Chromatography resins and uses thereof

Abstract

Affinity chromatography resins comprising anti-tissue factor antibodies or antigen-binding fragments thereof bound to a base resin, and methods of using the same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 982,002, filed February 26, 2020, and U.S. Provisional Patent Application No. 62 / 975,141, filed February 11, 2020, both of which are incorporated by reference in their entireties.

[0002] Technical Field The present disclosure relates to the field of biotechnology, and more specifically to antigen-binding molecules. [Background technology]

[0003] Tissue factor (TF), a 263-amino acid integral membrane glycoprotein with a molecular weight of approximately 46 kDa and the trigger protein of the extrinsic blood coagulation pathway, is the primary initiator of coagulation in vivo. Normally, tissue factor, not in contact with circulating blood, initiates the coagulation cascade upon exposure to circulating serine protease factors. Vascular injury exposes subendothelial cells expressing tissue factor, resulting in the formation of a calcium-dependent, high-affinity complex with pre-existing plasma factor VIIa (FVIIa). Binding of the serine protease FVIIa to tissue factor promotes rapid cleavage of FX to FXa and FIX to FIXa. The resulting proteolytic activity of FXa and the active membrane surface then inefficiently converts small amounts of prothrombin to thrombin. Thrombin generated by FXa initiates platelet activation, activating trace amounts of procofactors factor V (FV) and factor VIII (FVIII) to the active cofactors factor Va (FVa) and factor VIIIa (FVIIIa). FIXa complexes with FVIIIa on the platelet surface to form the intrinsic tenase complex, which leads to the rapid generation of FXa, and FXa complexes with FVa to form the prothrombinase complex on the surface of activated platelets, which leads to the rapid cleavage of prothrombin to thrombin.

[0004] In addition to the tissue factor-FVIIa complex, recent studies have shown that the tissue factor-FVIIa-FXa complex can activate FVIII, thereby providing additional levels of FVIIIa during the initiation phase. While the extrinsic pathway is most important in initiating coagulation by activating a limited amount of thrombin, the intrinsic pathway sustains coagulation by dramatically amplifying the initial signal.

[0005] Much of the tissue factor expressed on the cell surface is "encoded" and must be "decoded" to fully participate in coagulation. The mechanism by which cell surface tissue factor is "decoded" remains unclear at present, but exposure of anionic phospholipids plays a major role in this process. Healthy cells actively sequester anionic phospholipids, such as phosphatidylserine (PS), in the inner leaflet of the plasma membrane. Upon cell injury, activation, or cytoplasmic Ca 2+ After increased levels, this bilayer asymmetry is lost, resulting in increased PS exposure in the outer leaflet, which increases the specific activity of the cell surface tissue factor-FVIIa complex. Although PS exposure is known to reduce the apparent Km for activation of FIX and FX by the tissue factor-FVIIa complex, additional mechanisms may include conformational rearrangements of tissue factor or tissue factor-FVIIa and subsequent exposure of substrate-binding sites. Summary of the Invention

[0006] Provided herein are affinity chromatography resins comprising anti-tissue factor antibodies or antigen-binding fragments thereof bound to a base resin, and methods of using the same.

[0007] Provided herein is an affinity chromatography resin comprising an anti-tissue factor antibody or antigen-binding fragment thereof bound to a base resin, the antibody comprising a heavy chain variable domain comprising the CDRs of SEQ ID NOs: 1, 2 or 9, and 3, and a light chain variable domain comprising the CDRs of SEQ ID NOs: 4, 5, and 6.

[0008] In some embodiments of any of the affinity chromatography resins described herein, the heavy chain variable domain comprises a sequence at least 90% identical to SEQ ID NO: 7, and the light chain variable domain comprises a sequence at least 90% identical to SEQ ID NO: 8. In some embodiments of any of the affinity chromatography resins described herein, the heavy chain variable domain comprises SEQ ID NO: 7, and the light chain variable domain comprises SEQ ID NO: 8.

[0009] In some embodiments of any of the affinity chromatography resins described herein, the base resin is Sepharose. In some embodiments of any of the affinity chromatography resins described herein, the base resin is a support material. In some embodiments of any of the affinity chromatography resins described herein, the support material is an agarose resin or a Capto resin. In some embodiments of any of the affinity chromatography resins described herein, the anti-tissue factor antibody or antigen-binding fragment thereof is non-covalently bound to the base resin. In some embodiments of any of the affinity chromatography resins described herein, the anti-tissue factor antibody or antigen-binding fragment thereof is covalently bound to the base resin.

[0010] In some embodiments of any of the affinity chromatography resins described herein, the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin by formation of a disulfide bond between a cysteine ​​of the anti-tissue factor antibody or antigen-binding fragment thereof and a chemical group on the base resin. In some embodiments of any of the affinity chromatography resins described herein, the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin by formation of a covalent bond between a free amine of the anti-tissue factor antibody or antigen-binding domain and a chemical group on the base resin.

[0011] In some embodiments of any of the affinity chromatography resins described herein, the base resin is a CNBr-activated or N-hydroxysuccinimide (NHS)-activated solid support. In some embodiments of any of the affinity chromatography resins described herein, the covalent bond is represented by Formula I: TIFF0007758680000001.tif11128In the formula, R represents an anti-tissue factor antibody or an antigen-binding fragment thereof.

[0012] Also provided herein are kits that include any of the affinity chromatography resins described herein.

[0013] Also provided herein are methods for purifying tissue factor-containing proteins, comprising the use of any of the affinity chromatography resins described herein. Some embodiments of any of the methods described herein further comprise loading a liquid containing the tissue factor-containing protein onto the affinity chromatography resin, washing the affinity chromatography resin with one or more wash buffers, and eluting the tissue factor-containing protein with an elution buffer. In some embodiments of any of the methods described herein, the liquid containing the tissue factor-containing protein is a clarified liquid culture medium. In some embodiments of any of the methods described herein, the liquid containing the tissue factor-containing protein comprises a cell lysate.

[0014] In some embodiments of any of the methods described herein, the one or more wash buffers are (i) a first wash buffer comprising phosphate-buffered saline, and (ii) a second wash buffer comprising about 0.01 M to about 0.2 M citrate and having a pH of about 4.5 to about 5.5. In some embodiments of any of the methods described herein, (i) the first wash buffer is phosphate-buffered saline, and (ii) the second wash buffer is 0.1 M citrate, pH 5.0.

[0015] In some embodiments of any of the methods described herein, the elution buffer comprises 0.01 M to about 0.2 M acetate and has a pH of about 2.5 to about 3.5. In some embodiments of any of the methods described herein, the elution buffer comprises 0.1 M acetate and has a pH of about 2.9.

[0016] Also provided herein are methods for producing a tissue factor-containing protein, comprising: (i) purifying the tissue factor-containing protein using any of the methods described herein, including the use of any of the affinity chromatography resins described herein; and (ii) performing one or more additional unit operations on the eluate obtained from step (i). In some embodiments of any of the methods described herein, the one or more additional unit operations comprise, in sequential order, performing low pH viral inactivation, performing depth filtration, performing polishing chromatography, performing nanofiltration, and performing ultrafiltration and diafiltration (UF / DF).

[0017] In some embodiments of any of the methods described herein, the tissue factor-containing protein is a single-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide comprises (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a second target binding domain. In some embodiments of any of the methods described herein, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain. In some embodiments of any of the methods described herein, the soluble tissue factor domain and the second target binding domain are immediately adjacent to each other. In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises a linker sequence between the second target binding domain and the soluble tissue factor domain.

[0018] In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0019] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, C The antibody binds to a target selected from the group consisting of D155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

[0020] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein. In some embodiments of any of the methods described herein, the soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding protein.

[0021] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. In some embodiments of any of the methods described herein, the soluble interleukin receptor, the soluble cytokine receptor, or the soluble cell surface receptor is a soluble TGF-β receptor II (TGF-βRII), a soluble TGF-βRIII, a soluble NKG2D, a soluble NKp30, a soluble NKp44, a soluble NKp46, a soluble DNAM-1, a scMHCI, a scMHCII, a scTCR, a soluble CD155, or a soluble CD28.

[0022] In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO: 10. In some embodiments of any of the methods described herein, the soluble tissue factor domain does not comprise any of: a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein, an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein, a tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, an arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and a phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

[0023] In some embodiments of any of the methods described herein, the soluble tissue factor domain comprises one or more of: a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

[0024] In some embodiments of any of the methods described herein, the single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and / or C-terminus.

