useful in the context of DDpp-CAR. For example, the cytoplasmic domain of DDpp-CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to a portion of CAR that contains the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 41BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen 1 (LFA1), CD2, CD7, LIGHT, NKG2C, B7H3, TIM1, and LAG3.

[0169] A polypeptide linker can be placed between adjacent elements of a DDpp-CAR. For example, the linker can be placed between adjacent DDpps, or between a DDpp and a transmembrane domain, or between a transmembrane domain and a cytoplasmic domain, or between adjacent cytoplasmic domains. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of a DDpp-CAR can be linked to each other randomly or in a specified order. Optionally, a short linker, preferably a linker of 2 to 10 amino acids in length, can form the bond. A glycine-serine doublet provides a particularly suitable linker.

[0170] In further embodiments, the DDpp fusion protein is a chimeric antigen receptor (CAR) comprising a target binding domain comprising a DD disclosed herein (e.g., a DD comprising the amino acid sequence of SEQ ID NO: 11-949, or 950). In some embodiments, the DD binds to BCMA and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the DD binds to CD123 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the DD binds to CS1 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In some embodiments, the DD binds to HER2 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the DD binds to AFP and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the DD binds to AFP p26 and comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. 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 41BB or CD28 transmembrane domain. In some embodiments, the CAR comprises an intracellular signaling domain 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. In some embodiments, the CAR 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 (LFAl), 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 CS1 and a second target binding domain that binds BCMA. In some embodiments, the CAR is expressed on an immune cell. In some embodiments, the immune effector cell is a T cell (CAR-T cell) or a natural killer (NK) cell (CAR-NK cell). In some embodiments, the CAR is conjugated to a liposome. In some embodiments, the CAR comprises two, three, four, five, or more than five DD and / or other binding domains (e.g., scFv) that specifically bind to a target (e.g., BCMA, CS1, or CD123) expressed on the surface of a cancer cell. In further embodiments, the CAR comprises two, three, four, five, or more than five DD or other binding domains (e.g., scFv) that specifically bind to a second, different target expressed on the surface of a cancer cell. In further embodiments, the administered CAR further comprises two, three, four, five, or more than five DD or other binding domains (e.g., scFvs) that specifically bind to a second, different target expressed by a second, different cancer cell or vascular endothelial cell.

[0171] Additional DDpp fusion proteins In some embodiments, DDpp comprises a heterologous polypeptide comprising a fragment 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 of the extracellular domain of a cell surface receptor. In some embodiments, DDpp comprises a heterologous polypeptide comprising the extracellular domain, or a fragment of the extracellular domain, of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8). In some embodiments, DDpp comprises a heterologous polypeptide comprising the extracellular domain, or a fragment of the extracellular domain, of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965). In some embodiments, the DDpp comprises 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.

[0172] 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 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 acid fragment of a serum protein from the extracellular domain of a cell surface receptor. In some embodiments, the protein comprises a heterologous polypeptide comprising an intracellular protein (e.g., a nuclear protein) or an antigenic portion of an intracellular protein. In some embodiments, the DDpp comprises a heterologous polypeptide comprising a fragment of the extracellular domain of a cell surface receptor 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 of an intracellular protein. In some embodiments, the DDpp comprises a heterologous polypeptide having the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974.

[0173] In some embodiments, the DDpp fusion protein specifically binds to BCMA, CD123, CS1, HER2, AFP, and / or AFP p26, and further binds to one or more additional target molecules. The target specifically bound by the DDpp fusion protein can be any molecule to which it is desirable for DDpp to bind. For example, the target specifically bound by the DDpp fusion protein can be BCMA, CD123, CS1, HER2, AFP, and / or AFP p26, and can further be any additional target of manufacturing, prescription, therapeutic, diagnostic, or prognostic relevance or value. A number of representative additional targets are provided herein by way of example, but are intended to be illustrative and not limiting. The additional target bound by the DDpp fusion protein can be naturally occurring or synthetic. The additional target can be an extracellular or intracellular component, a soluble factor (e.g., an enzyme, hormone, cytokine, growth factor, toxin, venom, contaminant, etc.), or a transmembrane protein (e.g., a cell surface receptor). In some embodiments, the target bound by the DDpp fusion protein is a human protein. In one embodiment, DDpp (e.g., DDpp fusion protein) binds to a human protein target and its monkey (e.g., cynomolgus), mouse, rabbit, hamster, and / or rabbit orthologs.

[0174] In one embodiment, the DDpp fusion protein specifically binds to BCMA, CD123, CS1, HER2, AFP, and / or AFP p26, 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 improved pharmacodynamic properties to DDpp (or its fusion), including, but not limited to, improved tumor targeting, tumor penetration, intratumoral spread, and enhanced therapeutic action compared to DDpp fusion proteins lacking the protein-binding sequence (see, e.g., WO 01 / 45746, the contents of which are incorporated herein by reference in their entirety).

[0175] Antibody-based DDpp fusion proteins 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 other embodiments, the DDpp is an Fc fusion protein. In further embodiments, the Fc protein comprises a variable human Fc domain.

[0176] In some embodiments, DDpp fusion proteins comprise full-length antibodies or antibody fragments or subdomains (e.g., IgG1 antibodies, IgG3 antibodies, antibody variable regions, CDR3, scFv, Fc, FcRn-binding subdomains, and other antibody subdomains). DDpp proteins can be operably linked to each other and / or to one or more antibody termini, antibody chains, antibody fragments, or antibody subdomains to form DDpp fusion proteins.

[0177] 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 conventional 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 certain embodiments, the antibody is an Fc-optimized antibody. Antibodies can be derived from or derived from any animal origin, including birds and mammals, or can be artificially 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 techniques). In certain embodiments, the antibody component of the antibody-DDpp fusion has extended half-life, enhanced or reduced 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 certain embodiments, the antibody is human.

[0178] The constant region is generally understood to mediate several effector functions. For example, binding of the C1 component of complement to an antibody activates the complement system. Complement activation is important for opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and is 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 the cell. There are numerous Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of an antibody to an Fc receptor on the cell surface triggers a number of important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cellular cytotoxicity (ADCC)), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production.

[0179] In certain embodiments, the DDpp-Fc fusion protein has altered effector functions, which in turn affect 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 inhibit complement binding, thus shortening serum half-life and reducing nonspecific binding of conjugated cytotoxins. Still other modifications of the constant region can be used to remove disulfide bonds or oligosaccharide moieties that allow for enhanced localization by increasing antigen specificity or antibody flexibility. Similarly, modifications to the constant region according to the present disclosure can be readily made using biochemical or molecular engineering techniques known to those skilled in the art.

[0180] 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 genetic modifications of Fc sequences that reduce or eliminate ADCC and / or CDC activity and Fc receptor and / or complement factor binding, as well as assays and procedures for this testing, are described herein or are known in the art.

[0181] In some embodiments, DDpp-Fc fusion proteins are genetically 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 attached to the hinge region using a 5-20 amino acid spacer. Such spacers are added, for example, to ensure that regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. Amino acid spacers can, in some cases, prove immunogenic and elicit undesirable immune responses against the construct. Therefore, in certain embodiments, any spacers added to the construct are relatively non-immunogenic or can be removed entirely to maintain the desired biochemical properties of the modified DDpp-Fc fusion protein.

[0182] In further embodiments, the DDpp-Fc fusion protein is modified by partial deletion or substitution of a few or even a single amino acid in the constant region. For example, mutation of a single amino acid in a selected region 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) may be completely or partially deleted. Such partial deletion of the constant region can improve the selectivity of the DDpp-Fc fusion protein (e.g., serum half-life) while preserving other desired functions associated with the corresponding constant region domain. 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 improve the profile of the resulting construct. This can inhibit 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 that include the addition of one or more amino acids to the constant region to enhance a desired property, such as increasing or decreasing effector function or providing a binding site for one or more of a cytotoxin, label, or carbohydrate moiety. In such embodiments, it may be desirable to insert or repeat a particular sequence from a selected constant region domain.

[0183] In some embodiments, DDpp is operably linked to an antibody fragment or subdomain (e.g., an scFv, a diabody; EP 404,097; WO 93 / 111161; WO 14 / 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, WO 04 / 058820, WO 99 / 42077, and WO 05 / 017148, the contents of each of which are incorporated herein by reference in their entirety. For example, the DDpp fusion protein can comprise an antibody effector domain or an antibody effector domain derivative that confers one or more effector functions to 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 comprises an antigen-binding fragment of an antibody or a fragment thereof. In further embodiments, the DDpp-antibody fusion protein comprises 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 invention will be apparent to those skilled in the art and can be routinely selected and designed into the DDpp fusion proteins encompassed herein based on the desired effector functions.

[0184] In one embodiment, the DDpp fusion protein comprises 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 pathogen (e.g., bacterial cells (tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniasis), a fungal infection, a mold, a mycoplasma, or a prion antigen. In another embodiment, the DDpp fusion protein comprises an antibody or antibody fragment that specifically binds to a pathogen (e.g., bacterial cells (tuberculosis, smallpox, anthrax)), a virus (e.g., HIV), a parasite (e.g., malaria, leishmaniasis), a fungal infection, a mold, a mycoplasma, or a 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.

[0185] In preferred embodiments, DDpp fusion proteins comprising antibody fragments or domains retain the activity of the parent antibody. Thus, in certain embodiments, DDpp fusion proteins comprising antibody fragments or domains are capable of inducing complement-dependent cytotoxicity. In certain embodiments, DDpp fusion proteins comprising antibody fragments or domains are capable of inducing antibody-dependent cellular cytotoxicity (ADCC).

[0186] 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 to tumor sites, and an extended serum half-life compared to a DDpp fusion protein lacking said one or more DDpps. In certain embodiments, the DDpp fusion protein is at least as stable as the corresponding antibody in the absence of the bound DDpp. In certain embodiments, the DDpp fusion protein is more stable than the corresponding antibody in the absence of the bound DDpp. The activity of the DDpp fusion protein can be measured using established methods, such as, 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 the recited times). In one embodiment, the DDpp fusion comprises an immunoglobulin effector domain or half-life-affecting domain corresponding to an immunoglobulin domain or fragment in which at least a portion of one or more constant region domains has been modified to confer a desired property, such as reduced or increased effector function, ability to non-covalently dimerize, increased ability to localize to tumor sites, shortened serum half-life, or extended serum half-life, compared to an immunoglobulin fragment having a corresponding unmodified immunoglobulin sequence. Modifications of these constant region domains can be amino acid substitutions, insertions, or deletions.

[0187] In one embodiment, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or an immunoglobulin effector domain derivative that confers antibody-dependent cellular cytotoxicity (ADCC) to the DDpp fusion protein. In further embodiments, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain that has been modified to enhance 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); WO 06 / 020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO 04 / 074455, each of which is incorporated herein by reference in its entirety.Examples of immunoglobulin fragment genetic modifications included in the amino acid sequence in the DDpp fusion protein that enhance ADCC include IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S and S298D or V together or K290S alone; IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; and IgG1-F243L, R292P, Y300L, V305I, P396L (numbering of residues in the Fc region is that of the EU index of Kabat et al., Sequences of proteins of Immunological Included are immunoglobulin effector domain sequences having one or more modifications corresponding to those described in US Pat. No. 6,199,162, 5th Ed., the contents of which are incorporated herein by reference in their entirety).

[0188] In other embodiments, the DDpp fusion protein comprises the 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 (1999); Hutchins et al., J. Immunol. 200:16-26 (2000)). al., PNAS 92:11980-11984 (1995); Reddy et al., J. Immunol. 164:1925-1933 (2000); WO 97 / 11971; WO 07 / 106585; U.S. Patent Application Publication No. 2007 / 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 immunoglobulin fragment sequence genetic modifications included in the amino acid sequence in the DDpp fusion protein that 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; IgG4- IgG1-C220S, C226S, C229S, p268S; IgG1-C226S, C229S, E233P, L234V, L235A; or IgG1-L234F, L235E, P331S (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th Edition, the contents of which are incorporated herein by reference in their entirety)).

[0189] In a further embodiment, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or an immunoglobulin effector domain derivative that confers antibody-dependent cellular phagocytosis (ADCP) to the DDpp fusion protein. In further embodiments, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain that has been modified to enhance 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); WO 06 / 020114; Strohl, Curr. Op. Biotechnol. 20:685-691 (2009); and WO 04 / 074455, the contents of each of which are incorporated herein by reference in their entireties.Examples of immunoglobulin fragment genetic modifications included in the amino acid sequence in the DDpp fusion protein that reduce ADCP include IgG1-S298A, E333A, K334A; IgG1-S239D, I332E; IgG1-S239D, A330L, I332E; IgG1-P247I, A339D or Q; IgG1-D280H, K290S and S298D or IgG1-F243L, R292P, Y300L; IgG1-F243L, R292P, Y300L, P396L; IgG1-F243L, R292P, Y300L, V305I, P396L; and IgG1-G236A, S239D, I332E (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th Edition, the contents of which are incorporated herein by reference in their entirety)).