[0025] In some embodiments of any of the methods described herein, the tissue factor-containing protein is a multi-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the multi-chain chimeric polypeptide comprises: (a) a first chimeric polypeptide, the first chimeric polypeptide comprising (i) a first target-binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide, the second chimeric polypeptide comprising (i) a second domain of the pair of affinity domains and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate through binding between the first domain and the second domain of the pair of affinity domains. In some embodiments of any of the methods described herein, the first target-binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the soluble tissue factor domain and the first domain of the pair of affinity domains are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the pair of affinity domains in the first chimeric polypeptide. In some embodiments of any of the methods described herein, the second domain of the pair of affinity domains and the second target binding domain are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains and the second target binding domain in the second chimeric polypeptide.

[0026] In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0027] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is an antigen binding domain. In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD1 55, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

[0028] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein. In some embodiments of any of the methods described herein, the soluble interleukin, cytokine, or ligand protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L.

[0029] In some embodiments of any of the methods described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the methods described herein, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28.

[0030] In some embodiments of any of the methods described herein, the first chimeric polypeptide further comprises one or more additional target binding domains. In some embodiments of any of the methods described herein, the second chimeric polypeptide further comprises one or more additional target binding domains.

[0031] In some embodiments of any of the methods described herein, the soluble tissue factor domain is a soluble human tissue factor domain. In some embodiments of any of the methods described herein, the soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO: 10.

[0032] In some embodiments of any of the methods described herein, the soluble tissue factor domain does not comprise any of the following: a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein, an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein, a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and a phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

[0033] In some embodiments of any of the methods described herein, the soluble tissue factor domain comprises one or more of: a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein.

[0034] In some embodiments of any of the methods described herein, the pair of affinity domains is the sushi domain from human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. In some embodiments of any of the methods described herein, the pair of affinity domains is selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter / docking tag modules based on mutant RNase I fragments, and SNARE modules based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

[0035] Also provided are tissue factor-containing proteins produced by any of the methods described herein. Also provided herein are pharmaceutical compositions comprising any of the tissue factor-containing proteins produced as described herein.

[0036] As used herein, the term "chimeric" refers to a polypeptide that includes amino acid sequences (e.g., domains) that are originally derived from two different sources (e.g., two different naturally occurring proteins, from the same or different species). For example, a chimeric polypeptide can include domains that are derived from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can include a domain that is a synthetic sequence (e.g., an scFv) and a domain that is derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide can include at least two different domains that are synthetic sequences (e.g., two different scFvs).

[0037] An "antigen-binding domain" is one or more protein domains (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens. In some examples, an antigen-binding domain can bind to an antigen or epitope with similar specificity and affinity as a naturally occurring antibody. In some embodiments, the antigen-binding domain may be an antibody or fragment thereof. In some embodiments, the antigen-binding domain may comprise an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Additional examples of antigen-binding domains are known in the art.

[0038] A "soluble tissue factor domain" refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) with a segment of a wild-type mammalian tissue factor protein (e.g., a wild-type human tissue factor protein) lacking the transmembrane and intracellular domains. Non-limiting examples of soluble tissue factor domains are described herein.

[0039] The term "soluble interleukin protein" is used herein to refer to a mature secreted interleukin protein or a biologically active fragment thereof. In some examples, a soluble interleukin protein can comprise a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein) and retains its biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

[0040] The term "soluble cytokine protein" is used herein to refer to a mature secreted cytokine protein or a biologically active fragment thereof. In some examples, a soluble cytokine protein can comprise a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to a wild-type mature secreted mammalian interleukin protein (e.g., a wild-type human interleukin protein) and retains its biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

[0041] The term "soluble interleukin receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that can bind to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble interleukin receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type interleukin receptor and retains the ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks the intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0042] The term "soluble cytokine receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble cytokine receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type cytokine receptor and retains the ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks the intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0043] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. Additional examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0044] "Affinity" refers to the strength of the sum of non-covalent interactions between an antigen-binding site and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between a member of the antigen-binding domain and the antigen or epitope. The affinity of a molecule X for its partner Y is determined by the dissociation equilibrium constant (K D) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity of an antigen-binding domain for its corresponding antigen or epitope are known in the art.

[0045] As used herein, a "multi-chain polypeptide" refers to a polypeptide comprising two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), wherein the two or more protein chains are linked via non-covalent bonds to form a quaternary structure.

[0046] As used herein, a "single-chain polypeptide" refers to a single protein chain.

[0047] The term "affinity domain pair" refers to a -7 Less than M (e.g., 1 × 10 8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M or 1 x 10 -11 K (less than M) D A pair of affinity domains is two different protein domains that specifically bind to each other at a specific site. In some examples, the pair of affinity domains can be a pair of naturally occurring proteins. In some embodiments, the pair of affinity domains can be a pair of synthetic proteins. Non-limiting examples of paired affinity domains are described herein.

[0048] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen-binding domain. An epitope may consist of, for example, surface-accessible amino acid residues and / or sugar side chains, and may have specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former may be lost in the presence of denaturing solvents, but the binding to the latter may not be lost. An epitope may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. Methods for identifying the epitope bound by an antigen-binding domain are known in the art.

[0049] The term "treatment" refers to ameliorating at least one symptom of a disorder. In some examples, the disorder being treated is cancer, and ameliorating at least one symptom of cancer includes reducing abnormal proliferation, gene expression, signal transduction, translation, and / or secretion of a factor. Generally, treatment methods involve administering to a subject in need of, or determined to be in need of, such treatment a therapeutically effective amount of a composition that alleviates at least one symptom of the disorder.

[0050] The term "purifying" refers to a step taken to isolate a protein (e.g., any of the exemplary single-chain or multi-chain chimeric polypeptides described herein) from one or more other impurities (e.g., bulk impurities) or components present in a fluid containing the protein (e.g., liquid culture medium protein present in or secreted from mammalian cells or one or more other components (e.g., DNA, RNA, other proteins, endotoxins, viruses, etc.)). Purification can be carried out using a resin, membrane, or any other solid support that binds either the protein or the contaminants (e.g., using affinity chromatography, hydrophobic interaction chromatography, anion or cation exchange chromatography, or molecular sieve chromatography). Proteins can be purified from a fluid containing the protein using at least one chromatography column (e.g., any of the chromatography columns described herein, including any of the affinity chromatography resins described herein).

[0051] The term "polishing" is a term of the art and refers to a step performed to remove trace or minor contaminants or impurities from a fluid containing a protein (e.g., any of the single-chain or multi-chain chimeric polypeptides described herein) close to the desired final purity. For example, polishing can be performed by passing the protein-containing fluid through a chromatography column or membrane absorber that selectively binds either the protein or minor contaminants or impurities present in the protein-containing fluid. In such an example, the protein is contained in the eluate / filtrate of the chromatography column or membrane absorber.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials for use in the present invention are described herein, and other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In the event of any discrepancy, the present specification, including definitions, shall prevail.