[0190] In other embodiments, the DDpp fusion protein comprises the 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. 200:16-20 (2000)). al., J. Immunol. 164:1925-1933 (2000); WO 97 / 11971; WO 07 / 106585; U.S. Patent Application Publication No. 2007 / 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 entirety.By way of example, the DDpp fusion protein may contain one or more of the following modifications that reduce ADCC: IgG1-N297A; IgG1-L234A, L235A; IgG2-V234A, G237A; IgG4-L235A, G237A, E318A; IgG4-S228P, L236E; IgG2-EU sequence 118-260; 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 (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th ed., the contents of which are incorporated herein by reference in their entirety).

[0191] In a further embodiment, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or an immunoglobulin effector domain derivative that confers complement-dependent cytotoxicity (CDC) 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 enhance 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) each of which is incorporated herein by reference in its entirety). For example, a DDpp fusion protein can comprise an antibody fragment or domain containing one or more of the following modifications that enhance CDC: IgG1-K326A, E333A; IgG1-K326W, E333S, IgG2-E333S (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th Edition, the contents of which are incorporated herein by reference in their entirety)).

[0192] In a further embodiment, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or an immunoglobulin effector domain derivative that confers to the DDpp fusion the ability to bind to the FcγRIIb receptor. In a further embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain that has been modified to enhance inhibitory binding to the FcγRIIb receptor (see, e.g., Chu et al., Mol. Immunol. 45:3926-3933 (2008)). An example of an immunoglobulin fragment genetic modification contained in the amino acid sequence of the DDpp fusion protein that inhibitively enhances binding to the FcγRIIb receptor is IgG1-S267E, L328F.

[0193] In other embodiments, the DDpp fusion protein comprises a sequence of an immunoglobulin effector domain that has been modified to reduce CDC (see, e.g., WO 97 / 11971; WO 07 / 106585; U.S. Patent Application Publication No. 2007 / 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, a DDpp fusion protein may comprise an antibody fragment or domain 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 (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th Edition, the contents of which are incorporated herein by reference in their entirety)).

[0194] The half-life of IgG is mediated by pH-dependent binding to its neonatal receptor FcRn. In certain embodiments, the DDpp fusion protein comprises the amino acid sequence of an immunoglobulin effector domain or an immunoglobulin effector domain derivative that confers on the DDpp fusion the ability to bind to the neonatal receptor FcRn. In certain embodiments, the DDpp fusion protein comprises 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 See Metab. Dispos. 35:86-94 (2007); Datta-Mannan et al., J. Biol. Chem. 282:1709-1717 (2007); WO 06 / 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 entirety.

[0195] In a further embodiment, the DDpp fusion protein 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 no affinity at pH 7.4. For example, the DDpp fusion protein may comprise an antibody fragment or domain containing one or more of the following half-life-enhancing modifications: IgG1-M252Y, S254T, T256E; IgG1-T250Q, M428L; IgG1-H433K, N434Y; IgG1-N434A; and IgG1-T307A, E380A, N434A (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th ed.), the contents of which are incorporated herein by reference in their entirety).

[0196] 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). For example, a DDpp fusion protein may comprise 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 (residue numbering is that of the EU index of Kabat et al. (Kabat et al., Sequences of proteins of Immunological Interest, 1991 5th Edition, the contents of which are incorporated herein by reference in their entirety)).

[0197] In 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, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 38 The DDpp fusion protein comprises an amino acid sequence corresponding to an immunoglobulin effector domain that has been modified to include at least one substitution in its sequence corresponding to a position in the Fc region (e.g., Fc gamma) selected from the group consisting of 37, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, and 439 (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 5th Edition; the contents of which are incorporated herein by reference in their entirety). In certain embodiments, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain derivative in which at least one residue corresponding to position 434 is selected from A, W, Y, F, and H. In another embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector fragment derivative having the following respective substitutions: S298A / E333A / K334A. In an additional 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 fragment derivative having one or any combination of the following substitutions: K246H, H268D, E283L, S324G, S239D and I332E. In yet another embodiment, the DDpp fusion protein comprises the sequence of an immunoglobulin effector domain derivative having substitutions corresponding to D265A / N297A.

[0198] In certain embodiments, DDpp fusion proteins comprise immunoglobulin effector domain sequences that have been glycoengineered or mutated to enhance 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 enhancing tumor localization. In other cases, constant region modifications consistent with certain provided embodiments may slow complement binding, thereby shortening serum half-life and reducing nonspecific binding of conjugated cytotoxins. Still other modifications of the constant region may be used to modify disulfide bonds or oligosaccharide moieties to increase antigen specificity or antibody flexibility, thereby enabling enhanced localization. The resulting physiological profiles, such as tumor localization, biodistribution, and serum half-life, as well as other effects of biochemical modifications, can be readily measured and quantified using known immunological techniques without undue experimentation.

[0199] In some embodiments, immune effector cells comprise cell surface receptors for immunoglobulins or other peptide-binding molecules, e.g., receptors for immunoglobulin constant regions, and include the class of receptors commonly referred to as "Fc receptors" ("FcRs"). Numerous FcRs have been structurally and / or functionally characterized and are known in the art, including FcRs that have a particular ability to interact with restricted subsets of immunoglobulin heavy chain isotypes, or that interact with Fc domains with different affinities, and / or that may be expressed on restricted subsets of immune effector cells under specific 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 ... 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., 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 entireties.

[0200] 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 granulocytic cells, such as cells involved in allergic response mechanisms. Thus, immune effector cells include, but are not limited to, cells of hematopoietic origin, including cells at various stages of differentiation within the myeloid and lymphoid lineages, as well as T lymphocytes, B lymphocytes, NK cells, monocytes, macrophages, dendritic cells, neutrophils, basophils, eosinophils, mast cells, platelets, erythrocytes, and precursors, progenitor cells (e.g., hematopoietic stem cells), as well as cells that may (or may not) express one or more types of functional cell surface FcRs, such as resting, activated, and mature forms of these cells. 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 cells that result in the induction of apoptosis, or cells that result in microbial immunity or neutralization of microbial infection, or cells that mediate allergic, inflammatory, hypersensitivity, and / or autoimmune responses.

[0201] DDpp fusion proteins with extended half-lives The disclosed DDpp can be fused or conjugated to a second peptide domain to extend the half-life or increase the stability of the DDpp.

[0202] 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, proteins comprising DDpp linked to one or more polypeptides, polypeptide fragments, or amino acids not normally recognized as part of a protein sequence. In one embodiment, the fusion peptide comprises the entire amino acid sequences of two or more peptides, or alternatively, portions (fragments) of two or more peptides. In some embodiments, the peptide (e.g., a protein S-linked 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 facilitates the formation of a multimeric protein, or a fragment of any of the foregoing. Suitable fusion partners include, but are not limited to, a histidine tag, a FLAG tag, a strep tag, and a mic tag.