[0053] [The present invention 1001] An anti-tissue factor antibody or antigen-binding fragment thereof bound to a base resin, comprising a heavy chain variable domain comprising the CDRs of SEQ ID NOs: 1, 2 or 9, and 3, and a light chain variable domain comprising the CDRs of SEQ ID NOs: 4, 5, and 6. 1. An affinity chromatography resin comprising: [The present invention 1002] 1001. The affinity chromatography resin of the present invention, wherein said heavy chain variable domain comprises a sequence at least 90% identical to SEQ ID NO:7 and said light chain variable domain comprises a sequence at least 90% identical to SEQ ID NO:8. [The present invention 1003] 1002. The affinity chromatography resin of the present invention, wherein said heavy chain variable domain comprises SEQ ID NO:7 and said light chain variable domain comprises SEQ ID NO:8. [The present invention 1004] 1004. The affinity chromatography resin of any one of claims 1001 to 1003, wherein the base resin is Sepharose. [The present invention 1005] 1004. The affinity chromatography resin of any one of claims 1001 to 1003, wherein the base resin is any support material. [The present invention 1006] 1005. The affinity chromatography resin of the present invention, wherein said support material is an agarose resin or a Capto resin. [The present invention 1007] 1007. The affinity chromatography resin of any one of claims 1001 to 1006, wherein the anti-tissue factor antibody or antigen-binding fragment thereof is non-covalently bound to the base resin. [The present invention 1008] 1007. The affinity chromatography resin of any one of claims 1001 to 1006, wherein the anti-tissue factor antibody or antigen-binding fragment thereof is covalently bound to the base resin. [The present invention 1009] 1008. The affinity chromatography resin of the present invention, wherein the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin by formation of a disulfide bond between a cysteine ​​of the anti-tissue factor antibody or antigen-binding fragment thereof and a chemical group on the base resin. [The present invention 1010] 1008. The affinity chromatography resin of the present invention, wherein the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin by formation of a covalent bond between a free amine of the anti-tissue factor antibody or antigen-binding domain and a chemical group on the base resin. [The present invention 1011] 10. The affinity chromatography resin of claim 10, wherein the base resin is a CNBr-activated or N-hydroxysuccinimide (NHS)-activated solid support. [The present invention 1012] The covalent bond is represented by Formula I: TIFF0007758680000002.tif111281010. The affinity chromatography resin of the present invention, wherein R represents the anti-tissue factor antibody or an antigen-binding fragment thereof. [The present invention 1013] A kit comprising the affinity chromatography resin of any one of 1001 to 1012 of the present invention. [The present invention 1014] A method for purifying a tissue factor-containing protein, comprising the use of the affinity chromatography resin of any one of claims 1001 to 1012. [The present invention 1015] loading a liquid containing the tissue factor-containing protein onto the affinity chromatography resin; washing the affinity chromatography resin with one or more wash buffers; eluting the tissue factor-containing protein using an elution buffer; The method of the present invention 1014, comprising: [The present invention 1016] 1015. The method of claim 1015, wherein said liquid containing said tissue factor-containing protein is a clarified liquid culture medium. [The present invention 1017] 1016. The method of claim 1015, wherein said liquid containing said tissue factor-containing protein comprises a cell lysate. [The present invention 1018] the one or more wash buffers (i) a first wash buffer comprising phosphate-buffered saline; and (ii) a second wash buffer containing about 0.01 M to about 0.2 M citrate and having a pH of about 4.5 to about 5.5; Any of methods 1015 to 1017 of the present invention. [The present invention 1019] (i) the first wash buffer is phosphate buffered saline; (ii) the second wash buffer is 0.1 M citrate, pH 5.0; The method of the present invention 1018. [The present invention 1020] 1019. The method of any of claims 1015 to 1019, wherein said elution buffer contains 0.01 M to about 0.2 M acetate and has a pH of about 2.5 to about 3.5. [The present invention 1021] 1020. The method of claim 1020, wherein said elution buffer comprises 0.1 M acetate and has a pH of about 2.9. [The present invention 1022] 1. A method for producing a tissue factor-containing protein, comprising: (i) purifying a tissue factor-containing protein using any of the methods of the present invention 1014 to 1021; (ii) subjecting the eluate obtained from step (i) to one or more additional unit operations; A method comprising: [The present invention 1023] the one or more additional unit operations, in sequential order: Low pH viral inactivation Performing depth filtration, performing polishing chromatography; performing nanofiltration; and Performing ultrafiltration and diafiltration (UF / DF) The method of the present invention 1022, comprising: [The present invention 1024] The method of any one of claims 1014 to 1023, wherein the tissue factor-containing protein is a single-chain chimeric polypeptide. [The present invention 1025] the single-chain chimeric polypeptide (i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a second target binding domain; and The method of the present invention 1024, comprising: [The present invention 1026] 1026. The method of claim 1025, wherein said first target binding domain and said soluble tissue factor domain are immediately adjacent to each other. [The present invention 1027] 1026. The method of claim 1025, wherein said single-chain chimeric polypeptide further comprises a linker sequence between said first target binding domain and said soluble tissue factor domain. [The present invention 1028] 1028. The method of any of claims 1025 to 1027, wherein said soluble tissue factor domain and said second target binding domain are immediately adjacent to each other. [The present invention 1029] 1028. The method of any of claims 1025 to 1027, wherein said single-chain chimeric polypeptide further comprises a linker sequence between said soluble tissue factor domain and said second target binding domain. [The present invention 1030] 1029. The method of any of claims 1025 to 1029, wherein said first target-binding domain and said second target-binding domain specifically bind to the same antigen. [The present invention 1031] The method of claim 1030, wherein said first target binding domain and said second target binding domain specifically bind to the same epitope. [The present invention 1032] 1032. The method of claim 1031, wherein said first target binding domain and said second target binding domain comprise the same amino acid sequence. [The present invention 1033] 1029. The method of any of claims 1025 to 1029, wherein said first target-binding domain and said second target-binding domain specifically bind to different antigens. [The present invention 1034] The method of any of claims 1025 to 1033, wherein one or both of said first target-binding domain and said second target-binding domain is an antigen-binding domain. [This invention 1035] One or both of the first target binding domain and the second target binding domain are selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULB P2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BC MA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TG any of methods 1025 to 1034, wherein the antibody binds to a target selected from the group consisting of a TGF-βRIII (TGF-βRII) ligand, a TGF-βRIII ligand, a DNAM-1 ligand, an NKp46 ligand, an NKp44 ligand, an NKG2D ligand, an NKp30 ligand, a scMHCII ligand, a scTCR ligand, an IL-1 receptor, an IL-2 receptor, an IL-3 receptor, an IL-7 receptor, an IL-8 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-17 receptor, an IL-18 receptor, an IL-21 receptor, a PDGF-DD receptor, a stem cell factor (SCF) receptor, a stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, a MICA receptor, a MICB receptor, a ULP16 binding protein receptor, a CD155 receptor, a CD122 receptor, and a CD28 receptor. [The present invention 1036] The method of any one of claims 1025 to 1034, wherein one or both of said first target binding domain and said second target binding domain is a soluble interleukin, a soluble cytokine protein, or a soluble cell surface protein. [This invention 1037] 1036. The method of claim 1036, wherein said soluble interleukin, soluble cytokine protein, or soluble cell surface protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, FLT3L, MICA, MICB, and ULP16 binding protein. [The present invention 1038] The method of any of claims 1025 to 1034, wherein one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor, a soluble cytokine receptor, or a soluble cell surface receptor. [This invention 1039] 1038. The method of claim 1038, wherein the soluble interleukin receptor, the soluble cytokine receptor, or the soluble cell surface receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28. [The present invention 1040] 1039. The method of any of claims 1025 to 1039, wherein said soluble tissue factor domain is a soluble human tissue factor domain. [The present invention 1041] 104. The method of claim 1040, wherein said soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO:10. [The present invention 1042] the soluble tissue factor domain a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and Phenylanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein Any of the methods of 1025 to 1039 of the present invention, which does not include any of the above. [This invention 1043] the soluble tissue factor domain a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and Phenylanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein Any of the methods of 1025 to 1039 of the present invention, including one or more of the following. [This invention 1044] 1044. The method of any of claims 1025 to 1043, wherein said single-chain chimeric polypeptide further comprises one or more additional target binding domains at its N-terminus and / or C-terminus. [This invention 1045] The method of any one of claims 1014 to 1023, wherein the tissue factor-containing protein is a multi-chain chimeric polypeptide. [The present invention 1046] the multi-chain chimeric polypeptide (a)(i) a first target binding domain; (ii) a soluble tissue factor domain, and (iii) the first domain of the pair of affinity domains a first chimeric polypeptide comprising: (b)(i) a second domain of the pair of affinity domains, and (ii) a second target-binding domain and a second chimeric polypeptide comprising: Including, 1045. The method of claim 1045, wherein the first chimeric polypeptide and the second chimeric polypeptide associate via binding between the first domain and the second domain of the pair of affinity domains. [This invention 1047] 1047. The method of claim 1046, wherein said first target binding domain and said soluble tissue factor domain are immediately adjacent to each other in said first chimeric polypeptide. [This invention 1048] 1047. The method of claim 1046, wherein said first chimeric polypeptide further comprises a linker sequence between said first target binding domain and said soluble tissue factor domain within said first chimeric polypeptide. [This invention 1049] 1049. The method of any of claims 1046 to 1048, wherein said soluble tissue factor domain and said first domain of said pair of affinity domains are immediately adjacent to each other within said first chimeric polypeptide. [The present invention 1050] Any of the methods of claims 1046 to 1048, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain of the pair of affinity domains. [This invention 1051] 1050. The method of any of claims 1046 to 1050, wherein said second domain and said second target binding domain of said pair of affinity domains are immediately adjacent to each other within said second chimeric polypeptide. [This invention 1052] Any of the methods of claims 1046 to 1050, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the pair of affinity domains within the second chimeric polypeptide and the second target binding domain. [This invention 1053] 1053. The method of any of claims 1046 to 1052, wherein said first target-binding domain and said second target-binding domain specifically bind to the same antigen. [This invention 1054] The method of any of claims 1046 to 1052, wherein said first target-binding domain and said second target-binding domain specifically bind to different antigens. [This invention 1055] The method of any of claims 1046 to 1054, wherein one or both of said first target-binding domain and said second target-binding domain is an antigen-binding domain. [This invention 1056] One or both of the first target binding domain and the second target binding domain may be selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, and ULBP. 2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA , P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF- any of methods 1046 to 1055, wherein the antibody specifically binds to a target selected from the group consisting of a TGF-βRIII (TGF-βRII) ligand, a TGF-βRIII ligand, a DNAM-1 ligand, an NKp46 ligand, an NKp44 ligand, an NKG2D ligand, an NKp30 ligand, a scMHCII ligand, a scTCR ligand, an IL-1 receptor, an IL-2 receptor, an IL-3 receptor, an IL-7 receptor, an IL-8 receptor, an IL-10 receptor, an IL-12 receptor, an IL-15 receptor, an IL-17 receptor, an IL-18 receptor, an IL-21 receptor, a PDGF-DD receptor, a stem cell factor (SCF) receptor, a stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, a MICA receptor, a MICB receptor, a ULP16 binding protein receptor, a CD155 receptor, a CD122 receptor, and a CD28 receptor. [This invention 1057] 1056. The method of any of claims 1046 to 1055, wherein one or both of said first target binding domain and said second target binding domain is a soluble interleukin or cytokine protein. [This invention 1058] 1057. The method of claim 1057, wherein said soluble interleukin, cytokine, or ligand protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. [This invention 1059] The method of any of claims 1046 to 1055, wherein one or both of said first target binding domain and said second target binding domain is a soluble interleukin or cytokine receptor. [The present invention 1060] 1059. The method of the invention 1059, wherein the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, or soluble CD28. [The present invention 1061] 1061. The method of any of claims 1046 to 1060, wherein said first chimeric polypeptide further comprises one or more additional target binding domains. [The present invention 1062] 1062. The multi-chain chimeric polypeptide of any of claims 1046 to 1061, wherein said second chimeric polypeptide further comprises one or more additional target binding domains. [This invention 1063] 1063. The method of any one of claims 1046 to 1062, wherein said soluble tissue factor domain is a soluble human tissue factor domain. [This invention 1064] 1064. The method of claim 1063, wherein said soluble human tissue factor domain comprises a sequence at least 80% identical to SEQ ID NO:10. [This invention 1065] the soluble tissue factor domain a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and Phenylanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein Any of the methods of inventions 1046 to 1062, which does not include any of the above. [The present invention 1066] the soluble tissue factor domain a lysine at an amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and Phenylanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein Any of the methods of 1046 to 1062 of the present invention, comprising one or more of the following: [This invention 1067] The method of any of claims 1046 to 1066, wherein the pair of affinity domains is a sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. [The present invention 1068] 1066. The method of any of claims 1046 to 1066, wherein the pair of affinity domains is selected from the group consisting of barnase and barnstar, PKA and AKAP, adapter / docking tag modules based on mutant RNase I fragments, and SNARE modules based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. [The present invention 1069] A tissue factor-containing protein produced by any of the methods of the present inventions 1014 to 1068. [The present invention 1070] A pharmaceutical composition comprising the tissue factor-containing protein produced according to the present invention. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. [Brief explanation of the drawings]

[0054] [Figure 1]1 is a workflow schematic illustrating an exemplary workflow for producing tissue factor-containing protein, including providing a harvested cell culture (e.g., a substantially cell-free (e.g., at least 99% cell-free) liquid culture medium), purifying the tissue factor-containing protein using any of the affinity chromatography resins described herein, performing low pH viral inactivation, performing depth filtration, performing polishing chromatography, performing nanofiltration, and performing ultrafiltration / diafiltration (shown in the schematic as "TFF / Final Formulation"). [Figure 2] FIG. 10 is an elution profile of 18t15-12s protein from an exemplary affinity chromatography resin provided herein (αTF antibody CNBr-Sepharose affinity column) equilibrated with phosphate buffered saline, loaded, washed with phosphate buffered saline, and eluted using 0.1 M acetic acid, pH 2.9. [Figure 3] 1 is a graph of ligand density versus binding capacity for different affinity chromatography resin preparations, showing the relationship between anti-tissue factor antibody coupling density and the binding capacity of the resin for tissue factor-containing proteins, such as 18t15-12s protein. [Figure 4] 1 is a graph showing optimization of wash conditions for 18t15-12s purification using an exemplary affinity chromatography resin (anti-tissue factor CNBr-Sepharose affinity column) provided herein. Each experiment was performed using a column equilibrated with phosphate-buffered saline, loaded, washed with phosphate-buffered saline, and eluted using 0.1 M acetic acid, pH 2.9. Data shown in blue did not include an additional 5 CV wash with 0.1 M sodium citrate, pH 5.0, while data in orange included an additional 5 CV wash with 0.1 M sodium citrate, pH 5.0. [Figure 5]FIG. 1 shows the elution profile of the first run of a freshly prepared exemplary affinity chromatography resin (anti-tissue factor CNBr column) purified by equilibrating the resin with phosphate buffered saline, loading the resin, washing the resin with phosphate buffered saline, and eluting the resin with 0.1 M acetic acid, pH 2.9. [Figure 6] The elution profile of the 51st purification run using the same column used in Figure 5 is shown. The 51st purification run was performed by equilibrating the column with phosphate buffered saline, loading the column, washing the column with phosphate buffered saline, and eluting the column with 0.1 M acetic acid, pH 2.9. [Figure 7] 1 shows the partial structure of NHS-activated Sepharose high performance resin with an activated spacer arm. [Figure 8] Coupling of a protein ligand containing a primary amino group (R-NH2) to NHS-activated Sepharose resin is shown. [Figure 9] 1 shows a chromatogram of the elution profile of TGFRt15-TGFRs from an exemplary affinity chromatography resin (anti-tissue factor CNBr column) provided herein. The dark gray line is absorbance at 280 nm, and the light gray line represents the use of elution buffer (0.1 M acetic acid, pH 2.9). [Figure 10] 1 shows a chromatogram of the 2t2 elution profile from an exemplary affinity chromatography resin (anti-tissue factor CNBr column) provided herein. The dark gray line is absorbance at 280 nm, and the light gray line represents the use of elution buffer (0.1 M acetic acid, pH 2.9). DETAILED DESCRIPTION OF THE INVENTION

[0055] Provided herein is an affinity chromatography resin comprising an anti-tissue factor antibody or antigen-binding fragment thereof bound to a base resin, the antibody comprising a heavy chain variable domain comprising the CDRs of SEQ ID NOs: 1, 2 or 9, and 3, and a light chain variable domain comprising the CDRs of SEQ ID NOs: 4, 5, and 6.

[0056] Also provided herein are kits comprising any of the affinity chromatography resins described herein. Also provided herein are methods for purifying tissue factor-containing proteins comprising the use of any of the affinity chromatography resins described herein. Also provided herein are methods for producing tissue factor-containing proteins, comprising: (i) purifying tissue factor-containing proteins using any of the methods for purifying tissue factor-containing proteins described herein; and (ii) performing one or more additional unit operations on the eluate obtained from step (i). Also provided herein are tissue factor-containing proteins produced by any of the methods described herein. Also provided herein are pharmaceutical compositions comprising any of the tissue factor-containing proteins produced as described herein.