[0203] In some embodiments, DDpp is fused to one or more moieties that extend the half-life of the polypeptide. Half-life can be extended, for example, by increasing the molecular weight of DDpp to avoid renal clearance and / or by incorporating a binding domain for FcRn-mediated recycling. In one embodiment, DDpp is fused or chemically conjugated to an albumin polypeptide or a fragment thereof (e.g., human serum albumin (HSA)). In specific 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 or conjugated to 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)).

[0204] In one embodiment, DDpp is fused or chemically conjugated to a transferrin polypeptide or a fragment thereof (e.g., human transferrin). In specific 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 or conjugated to a transferrin binding domain that binds to albumin when administered in vivo.

[0205] In some embodiments, DDpp is fused or chemically conjugated to a proline-alanine-serine polymer (PASylation; XL-Protein GmbH), a non-exact 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).

[0206] Additional DDpp fusion proteins In some embodiments, the DDpp fusion protein specifically binds to BCMA, CD123, CS1, HER2, AFP, and / or AFP p26. The disease-associated antigen can be an antigen characteristic of a 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 leishmaniasis), 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.

[0207] In additional embodiments, the DDpp fusion protein is conjugated 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 conjugates, enzymes, toxins, and cytotoxic agents. Photosensitizers include, for example, Photofrin, Visudyne, Levlan, Foscan, Metobix, Hexvix (registered trademark, Cysview™), Laserphyrin, Antolin, Photochlor, Photosens, Photrex, Lumacan, Cevira, Visonac, BF-200ALA, and Amphinex. In further embodiments, a histidine tag, FLAG tag, Strep tag, or Mikal tag is conjugated to DDpp.

[0208] In another embodiment, the DDpp fusion protein comprises a DD that binds to BCMA, CD123, CS1, HER2, AFP, AFP p26, or a fragment thereof. Such peptide tags provide a useful means for detecting and / or binding to targets containing the peptide tag. In one embodiment, the DDpp fusion protein specifically binds to a peptide tag selected from the group: hexahistidyl (His6) tag, mic tag, and FLAG tag. Other peptide tags are described herein or known in the art.

[0209] DDpp fusion proteins with epitope tags 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 mic tag, and a FLAG tag. In further embodiments, peptide tags include, but are not limited to, avitag (which allows the tag to be biotinylated and isolated with streptavidin), calmodulin, E-tag, hemagglutinin (HA), S-tag, SBP-tag, softag1, streptavidin, tetra- or polycysteine, V5, VSV, and Xpress tags. Additionally, polyhistidine tags (other than six residues) can be used. In further embodiments, covalently linked peptide tags, protein tags, and the like can be used. Covalently linked peptide tags include, but are not limited to, isopeptag (covalently linked to the pyrin C protein), Spytag (covalently linked to the spycatcher protein), and snooptag (covalently linked to the spycatcher protein). In still further embodiments, protein tags may optionally be used, including but not limited to biotin carboxyl carrier protein (BCCP), glutathione-S-transferase, green fluorescent protein (or other fluorophores), halotag, Nus tag, thioredoxin, and Fc tag. In still further embodiments, polymorphic tags may be used. In still further embodiments, no tags are used. In still further embodiments, the DDpp fusion protein comprises a removable tag. Depending on the embodiment, any combination of extracellular, transmembrane, and intracellular domains disclosed herein may be used.

[0210] DDpp linker The terms "linker" and "spacer" are used interchangeably herein and refer to a peptide bond or other chemical bond that functions to link otherwise independent functional domains. In one embodiment, the linker in a DDpp is positioned between the DDpp and another polypeptide component that would otherwise comprise an independent functional domain. Suitable linkers for linking two or more linked DDpps will be apparent to those skilled in the art and may be any linker typically used in the art to link peptides, proteins or other organic molecules. In certain embodiments, such linkers are suitable for constructing proteins or polypeptides intended for pharmaceutical use.

[0211] Suitable linkers for operably linking DDpp and additional components of a DDpp fusion protein 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 of mostly polar polypeptide fragments.

[0212] In one embodiment, the linker is composed mostly of amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In one embodiment, the linker is composed mostly of amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In one embodiment, the DDpp fusion protein linker is composed mostly of one or more amino acids selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In one embodiment, the DDpp fusion protein linker is composed mostly of one or more amino acids selected from glycine, alanine, proline, asparagine, aspartic acid, threonine, glutamine, and lysine. In another embodiment, the DDpp fusion protein linker is composed mostly of amino acids that are sterically unhindered. In another embodiment, the linker has a majority of amino acids therein that are glycine, serine, and / or alanine. In some embodiments, the peptide linker is selected from polyglycines, e.g., (Gly)5 (SEQ ID NO: 975), and (Gly)8 (SEQ ID NO: 976), poly(Gly-Ala), and polyalanines. In some embodiments, the peptide linker comprises the sequence Gly-Gly-Gly-Gly-Gly-Thr-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 4). In some embodiments, the peptide linker comprises the sequence Gly-Gly-Gly-Gly-Asp-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 5).

[0213] In one embodiment, the DDpp fusion comprises a DDpp directly linked (i.e., without a linker) to another component of the DDpp fusion protein, hi one embodiment, the DDpp fusion comprises at least two, at least three, at least four DDpps directly linked to another component of the DDpp fusion.

[0214] In another embodiment, the DDpp can be operably linked to another component of the DDpp fusion protein via a linker. The DDpp fusion protein can include a single linker, multiple linkers, or no linker. In one embodiment, the DDpp fusion comprises a DDpp operably linked to another component of the DDpp fusion protein via a linker peptide. In one embodiment, the DDpp fusion comprises two, three, four, or five DDs operably linked to another component of the DDpp fusion protein via a linker peptide.

[0215] The linker can be of any size or composition, so long as it operably links the DDpp to bind the target of interest, such as BCMA, CD123, CS1, HER2, AFP, or AFP p26. In some embodiments, the linker is about 1-100 amino acids, about 1-50 amino acids, about 1-20 amino acids, about 1-15 amino acids, about 1-10 amino acids, about 1-5 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. It should be noted that the length, degree of flexibility, and / or other characteristics of the linker may affect the properties of the resulting DD-containing protein, including, but not limited to, affinity, specificity, or avidity for the target of interest or for one or more other protein targets of interest. When two or more linkers are used in a DDpp fusion protein, these linkers may be the same or different. In the context and disclosure provided herein, one of skill in the art would be able to routinely determine the optimal linker composition and length to operably link the DDpp and other components of the DDpp fusion protein.