[0057] In some embodiments, the affinity chromatography resins described herein have a concentration of about 0.5 mg / mL to about 6.0 mg / mL (e.g., about 0.5 mg / mL to about 5.5 mg / mL, about 0.5 mg / mL to about 5.0 mg / mL, about 0.5 mg / mL to about 4.5 mg / mL, about 0.5 mg / mL to about 4.0 mg / mL, about 0.5 mg / mL to about 3.5 mg / mL, about 0.5 mg / mL to about 3.0 mg / mL, about 0.5 mg / mL to about 2.5 mg / mL, about 0.5 mg / mL to about 2.0 mg / mL, about 0.5 mg / mL to about 1.5 ... L ~ about 1.0 mg / mL, about 1.0 mg / mL - about 6.0 mg / mL, about 1.0 mg / mL - about 5.5 mg / mL, about 1.0 mg / mL - about 5.0 mg / mL, about 1.0 mg / mL - about 4.5 mg / mL, about 1.0 mg / mL - about 4.0 mg / mL, about 1.0 mg / mL - about 3 .5mg / mL, about 1.0mg / mL to about 3.0mg / mL, about 1.0mg / mL to about 2.5mg / mL, about 1.0mg / mL to about 2.0mg / mL, about 1.0mg / mL to about 1.5mg / mL, about 1.5mg / mL to about 6.0mg / mL, about 1.5mg / mL to about 5.5mg / mL, approximately 1.5 mg / mL to approximately 5.0 mg / mL, approximately 1.5 mg / mL to approximately 4.5 mg / mL, approximately 1.5 mg / mL to approximately 4.0 mg / mL, approximately 1.5 mg / mL to approximately 3.5 mg / mL, approximately 1.5 mg / mL to approximately 3.0 mg / mL, approximately 1.5 mg / mL to approximately 2.5 mg / mL, Approximately 1.5 mg / mL to approximately 2.0 mg / mL, approximately 2.0 mg / mL to approximately 6.0 mg / mL, approximately 2.0 mg / mL to approximately 5.5 mg / mL, approximately 2.0 mg / mL to approximately 5.0 mg / mL, approximately 2.0 mg / mL to approximately 4.5 mg / mL, approximately 2.0 mg / mL to approximately 4.0 mg / mL, approximately 2.0 mg / mL~about 3.5mg / mL, about 2.0mg / mL~about 3.0mg / mL, about 2.0mg / mL~about 2.5mg / mL, about 2.5mg / mL~about 6.0mg / mL, about 2.5mg / mL~about 5.5mg / mL, about 2.5mg / mL~about 5.0mg / mL, about 2.5mg / m L~4.5mg / mL, 2.5mg / mL~4.0mg / mL, 2.5mg / mL~3.5mg / mL, 2.5mg / mL~3.0mg / mL, 3.0mg / mL~6.0mg / mL, 3.0mg / mL~5.5mg / mL, 3.0mg / mL~5.0mg / mL, about 3.0mg / mL to about 4.5mg / mL, about 3.0mg / mL to about 4.0mg / mL, about 3.0mg / mL to about 3.5mg / mL, about 3.5mg / mL to about 6.0mg / mL, about 3.5mg / mL to about 5 .5mg / mL, about 3.5mg / mL to about 5.0mg / mL, about 3.5mg / mL to about 4.5mg / mL, about 3.5mg / mL to about 4.0mg / mL, about 4.0mg / mL to about 6.0mg / mL, about 4.0mg / mL to about The tissue factor-containing protein may have a binding ability of about 5.5 mg / mL, about 4.0 mg / mL to about 5.0 mg / mL, about 4.0 mg / mL to about 4.5 mg / mL, about 4.5 mg / mL to about 6.0 mg / mL, about 4.5 mg / mL to about 5.5 mg / mL, about 4.5 mg / mL to about 5.0 mg / mL, about 5.0 mg / mL to about 6.0 mg / mL, about 5.0 mg / mL to about 5.5 mg / mL, or about 5.5 mg / mL to about 6.0 mg / mL.

[0058] As used herein, "affinity chromatography" refers to a method for separating a solution or mixture based on highly specific interactions between antigens and antibodies, enzymes and substrates, receptors and ligands, or proteins and nucleic acids. Affinity chromatography (e.g., Protein A, Protein G, Protein L, or metal chelate affinity chromatography) is an effective method for specifically purifying target proteins in starting mixtures such as cell culture supernatants, serum, and plasma with high recovery rates and low levels of contaminating proteins and other components. Once a clarified liquid or mixture containing the target protein is obtained, affinity chromatography can be used in conjunction with a combination of one or more different purification techniques (e.g., ion exchange separation, hydrophobic interaction separation) for the purification of the target protein.

[0059] Antibody (e.g., monoclonal or polyclonal)-based immunoaffinity chromatography is widely used in research, development, and manufacturing as a method / process for purifying specific antigens or antigen-containing proteins (e.g., human tissue factor-containing proteins). In immunoaffinity methods, antibodies (e.g., monoclonal) or antibody mixtures (e.g., polyclonal) are covalently immobilized (e.g., coupled) onto solid supports such as CNBr-activated Sepharose, NHS-Sepharose, and other related beads / resins suitable for protein coupling or immobilization. The solid support can be any material to which a biospecific ligand can be covalently attached. Examples of materials used in affinity chromatography include agarose, cellulose, dextran, polyacrylamide, latex, and controlled-pore glass. Useful supports can have properties such as a high surface area-to-volume ratio, chemical groups that are easily modified for covalent attachment of ligands, minimal nonspecific binding properties, good flow properties, and mechanical and chemical stability.

[0060] tissue factor Human tissue factor is a 263-amino acid transmembrane protein containing three domains: (1) a 219-amino acid N-terminal extracellular domain (residues 1–219), (2) a 22-amino acid transmembrane domain (residues 220–242), and (3) a 21-amino acid cytoplasmic C-terminal tail (residues 242–263) (UniProtKB Identifier Number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258 and one S-palmitoylation site at Cys245. Deletions or mutations in the cytoplasmic domain have not been found to affect tissue factor clotting activity. Tissue factor has one S-palmitoylation site within the intracellular domain of the protein at Cys245. Cys245 is located at the amino acid terminus of the intracellular domain, near the membrane surface. The tissue factor transmembrane domain is composed of a single transmembrane α-helix.

[0061] The extracellular domain of tissue factor, consisting of two fibronectin type III domains, is linked to the transmembrane domain via a six-amino acid linker. This linker provides conformational flexibility to separate the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping β-sheets, with the upper sheet domain containing three antiparallel β-strands and the lower sheet containing four β-strands. The β-strands are connected by β-loops between strands βA and βB, βC and βD, and βE and βF, all of which conformations are conserved within the two modules. Three short α-helical segments connect the β-strands. A unique feature of tissue factor is the 17-amino acid β-hairpin between strands β10 and β11, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is unique to tissue factor and absent in the C-terminal domain. Such a fibronectin type III domain structure is characteristic of the immunoglobulin-like protein family fold and is conserved among a wide variety of extracellular proteins.

[0062] Zymogen FVII is rapidly converted to FVIIa by limited proteolysis upon binding to tissues to form the active tissue factor-FVIIa complex. FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa on the tissue factor-FVIIa complex significantly increases the enzymatic activity of FVIIa, increasing the hydrolysis rate of small chromogenic peptidyl substrates by approximately 20- to 100-fold and the activation rate of the natural polymer substrates FIX and FX by nearly one million-fold. In concert with the allosteric activation of the FVIIa active site upon binding to tissue factor, the formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface) induces Ca + ... 2+It further increases the activation rate of FIX or FX by 1,000-fold in a tissue factor-dependent manner. The approximately 1,000,000-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complexes compared to free FVIIa is a critical regulatory point in the coagulation cascade.

[0063] FVII is a single polypeptide chain of approximately 50 kDa, consisting of 406 amino acid residues, with an N-terminal gamma-carboxyglutamic acid-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EGFR2), and a C-terminal serine protease domain. FVII is characterized by the Ile-Ile linker region between the EGF2 and protease domains. 154 -Arg 152 Activation to FVIIa occurs by specific proteolytic cleavage of the bond between the light and heavy chains. 135 and Cys 262 FVIIa binds to Ca via its N-terminal GLA domain. 2+ Immediately C-terminal to the GLA domain is an aromatic stack and two EGF domains. The aromatic stack binds the GLA domain to a single Ca 2+ This Ca 2+ Occupation of the binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consists of His 193 , Asp 242 , and Ser 344 and a single Ca domain in the FVIIa protease domain. 2+ Ion binding is essential for its catalytic activity. The proteolytic activity of FVII to FVIIa is mediated by Ile 153 releases the newly formed amino terminus, folds back, and inserts into the activation pocket, forming Asp 343It forms a salt bridge with the carboxylate of FVIIa to generate an oxyanion hole. This salt bridge formation is essential for FVIIa activity. However, the formation of the oxyanion hole does not occur in free FVIIa upon proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

[0064] Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is important for FVIIa to its cognate substrate. Free FVIIa adopts a stable extended conformation upon membrane binding, with its active site positioned approximately 80 Å above the membrane surface. Upon FVIIa binding to tissue factor, the FVIIa active site repositions approximately 6 Å closer to the membrane. This adjustment may aid in the proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLA domain-less FVIIa, it was shown that the active site was still positioned at a similar distance from the membrane, indicating that tissue factor can fully support FVIIa active site positioning even in the absence of FVIIa-membrane interactions. Additional data showed that tissue factor supports full FVIIa proteolytic activity as long as the tissue factor extracellular domain is somehow tethered to the membrane surface. However, elevating the active site of FVIIa more than 80 Å from the membrane surface significantly reduced the ability of the tissue factor-FVIIa complex to activate FX, but did not reduce tissue factor-FVIIa amidolytic activity.

[0065] Alanine-scanning mutagenesis has been used to evaluate the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitutions identified a limited number of residue positions where alanine substitutions caused a 5- to 10-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be well exposed to solvent in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand-binding site spans an extensive region of the interface between the two modules. In the C-module, residues Arg, located on the protruding BC loop, 135 and Phe 140 provides independent contact with FVIIa. 133 is located at the base of the finger-like structure and is packed into the gap between the two modules. 20 , Thr 60 , Asp 58 , and Ile 22 Continuity is provided for the major cluster of important binding residues consisting of Thr 60 is only partially solvent exposed and may play a local structural role rather than making significant contact with the ligand. 24 and Gln 110 , as well as potentially more distal residue Val 207 The binding region extends from Asp58 to Lys 48 , Lys 46 , Gln 37 , Asp 44 , and Trp 45 The convex area formed by the Trp 45 and Asp 44 does not interact independently with FVIIa, and Trp 45 The effect of mutation at position 1 is similar to that of the adjacent Asp 44 and Gln 37The interaction region is located between two surface-exposed aromatic residues, Phe, which form part of a hydrophobic cluster within the N-module. 76 and Tyr 78 Further includes:

[0066] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as certain proteinase-activated receptors. Mutational analysis has identified several residues that, when mutated, support full FVIIa amidolytic activity on small peptidyl substrates but lack the ability to support activation of macromolecular substrates (i.e., FVII, FIX, and FX) (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9):6375-6381, 1992; Huang et al., J Biol Chem 271(36):21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region from residues 159 to 165, and residues within or adjacent to this flexible loop, have been shown to be essential for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate-binding exosite region of tissue factor, located far from the FVIIa active site. Substitution of the glycine residue with a slightly more bulky alanine residue significantly impaired the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility afforded by glycine is essential for the loop from residues 159 to 165 for tissue factor macromolecular substrate recognition.

[0067] Residue Lys 165 and Lys 166 It has also been demonstrated that Lys is important for substrate recognition and binding. Mutation of either of these residues to alanine significantly reduces tissue factor cofactor function. 165 and Lys 166 are facing outwards, and Lys165 refers to FVIIa in most tissue factor-FVIIa structures, and Lys 166 refers to the substrate-binding exosite region in the crystal structure. Lys of FVIIa 165 and FVIIa Gla 35 The putative salt bridge formation between tissue factor and the GLA domain of FVIIa would support the notion that tissue factor interaction with the GLA domain of FVIIa regulates substrate recognition. These results suggest that the C-terminal part of the tissue factor ectodomain interacts directly with the GLA domains of FIX and FX, and with the possible adjacent EGF1 domain, and that the presence of the FVIIa GLA domain may regulate these interactions either directly or indirectly.

[0068] Anti-tissue factor antibodies and antigen-binding antibody fragments In some examples, the anti-tissue factor antibody is a humanized or fully human anti-tissue factor antibody (see, eg, the anti-tissue factor antibodies described in US Pat. Nos. 7,968,094 and 8,007,795).

[0069] In some embodiments, the anti-tissue factor antibody may comprise a heavy chain variable domain comprising the following series of CDR sequences: CDR1 comprising DYNVY (SEQ ID NO: 1), CDR2 comprising YIDPYNGITIYDQNFKG (SEQ ID NO: 2), and CDR3 comprising DVTTALDF (SEQ ID NO: 3).

[0070] In some embodiments, the anti-tissue factor antibody may comprise a heavy chain variable domain comprising the following series of CDR sequences: CDR1 comprising DYNVY (SEQ ID NO: 1), CDR2 comprising YIDPYNGITIYDQNLKG (SEQ ID NO: 9), and CDR3 comprising DVTTALDF (SEQ ID NO: 3). Exemplary Heavy Chain Variable Domain (SEQ ID NO: 7) QIQLVQSGGEVKKPGASVRVSCKASGYSFTDYNVYWVRQSPGKGLEWIGYIDPYNGITIYDQNFKGKATLTVDKSTSTAYMELSSLRSEDTAVYFCARDVTTALDFWGQGTTVTVSS Exemplary Heavy Chain (SEQ ID NO: 127) QIQLVQSGGEVKKPGASVRVSCKASGYSFTDYNVYWVRQSPGKGLEWIGYIDPYNGITIYDQNFKGKATLTVDKSTSTAYMELSSLRSEDTAVYFCARDVTTALDFWGQGTT VTVSSEFASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK cDNA encoding an exemplary heavy chain (SEQ ID NO: 128)

[0071] In some embodiments, the anti-tissue factor antibody may comprise a heavy chain variable domain comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:7.

[0072] In some embodiments, the anti-tissue factor antibody may comprise a heavy chain variable domain comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 127.

[0073] In some embodiments, the anti-tissue factor antibody may comprise a light chain variable domain comprising the following series of CDR sequences: CDR1 comprising LASQTIDTWLA (SEQ ID NO: 4), CDR2 comprising AATNLAD (SEQ ID NO: 5), and CDR3 comprising QQVYSSPFT (SEQ ID NO: 6). Exemplary Light Chain Variable Domain (SEQ ID NO: 8) DIQMTQSPASLSASVGDRVTITCLASQTIDTWLAWYLQKPGKSPQLLIYAATNLADGVPSRFSGSGSGTDFSFTISSLQPEDFATYYCQQVYSSPFTFGQGTKLEIK Exemplary Light Chain (SEQ ID NO: 129) DIQMTQSPASLSASVGRVTITCLASQTIDTWLAWLQKPGKSPQLLIYAATNLDGVPSRFSGSGSGTDFSFISSLQPEDFATYYCQQVYSSPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC cDNA encoding an exemplary light chain (SEQ ID NO: 130) GACATCCAGATGACCCAGAGCCCCGCCAGCCTGAGCGCCAGCGTGGGCGACAGGGTGACCATCACCTGCCTGGCCAGCCAGACCATCGACACCTGGCTGGCCTGGTACCTGCAGAAGCCCGGCAAGAGCCCCCAGCTGCTGATCTACGCCGCCACCAACCTGGCCGACGGCGTGCCCAGCAGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCAGCTTCAACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGGTGTACTAGCAGCCCCTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGAGGACCGTGGCCGCCCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCAGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACAGCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGAGCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGCTGA

[0074] In some embodiments, the anti-tissue factor antibody may comprise a light chain variable domain comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:8.

[0075] In some embodiments, the anti-tissue factor antibody may comprise a light chain variable domain comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 129.

[0076] Non-limiting examples of anti-tissue factor antibodies include heavy chain variable domain antibodies comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:7. Examples of such a domain include a main and a light chain variable domain comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO:8.

[0077] Non-limiting examples of anti-tissue factor antibodies include those comprising a heavy chain variable domain comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 127. Examples of such a domain include a main and a light chain variable domain comprising a sequence at least 70% identical (e.g., at least 72%, at least 74%, at least 75%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 85%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to SEQ ID NO: 129.