[0216] 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)C(O)- (s = 2 to 20) can be used. These alkyl linkers may be further substituted with a non-sterically hindering group such as lower alkyl (e.g., C-C), lower acyl, halogen (e.g., Cl, Br), CN, NH, phenyl, etc. A representative 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.

[0217] Suitable linkers for joining DDpp fusion protein components by chemical cross-linking include, but are not limited to, homobifunctional chemical cross-linking 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 joining DDpp fusion protein components of heterobifunctional reagents for cross-linking include, but are not limited to, cross-linkers with one amine-reactive end and a sulfhydryl-reactive moiety at the other end, or cross-linkers with an NHS ester and an SH-reactive group (e.g., maleimide or pyridyl) at one end.

[0218] In further embodiments, one or more linkers in the DDpp fusion protein are cleavable. Examples of cleavable linkers include, but are not limited to, various types of peptide sequences recognized by proteases (in vitro or in vivo), such as Tev, thrombin, Factor Xa, plasmin (a blood protease), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other body compartments.

[0219] In some embodiments, the linker is a "cleavable linker" that facilitates release of the DDpp or cytotoxic agent in cells. 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 (Chari, Can. Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020; U.S. Patent Application Publication No. 20090110753; the contents of which are incorporated herein by reference in their entireties) can be used, and it is desirable that the covalent bond between the DDpp or cytotoxic agent and the fusion partner is cleaved intracellularly upon internalization of the composition into the cell. The terms "cleaved intracellularly" and "intracellular cleavage" refer to a metabolic process or intracellular reaction on a DDpp-drug conjugate, whereby a covalent bond, i.e., a bond between a DDpp and a cytotoxic agent, a DDpp and a fusion, or a bond via a linker between two DDpps, is broken, resulting in free DDpp and / or a cytotoxic agent within the cell.

[0220] Linker optimization can be assessed using techniques described herein and / or other techniques known in the art. In some embodiments, the linker does not interfere with the ability of the DDpp to bind to another DDpp fusion protein component, such as an antibody domain or fragment that binds to a target molecule and / or antigen.

[0221] DDpp as a chemical complex DDpp fusion proteins that promote specific binding to a target 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 conjugates have uses including, but not limited to, diagnostic, analytical, manufacturing, and therapeutic applications.

[0222] The inherent lack of cysteines in the DD sequence provides an opportunity for the introduction of a unique cysteine ​​for site-specific conjugation purposes.

[0223] In some embodiments, a DDpp (e.g., a DDpp fusion protein) comprises at least one reactive residue. The reactive residue is useful, for example, as a site for attachment of a conjugate such as 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 the N- or C-terminus of a DDpp or within the DDpp sequence. A cysteine ​​can replace another amino acid in the DDpp sequence. Additionally, a lysine can be added to either end of a DDpp or within the DDpp sequence and / or a lysine can replace another amino acid in the DDpp sequence. In one embodiment, a reactive residue (e.g., a cysteine, a lysine, etc.) is located in the loop sequence of a DD (e.g., amino acid residues 22-24 and 46-49 of SEQ ID NOS: 11-949 and 950). In one embodiment, the reactive residue is located in a linker located between components of a DDpp fusion, e.g., 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 the DDpp fusion protein. In one embodiment, the DDpp or DDpp fusion protein comprises at least one, at least two, or at least three reactive residues. In one embodiment, the DDpp, such as the DDpp fusion protein, comprises at least one, at least two, or at least three cysteine ​​residues.

[0224] BCMA-bound DDpp In some embodiments, the DD of the DDpp specifically binds to BCMA. In further embodiments, the DD of the DDpp specifically binds to BCMA having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In other embodiments, the BCMA-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306.

[0225] In some embodiments, the BCMA-binding DDpp comprises multiple target binding domains that bind to a single target (e.g., dimer, trimer, etc.). In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs that specifically bind to BCMA having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs having 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 BCMA and have amino acid sequences selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the DDpp comprises a DD that specifically binds BCMA and further comprises two, three, four, five, or more than five additional, different DDs or target-binding domains (e.g., scFvs) that specifically bind to BCMA or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to BCMA (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306) 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 cells. In some embodiments, the DDpp comprises a DD that specifically binds to BCMA (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306) 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 BCMA (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306) 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 BCMA (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306) 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 BCMA (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306) 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 BCMA (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells.

[0226] In some embodiments, the DDpp comprises a variant of a BCMA-binding DD disclosed herein (reference DD) that retains the ability to specifically bind BCMA. In some embodiments, the sequence of the BCMA-binding DD variant comprises 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 BCMA-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the sequence of the BCMA-binding DD variant comprises 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 BCMA-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the sequence of the BCMA-binding DD variant comprises 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 BCMA-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306.

[0227] In some embodiments, the sequence of the BCMA-binding DD variant comprises 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 BCMA-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the sequence of the BCMA-binding DD variant comprises 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 BCMA-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the sequence of the BCMA-binding DD variant comprises 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 BCMA-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 305, and 306.

[0228] In some embodiments, the sequence of the BCMA-binding DD variant comprises 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 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 BCMA-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the sequence of the BCMA-binding DD variant comprises 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: 11-305, and 306. In some embodiments, the sequence of the BCMA-binding DD variant comprises a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 non-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: 11-305, and 306.

[0229] In some embodiments, the present disclosure provides a BCMA-binding DDpp that completely or partially (e.g., overlaps with the epitope) blocks the binding of a reference DD to BCMA, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 11-305, and 306. In other embodiments, the present disclosure provides a BCMA-binding DDpp that binds to the same epitope of BCMA as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs: 11-305, and 306.

[0230] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to BCMA. In some embodiments, the DD specifically binds to a BCMA protein having the amino acid sequence of SEQ ID NO:7. In some embodiments, the BCMA-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:11-305, and 306. In other embodiments, the DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:11-305, and 306. 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 BCMA-binding DDpp is an Fc fusion protein. In further embodiments, the Fc protein comprises a variable human Fc domain.

[0231] In some embodiments, the DDpp fusion protein comprises a BCMA-binding DD operably linked to a serum protein. In further embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP or AFP p26, or a fragment thereof. In some embodiments, the DDpp fusion protein comprises AFP (e.g., SEQ ID NO: 9), or a fragment thereof. In other embodiments, the DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974. In some embodiments, the DDpp fusion protein comprises 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 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 of the serum protein.

[0232] In some embodiments, the BCMA-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: 11-305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In further embodiments, the BCMA-binding DDpp fusion protein comprises the extracellular domain of CD123 (e.g., SEQ ID NO: 8), or a fragment thereof. In some embodiments, the BCMA-binding DDpp fusion protein comprises the extracellular domain of a receptor, or a fragment thereof, selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, TSLPR, IL7R, and gp96.