[0078] In some embodiments of any of the anti-tissue factor antibodies described herein, the anti-tissue factor antibody is present in a concentration of about 0.1 nM to about 1 μM (e.g., about 0.1 nM to about 800 nM, about 0.1 nM to about 750 nM, about 0.1 nM to about 700 nM, about 0.1 nM to about 650 nM, about 0.1 nM to about 600 nM, about 0.1 nM to about 550 nM, about 0.1 nM to about 500 nM, about 0.1 nM to about 450 nM, about 0.1 nM to about 400 nM, about 0.1 nM to about 350 nM, about 0.1 nM to about 300 nM, about 0.1 nM to about 250 nM, about 0.1 nM to about 200 nM, about 0.1 nM to about ~about 150nM, about 0.1nM to about 100nM, about 0.1nM to about 80nM, about 0.1nM to about 60nM, about 0.1nM to about 50nM, Approximately 0.1nM to approximately 40nM, approximately 0.1nM to approximately 25nM, approximately 0.1nM to approximately 20nM, approximately 0.1nM to approximately 15nM, approximately 0.1nM to approximately 10 nM, about 0.1nM to about 8nM, about 0.1nM to about 6nM, about 0.1nM to about 5nM, about 0.1nM to about 4nM, about 0.1nM to about 3nM , about 0.1nM to about 2nM, about 0.1nM to about 1nM, about 0.1nM to about 0.8nM, about 0.1nM to about 0.6nM, about 0.1nM to about 0 .4nM, about 0.1nM to about 0.2nM, about 0.2nM to about 1μM, about 0.2nM to about 800nM, about 0.2nM to about 750nM, about 0 .2nM to about 700nM, about 0.2nM to about 650nM, about 0.2nM to about 600nM, about 0.2nM to about 550nM, about 0.2nM to about 500nM, about 0.2nM to about 450nM, about 0.2nM to about 400nM, about 0.2nM to about 350nM, about 0.2nM to about 300, about 0 .2nM to about 250nM, about 0.2nM to about 200nM, about 0.2nM to about 150nM, about 0.2nM to about 100nM, about 0.2nM to about 80nM, about 0.2nM to about 60nM, about 0.2nM to about 50nM, about 0.2nM to about 40nM, about 0.2nM to about 25nM, about 0.2n M ~ about 20nM, about 0.2nM - about 15nM, about 0.2nM - about 10nM, about 0.2nM - about 8nM, about 0.2nM - about 6nM, about 0.2 nM to about 5nM, about 0.2nM to about 4nM, about 0.2nM to about 3nM, about 0.2nM to about 2nM, about 0.2nM to about 1nM, about 0.2nM ~about 0.8nM, about 0.2nM to about 0.6nM, about 0.2nM to about 0.4nM, about 0.2nM to about 0.2nM, about 0.4nM to about 1μM,about 0.4 nM to about 800 nM, about 0.4 nM to about 750 nM, about 0.4 nM to about 700 nM, about 0.4 nM to about 650 nM, about 0.4 nM to about 600 nM, about 0.4 nM to about 550 nM, about 0.4 nM to about 500 nM, about 0.4 nM to about 450 nM, about 0.4 nM to about 400 nM M, about 0.4 nM to about 350 nM, about 0.4 nM to about 300, about 0.4 nM to about 250 nM, about 0.4 nM to about 200 nM, about 0.4 nM to about 150 nM, about 0.4 nM to about 100 nM, about 0.4 nM to about 80 nM, about 0.4 nM to about 60 nM, about 0.4 nM to about 50 nM, about 0.05 nM to about 100 nM, about 0.06 nM to about 150 nM, about 0.07 nM to about 100 nM, about 0.08 nM to about 150 nM, about 0.09 nM to about 100 nM, about 0.09 nM to about 150 nM, about 0.09 nM to about 1 .4 nM to about 40 nM, about 0.4 nM to about 25 nM, about 0.4 nM to about 20 nM, about 0.4 nM to about 15 nM, about 0.4 nM to about 10 nM, about 0.4 nM to about 8 nM, about 0.4 nM to about 6 nM, about 0.4 nM to about 5 nM, about 0.4 nM to about 4 nM, about 0.4 nM to about 3 nM, about 0.4 nM to about 4 nM, about 0.4 nM to about 3 nM, about 0.4 nM to about 5 nM, about 0.4 nM to about 6 nM, about 0.4 nM to about 5 nM, about 0.4 nM to about 4 nM, about 0.4 nM to about 3 nM, about 0 0.4 nM to about 2 nM, about 0.4 nM to about 1 nM, about 0.4 nM to about 0.8 nM, about 0.4 nM to about 0.6 nM, about 0.5 nM to about 1 μM, about 0.5 nM to about 800 nM, about 0.5 nM to about 750 nM, about 0.5 nM to about 700 nM, about 0.5 nM to about 650 nM, about 0.5 nM to about about 600 nM, about 0.5 nM to about 550 nM, about 0.5 nM to about 500 nM, about 0.5 nM to about 450 nM, about 0.5 nM to about 400 nM, about 0.5 nM to about 350 nM, about 0.5 nM to about 300, about 0.5 nM to about 250 nM, about 0.5 nM to about 200 nM, about 0.5 nM to about about 150 nM, about 0.5 nM to about 100 nM, about 0.5 nM to about 80 nM, about 0.5 nM to about 60 nM, about 0.5 nM to about 50 nM, about 0.5 nM to about 40 nM, about 0.5 nM to about 25 nM, about 0.5 nM to about 20 nM, about 0.5 nM to about 15 nM, about 0.5 nM to about 10 nM, about 0.5 nM to about 8 nM, about 0.5 nM to about 6 nM, about 0.5 nM to about 5 nM, about 0.5 nM to about 4 nM, about 0.5 nM to about 3 nM, about 0.5 nM to about 2 nM, about 0.5 nM to about 1 nM, about 0.5 nM to about 0.8 nM, about 0.5 nM to about 0.6 nM, about 0.6 nM to about 1 μM, about 0.6 nM to about 800 nM, about 0.6 nM to about 750 nM, about 0.6 nM to about 700 nM, about 0.6 nM to about 650 nM, about 0.6 nM to about 600 nM, about 0.6 nM to about 550 nM, about 0.6 nM to about 500 nM, about 0.6 nM to about 450 nM, about 0.6 nM to about 400 nM,about 0.6 nM to about 350 nM, about 0.6 nM to about 300, about 0.6 nM to about 250 nM, about 0.6 nM to about 200 nM, about 0.6 nM to about 150 nM, about 0.6 nM to about 100 nM, about 0.6 nM to about 80 nM, about 0.6 nM to about 60 nM, about 0.6 nM to about 50 nM, about 0. 6nM to about 40nM, about 0.6nM to about 25nM, about 0.6nM to about 20nM, about 0.6nM to about 15nM, about 0.6nM to about 10nM, about 0.6nM to about 8nM, about 0.6nM to about 6nM, about 0.6nM to about 5nM, about 0.6nM to about 4nM, about 0.6nM to about 3 ...5nM, about 0.6nM to about 6nM, about 0.6nM to about 5nM, about 0.6nM to about 4nM, about 0.6nM to about 3nM, about 0.6nM to about 5nM, about .6nM to about 2nM, about 0.6nM to about 1nM, about 0.6nM to about 0.8nM, about 1nM to about 1 μM, about 1nM to about 800nM, about 1nM to about 750nM, about 1nM to about 700nM, about 1nM to about 650nM, about 1nM to about 600nM, about 1nM to about 550nM, about 1nM to about 500nM, about 1nM to about 450nM, about 1nM to about 400nM, about 1nM to about 350nM, about 1nM to about 300, about 1nM to about 250nM, about 1nM to about 200nM, about 1nM to about 150nM, about 1nM to about 100nM, about 1nM to about 80nM, about 1nM to about 60nM, about 1 nM to about 50 nM, about 1 nM to about 40 nM, about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 8 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2nM to about 1 μM, about 2nM to about 800nM, about 2nM to about 750nM, about 2nM to about 700nM, about 2nM to about 650nM, about 2nM to about 600nM, about 2nM to about 550nM, about 2nM to about 500nM, about 2nM to about 450nM, about 2nM to about 400nM, about 2nM to about 350nM, about 2nM to about 300, about 2nM to about 250nM, about 2nM to about 200nM, about 2nM to about 150nM, about 2nM to about 100nM, about 2nM to about 80nM, about 2nM to about 60nM, about 2nM to about 50nM, about 2nM to about 40nM, about 2nM to about 25nM, about 2nM to about about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 8 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 5 nM to about 1 μM, about 5 nM to about 800 nM, about 5 nM to about 750 nM, about 5 nM to about 700 nM,about 5 nM to about 650 nM, about 5 nM to about 600 nM, about 5 nM to about 550 nM, about 5 nM to about 500 nM, about 5 nM to about 450 nM, about 5 nM to about 400 nM, about 5 nM to about 350 nM, about 5 nM to about 300, about 5 nM to about 250 nM, about 5 nM to about 200 nM, about 5 nM to about 150nM, about 5nM to about 100nM, about 5nM to about 80nM, about 5nM to about 60nM, about 5nM to about 50nM, about 5nM to about 40nM, about 5nM to about 25nM, about 5nM to about 20nM, about 5nM to about 15nM, about 5nM to about 10nM, about 5nM to about 8nM, about 5nM to about 6nM M, about 10 nM to about 1 μM, about 10 nM to about 800 nM, about 10 nM to about 750 nM, about 10 nM to about 700 nM, about 10 nM to about 650 nM, about 10 nM to about 600 nM, about 10 nM to about 550 nM, about 10 nM to about 500 nM, about 10 nM to about 450 nM, about 10 nM to about 400nM, about 10nM to about 350nM, about 10nM to about 300, about 10nM to about 250nM, about 10nM to about 200nM, about 10nM to about 150nM, about 10nM to about 100nM, about 10nM to about 80nM, about 10nM to about 60nM, about 10nM to about 50nM, about 10nM to about 40nM, about 10nM to about 25nm, about 10nM to about 20nM, about 10nM to about 15nM, about 20nM to about 1μM, about 20nM to about 800nM, about 20nM to about 750nM, about 20nM to about 700nM, about 20nM to about 650nM, about 20nM to about 600nM, about 20nM to about 550nM, about 20nM to about 500nM, about 20nM to about 450nM, about 20nM to about 400nM, about 20nM to about 350nM, about 20nM to about 300, about 20nM to about 250nM, about 20nM to about 200nM, about 20nM to about 150nM, about 20nM to about 100nM, about 20 nM to about 80 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM, about 50 nM to about 1 μM, about 50 nM to about 800 nM, about 50 nM to about 750 nM, about 50 nM to about 700 nM, about 50 nM to about 650 nM, about 50 nM to about 600 nM, about 50 nM to about 550 nM, about 50 nM to about 500 nM, about 50 nM to about 450 nM, about 50 nM to about 400 nM, about 50 nM to about 350 nM, about 50 nM to about 300, about 50 nM to about 250 nM, about 50 nM to about 200 nM, about 50 nM to about 150 nM, about 50 nM to about 100 nM,Approximately 50nM to approximately 80nM, approximately 100nM to approximately 1μM, approximately 100nM to approximately 800nM, approximately 100nM to approximately 750nM, approximately 100nM to approximately 700nM, approximately 100nM to approximately 650nM, approximately 100nM to approximately 600 nM, about 100nM to about 550nM, about 100nM to about 500nM, about 100nM to about 450nM, about 100nM to about 400nM, about 100nM to about 350nM, about 100nM to about 300, about 100nM ~about 250nM, about 100nM to about 200nM, about 100nM to about 150nM, about 250nM to about 1μM, about 250nM to about 800nM, about 250nM to about 750nM, about 250nM to about 700nM, about 250nM to about 650nM, about 250nM to about 600nM, about 250nM to about 550nM, about 250nM to about 500nM, about 250nM to about 450nM, about 250nM to about 400nM, about 250nM to about 3 50nM, about 250nM to about 300, about 400nM to about 1μM, about 400nM to about 800nM, about 400nM to about 750nM, about 400nM to about 700nM, about 400nM to about 650nM, about 400nM ~about 600nM, about 400nM to about 550nM, about 400nM to about 500nM, about 400nM to about 450nM, about 500nM to about 1μM, about 500nM to about 800nM, about 500nM to about 750nM, about 5 a binding constant (K, of about 00 nM to about 700 nM, about 500 nM to about 650 nM, about 500 nM to about 600 nM, about 500 nM to about 550 nM, about 600 nM to about 1 μM, about 600 nM to about 800 nM, about 600 nM to about 750 nM, about 600 nM to about 700 nM, about 600 nM to about 650 nM, about 750 nM to about 1 μM, about 750 nM to about 800 nM, about 800 nM to about 1 μM, or about 950 nM to about 1 μM) D ) binds to tissue factor.