[0233] In some embodiments, the BCMA-binding DDpp fusion protein comprises 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 comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965). In some embodiments, the DDpp comprises 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.

[0234] In further embodiments, the BCMA-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: 11-305, and 306. In other embodiments, the BCMA-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-305, and 306. In some embodiments, the BCMA-binding DDpp fusion protein comprises a fragment 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 acid residues of an intracellular protein (e.g., a nuclear protein).

[0235] CD123 binding DDpp In some embodiments, DD of DDpp specifically binds to CD123. In further embodiments, DD specifically binds to CD123 having the amino acid sequence of SEQ ID NO: 8. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740.

[0236] In some embodiments, the CD123-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 CD123 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 307-739 and 740. 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 CD123 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 307-739 and 740. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 and further comprises two, three, four, five, or more than five additional, different DDs or target-binding domains (e.g., scFvs) that specifically bind to CD123 or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740) 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 cells. In some embodiments, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740) 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 CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740) and specifically binds to two, three, four, five, or more than five different targets. In a further embodiment, the DDpp comprises a DD that specifically binds to CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740) 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 CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740) 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 CD123 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells.

[0237] In some embodiments, the DDpp comprises a variant of a CD123-binding DD disclosed herein (reference DD) that retains the ability to specifically bind to CD123. In some embodiments, the sequence of the CD123-binding DD variant comprises 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 CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the sequence of the CD123-binding DD variant comprises 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 CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the sequence of the CD123-binding DD variant comprises 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 CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740.

[0238] In some embodiments, the sequence of the CD123-binding DD variant comprises a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 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 CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the sequence of the CD123-binding DD variant comprises a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or 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 CD123-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the sequence of the CD123-binding DD variant comprises 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 CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740.

[0239] In some embodiments, the sequence of the CD123-binding DD variant comprises 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 CD123-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 739, and 740. In some embodiments, the sequence of the CD123-binding DD variant comprises 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 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: 307-739, and 740. In some embodiments, the sequence of the CD123-binding DD variant comprises 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: 307-739, and 740.

[0240] In some embodiments, the present disclosure provides a CD123-binding DDpp that completely or partially (e.g., overlaps with the epitope) blocks binding of a reference DD to CD123, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 307-739, and 740. In other embodiments, the present disclosure provides a CD123-binding DDpp that binds to the same epitope of CD123 as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs: 307-739, and 740.

[0241] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to CD123. In some embodiments, the DD of the DDpp fusion protein specifically binds to CD123 having the amino acid sequence of SEQ ID NO:8. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:307-739, and 740. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs:307-739, and 740. 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 CD123-binding DDpp is an Fc-fusion protein.

[0242] In some embodiments, the DDpp is a fusion protein comprising a CD123-binding DD operably linked to a serum protein. In further embodiments, the CD123-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the CD123-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: 9), or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof. In some embodiments, the CD123-binding DDpp fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974. In some embodiments, the DDpp fusion protein comprises 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 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 of the serum protein.

[0243] In some embodiments, the CD123-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: 307-739, and 740. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In further embodiments, the CD123-binding DDpp fusion protein comprises the extracellular domain of CD123 (e.g., SEQ ID NO: 8), or a fragment thereof. In some embodiments, the CD123-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, and gp96, or a fragment thereof.

[0244] In some embodiments, the CD123-binding DDpp fusion protein comprises a fragment 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 of the extracellular domain of a cell surface receptor. In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of CS1 (SEQ ID NO: 965). In some embodiments, the DDpp comprises 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.

[0245] In further embodiments, the CD123-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: 307-739, and 740. In other embodiments, the CD123-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 307-739, and 740. In some embodiments, the CD123-binding DDpp fusion protein comprises a 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 acid residues of an intracellular protein (e.g., a nuclear protein).

[0246] CS1 combined DDpp In some embodiments, DD of DDpp specifically binds to CS1. In further embodiments, DD specifically binds to CS1 having the amino acid sequence of SEQ ID NO: 965. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910.

[0247] 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: 896-909 and 910. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with 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: 896-909 and 910. 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 target-binding domains (e.g., scFvs) that specifically bind to CS1 or different target 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: 896-909, and 910) 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 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: 896-909, and 910) 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: 896-909, and 910) 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: 896-909, and 910) 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: 896-909, and 910) 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: 896-909, and 910) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells.

[0248] In some embodiments, the DDpp comprises a variant of a CS1-binding DD disclosed herein (reference DD) that retains the ability to specifically bind to CS1. In some embodiments, the sequence of the CS1-binding DD variant comprises 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: 896-909, and 910. In some embodiments, the sequence of the CS1-binding DD variant comprises 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: 896-909, and 910. In some embodiments, the sequence of the CS1-binding DD variant comprises 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: 896-909, and 910.

[0249] In some embodiments, the sequence of the CS1-binding DD variant comprises 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: 896-909, and 910. In some embodiments, the sequence of the CS1-binding DD variant comprises 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-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In some embodiments, the sequence of the CS1-binding DD variant comprises 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: 896-909, and 910.

[0250] In some embodiments, the sequence of the CS1-binding DD variant comprises 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: 896-909, and 910. In some embodiments, the sequence of the CS1-binding DD variant comprises 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 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: 896-909, and 910. In some embodiments, the sequence of the CS1-binding DD variant comprises 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 2-6, 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: 896-909, and 910.

[0251] In some embodiments, the present disclosure provides a CS1-binding DDpp that completely or partially (e.g., overlaps with the epitope) blocks the binding of a reference DD to CS1, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 896-909, and 910. 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: 896-909, and 910.

[0252] 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: 965. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 896-909, and 910. 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.

[0253] 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: 896-909, and 910. 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: 896-909, and 910. 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: 9), or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974. In some embodiments, the DDpp fusion protein comprises 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 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 of a serum protein.

[0254] 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: 896-909, and 910. 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: 896-909, and 910. In further embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of CS1 (SEQ ID NO: 965), or a fragment thereof. In some embodiments, the CS1-binding DDpp fusion protein comprises the extracellular domain of a receptor, or a fragment thereof, selected from the group consisting of CD19, CD20, CD22, HVEM, BTLA, DR3, CD37, TSLPR, IL7R, and gp96.

[0255] In some embodiments, the CS1-binding DDpp fusion protein comprises a fragment 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 of the extracellular domain of a cell surface receptor. In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of CS1 (SEQ ID NO: 965). In some embodiments, the DDpp comprises 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.

[0256] In further 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: 896-909, and 910. 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: 896-909, and 910. In some embodiments, the CS1-binding DDpp fusion protein comprises a 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 acid residues of an intracellular protein (e.g., a nuclear protein).

[0257] HER2 binding DDpp In some embodiments, DD of DDpp specifically binds to HER2. In further embodiments, DD specifically binds to HER2 having the amino acid sequence of SEQ ID NO: 967. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950.

[0258] In some embodiments, the HER2-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 HER2 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 911-949 and 950. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with 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 HER2 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 911-949 and 950. In some embodiments, the DDpp comprises a DD that specifically binds to HER2 and further comprises two, three, four, five, or more than five additional, different DDs or target-binding domains (e.g., scFvs) that specifically bind to HER2 or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to HER2 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950) 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 cancer cells. In some embodiments, the DDpp comprises a DD that specifically binds to HER2 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950) 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 HER2 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950) 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 HER2 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950) 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 HER2 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950) 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 HER2 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells.

[0259] In some embodiments, the DDpp comprises a variant of a HER2-binding DD disclosed herein (reference DD) that retains the ability to specifically bind to HER2. In some embodiments, the sequence of the HER2-binding DD variant comprises 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 HER2-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the sequence of the HER2-binding DD variant comprises 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 HER2-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the sequence of the HER2-binding DD variant comprises 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 HER2-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950.

[0260] In some embodiments, the sequence of the HER2-binding DD variant comprises 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 HER2-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the sequence of the HER2-binding DD variant comprises 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 HER2-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the sequence of the HER2-binding DD variant comprises 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 HER2-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950.

[0261] In some embodiments, the sequence of the HER2-binding DD variant comprises 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 HER2-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the sequence of the HER2-binding DD variant comprises 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 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: 911-949, and 950. In some embodiments, the sequence of the HER2-binding DD variant comprises 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 2-6, 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: 911-949, and 950.

[0262] In some embodiments, the present disclosure provides a HER2-binding DDpp that completely or partially (e.g., overlaps with the epitope) blocks the binding of a reference DD to HER2, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 911-949, and 950. In other embodiments, the present disclosure provides a HER2-binding DDpp that binds to the same epitope on HER2as as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs: 911-949, and 950.

[0263] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to HER2. In some embodiments, the DD of the DDpp fusion protein specifically binds to HER2 having the amino acid sequence of SEQ ID NO: 967. In some embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. 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 HER2-binding DDpp is an Fc-fusion protein.

[0264] In some embodiments, the DDpp is a fusion protein comprising a HER2-binding DD operably linked to a serum protein. In some embodiments, the HER2-binding DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In other embodiments, the HER2-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the HER2-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: 9), or a fragment thereof. In some embodiments, the HER2-binding DDpp fusion protein comprises AFP p26 (SEQ ID NO: 10), or a fragment thereof. In some embodiments, the HER2-binding DDpp fusion protein comprises a polypeptide having the sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974. In some embodiments, the DDpp fusion protein comprises 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 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 of a serum protein.

[0265] In some embodiments, the HER2-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: 911-949, and 950. In other embodiments, the HER2-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In further embodiments, the HER2-binding DDpp fusion protein comprises the extracellular domain of HER2 (SEQ ID NO: 967), or a fragment thereof. In some embodiments, the HER2-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, and gp96, or a fragment thereof.

[0266] In some embodiments, the HER2-binding DDpp fusion protein comprises a fragment 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 of the extracellular domain of a cell surface receptor. In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of CS1 (SEQ ID NO: 965). In some embodiments, the DDpp comprises 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.

[0267] In further embodiments, the HER2-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: 911-949, and 950. In other embodiments, the HER2-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 911-949, and 950. In some embodiments, the HER2-binding DDpp fusion protein comprises a 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 acid residues of an intracellular protein (e.g., a nuclear protein).

[0268] AFP binding DDpp In some embodiments, the DD of the DDpp specifically binds to an AFP or a fragment thereof. In further embodiments, the DD of the DDpp specifically binds to an AFP or a fragment thereof having the amino acid sequence of SEQ ID NO: 9. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the AFP-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895.

[0269] In some embodiments, the AFP-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 AFP and have amino acid sequences selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with 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 AFP and have amino acid sequences selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the DDpp comprises a DD that specifically binds to AFP and further comprises two, three, four, five, or more than five additional, different DDs or target-binding domains (e.g., scFvs) that specifically bind to AFP or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to AFP (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) 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 cells. In some embodiments, the DDpp comprises a DD that specifically binds to AFP (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) 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 AFP (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) and specifically binds to two, three, four, five, or more than five different targets. In a further embodiment, the DDpp comprises a DD that specifically binds to AFP (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895) 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 AFP (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) 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 AFP (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells.

[0270] In some embodiments, the DDpp comprises a variant of an AFP-binding DD disclosed herein (reference DD) that retains the ability to specifically bind to AFP. In some embodiments, the sequence of the AFP-binding DD variant comprises 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 AFP-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the sequence of the AFP-binding DD variant comprises 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 AFP-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the sequence of the AFP-binding DD variant comprises 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 AFP-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895.

[0271] In some embodiments, the sequence of the AFP-binding DD variant comprises 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 AFP-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the sequence of the AFP-binding DD variant comprises 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 AFP-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the sequence of the AFP-binding DD variant comprises 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 AFP-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895.

[0272] In some embodiments, the sequence of the AFP-binding DD variant comprises 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 AFP-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741 to 874, and 886 to 895. In some embodiments, the sequence of the AFP-binding DD variant comprises 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: 741-874, and 886-895. In some embodiments, the sequence of the AFP-binding DD variant comprises a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 non-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: 741-874, and 886-895.

[0273] In some embodiments, the present disclosure provides an AFP-binding DDpp that completely or partially (e.g., overlaps with) the binding of a reference DD to AFP, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 741-873, and 874. In other embodiments, the present disclosure provides an AFP-binding DDpp that binds to the same epitope of AFP as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs: 741-873, and 874.

[0274] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to AFP. In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to AFP having the amino acid sequence of SEQ ID NO: 9. In further embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the DDpp is a fusion protein comprising an AFP-binding DD that is a variant of a DD comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895.

[0275] In some embodiments, the DDpp is a fusion protein comprising a DD that specifically binds to AFP operably linked to a full-length antibody or a portion (fragment) of an antibody. In some embodiments, the DDpp is an Fc fusion protein. 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 AFP-binding DDpp is an Fc fusion protein.

[0276] In some embodiments, DDpp is a fusion protein comprising an AFP-binding DD operably linked to a serum protein. In some embodiments, the DDpp fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In other embodiments, the AFP-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the DDpp fusion protein comprises human serum albumin or a fragment thereof. In some embodiments, the DDpp fusion protein comprises a fragment of a serum protein or an antigenic fragment of a serum protein. In some embodiments, the DDpp fusion protein comprises a fragment 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 of a serum protein.

[0277] In some embodiments, the AFP-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: 741-874 and 886-895. In other embodiments, the AFP-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the AFP-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8), or a fragment thereof. In further embodiments, the AFP-binding DDpp fusion protein comprises the extracellular domain of BCMA (SEQ ID NO: 7), CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965), or a fragment thereof. In some embodiments, the AFP-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, and gp96, or a fragment thereof.

[0278] In some embodiments, the AFP-binding DDpp fusion protein comprises 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 comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7) or CD123 (SEQ ID NO: 8). In some embodiments, the DDpp fusion protein comprises a fragment of the extracellular domain of BCMA (SEQ ID NO: 7), or CD123 (SEQ ID NO: 8), or CS1 (SEQ ID NO: 965). In some embodiments, the DDpp comprises 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.

[0279] In further embodiments, the AFP-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: 741-874 and 886-895. In other embodiments, the AFP-binding DDpp fusion protein comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the AFP-binding DDpp fusion protein comprises a 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 acid residues of an intracellular protein (e.g., a nuclear protein).

[0280] AFP p26 binding DDpp In some embodiments, the DD of the DDpp specifically binds to AFP p26. In further embodiments, the DD of the DDpp specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO: 10. In some embodiments, the DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In further embodiments, the AFP p26-binding DDpp comprises a variant of an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895.

[0281] In some embodiments, the AFP p26-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 AFP p26 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the DDpp comprises two, three, four, five, or more than five DDs with 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 AFP p26 and have amino acid sequences selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 and further comprises two, three, four, five, or more than five additional, different DDs or target-binding domains (e.g., scFvs) that specifically bind to AFP p26 or different target antigens. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) and further comprises one or more additional DDs or other target-binding domains that bind to one or more antigens expressed on the surface of B cells. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) and further comprises one or more additional DDs or other target-binding domains that bind to one or more cancer antigens. In some embodiments, the DDpp comprises a DD that specifically binds to AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) 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 AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) 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 AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) 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 AFP p26 (e.g., a DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895) and specifically binds to two, three, four, five, or more than five different cancer antigens expressed on the surface of cancer cells.

[0282] In some embodiments, the DDpp comprises a variant of an AFP p26-binding DD (reference DD) disclosed herein that retains the ability to specifically bind to AFP p26. In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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 AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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 AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874 and 886-895. In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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 AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895.

[0283] In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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 AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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 AFP p26-DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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 AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895.

[0284] In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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 2-6, 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 AFP p26-binding DD having an amino acid sequence selected from the group consisting of SEQ ID NOs: 741-874, and 886-895. In some embodiments, the sequence of the AFP p26-binding DD variant comprises 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: 741-874, and 886-895. In some embodiments, the sequence of the AFP p26-binding DD variant comprises a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 1-3, 1-5, or 1-10 non-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: 741-874, and 886-895.

[0285] In some embodiments, the present disclosure provides an AFP p26-binding DDpp that completely or partially (e.g., overlaps with the epitope) blocks the binding of a reference DD to AFP p26, wherein the reference DD has an amino acid sequence selected from SEQ ID NOs: 741-873, and 874. In other embodiments, the present disclosure provides an AFP p26-binding DDpp that binds to the same epitope on AFP p26 as a reference DD consisting of an amino acid sequence selected from SEQ ID NOs: 741-873, and 874.

[0286] In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to AFP p26. In some embodiments, DDpp is a fusion protein comprising a DD that specifically binds to AFP p26 having the amino acid sequence of SEQ ID NO:9. In further embodiments, DDpp comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:741-874 and 886-895. In other embodiments, DDpp is a fusion protein comprising an AFP p26-binding DD that is a variant of DD comprising an amino acid sequence selected from the group consisting of...

Claims

1. 1. A protein comprising a D domain (DD) target binding domain and an AFP p26 polypeptide, wherein the DD target binding domain specifically binds to CD123 and comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 344, wherein the DD target binding domain does not comprise any one of the amino acid sequences of SEQ ID NOs: 337, 339, 340, 341, 343, 344, 345, 346, 347, 348, 349, 351, 352, 353, 354, 355, and 356, and the AFP p26 polypeptide consists of the amino acid sequence of SEQ ID NO: 10, 968, 969, 970, 971, 972, 973, or 974.

2. The protein of claim 1, wherein the DD target binding domain comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

344.

3. The protein of claim 2, wherein the DD target binding domain comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:

344.

4. The protein of claim 1, wherein the DD target binding domain comprises an amino acid sequence having 1, 2, 3, 4, or 5 substitutions relative to the amino acid sequence of SEQ ID NO:

344.

5. The protein of claim 1, wherein the DD target binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 338, 342, 350 and 357-360.

6. The protein of claim 1, wherein the DD target binding domain comprises an amino acid sequence having one, two, or three substitutions relative to the amino acid sequence of SEQ ID NO:

344.

7. The protein of claim 1 , wherein the AFP p26 polypeptide consists of the amino acid sequence of SEQ ID NO:

969.

8. The protein of claim 1 , wherein the DD target binding domain is fused to the AFP p26 polypeptide via a linker.

9. The protein according to any one of claims 1 to 8, which is labeled.

10. 10. The protein of claim 9, 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.

11. The protein of any one of claims 1 to 10, conjugated to a therapeutic or cytotoxic agent.

12. 12. The protein of any one of claims 1 to 11, further comprising a second target binding domain having the same or a different target than the DD target binding domain.

13. An isolated nucleic acid encoding the protein of any one of claims 1 to 12.

14. A vector comprising the nucleic acid of claim 13.

15. 15. The vector of claim 14, wherein the nucleic acid is operably linked to a nucleotide sequence that regulates expression of the protein encoded by the nucleic acid.

16. A host cell comprising the nucleic acid of claim 13 or the vector of claim 14 or claim 15.

17. A cell genetically modified to express the protein according to any one of claims 1 to 12.

18. A pharmaceutical composition comprising the protein according to any one of claims 1 to 12, the nucleic acid according to claim 13, the vector according to claim 14 or claim 15, or the cell according to claim 16 or claim 17.

19. 19. The pharmaceutical composition of claim 18 for treating myeloid malignancies.

20. 20. The pharmaceutical composition of claim 19, wherein the myeloid malignancy is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, leukemia, plasmacytoma, and myeloma.

21. 20. The pharmaceutical composition of claim 19, wherein the myeloid malignancy is acute myeloid leukemia.

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