[0079] In some embodiments, the anti-tissue factor antibody can be IgG (e.g., IgG1, IgG2, IgG3, and IgG4) (e.g., human IgG1, human IgG2, human IgG3, and human IgG4), IgA (e.g., human IgA), IgE (e.g., human IgE), or IgM (e.g., human IgM).

[0080] In some embodiments, the anti-tissue factor antibody is a human or humanized IgG1 antibody. In some embodiments, the anti-tissue factor antibody is a human or humanized IgG4 antibody.

[0081] In some embodiments, the anti-tissue factor antibody may comprise an IgG1 Fc region. In some embodiments, the anti-tissue factor antibody may comprise a heavy chain constant domain comprising a sequence at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to SEQ ID NO: 102 or SEQ ID NO: 103. Heavy chain constant region (SEQ ID NO: 102) EFASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Heavy chain constant region (SEQ ID NO: 103) EFASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0082] Affinity Chromatography Resins Provided herein is an affinity chromatography resin comprising an anti-tissue factor antibody or antigen-binding fragment thereof bound to a base resin, the base resin comprising a heavy chain variable domain comprising the CDRs of SEQ ID NOs: 1, 2 or 9, and 3, and a light chain variable domain comprising the CDRs of SEQ ID NOs: 4, 5, and 6. In some embodiments, the heavy chain variable domain comprises a sequence at least 90% identical to SEQ ID NO: 7, and the light chain variable domain comprises a sequence at least 90% identical to SEQ ID NO: 8. In some embodiments, the heavy chain variable domain comprises SEQ ID NO: 7, and the light chain variable domain comprises SEQ ID NO: 8.

[0083] In some embodiments, the base resin is Sepharose. In some embodiments, the base resin is Capto or agarose. In some embodiments, the base resin (or matrix or stationary phase) is a natural polymer, including but not limited to, cellulose, agarose, or chitosan; a synthetic polymer, including but not limited to, acrylomide and vinyl copolymers, acrylic polymers or polymethacrylates, poly(styrene-divinyl-benzene) copolymers; or an inorganic material, including but not limited to, hydroxyapatite, silica, glass, or zirconia. In some embodiments, the anti-tissue factor antibody or antigen-binding fragment thereof is non-covalently bound to the base resin. In some embodiments, the anti-tissue factor antibody or antigen-binding fragment thereof is covalently bound to the base resin. In some embodiments, the anti-tissue factor antibody or antigen-binding fragment thereof is covalently bound to the base resin by disulfide bond formation between a cysteine ​​of the anti-tissue factor antibody or antigen-binding fragment thereof and a chemical group on the base resin. In some embodiments, the anti-tissue factor antibody or antigen-binding fragment thereof is covalently attached to the base resin by formation of a covalent bond between a free amine of the anti-tissue factor antibody or antigen-binding domain and a chemical group on the base resin. In some embodiments, the base resin is a CNBr-activated or N-hydroxysuccinimide (NHS)-activated solid support. In some embodiments, the covalent bond is represented by Formula I below: TIFF0007758680000003.tif11128In the formula, R represents an anti-tissue factor antibody or an antigen-binding fragment thereof.

[0084] In some embodiments, NHS-activated Sepharose is designed for covalent coupling of ligands containing primary amino groups (often antigens or antibodies), the most common form of attachment. In some embodiments, the matrix of NHS-activated Sepharose is based on highly cross-linked agarose beads with a 10-atom spacer arm (6-aminohexanoic acid) attached by epichlorohydrin and activated by N-hydroxysuccinimide (Figure 7). In some embodiments, the matrix of NHS-activated Sepharose 4 Fast Flow is based on highly cross-linked agarose beads with a 14-atom spacer arm. In some embodiments, nonspecific adsorption of proteins to NHS-activated Sepharose (which may reduce the binding capacity of target proteins) is negligible due to the excellent hydrophilicity of the base matrix. In some embodiments, the matrix is ​​stable at high pH to allow for rigorous washing procedures (following the pH stability of the coupled ligand). In some embodiments, amino-containing ligands, such as proteins, rapidly and spontaneously couple via nucleophilic attack at the ester bond, resulting in a highly stable amide bond (Figure 8). In some embodiments, the amide bond is stable up to pH 13.0, making NHS-activated Sepharose suitable for applications requiring high pH conditions.

[0085] In some embodiments, CNBr-activated Sepharose is a pre-activated resin without an intermediate spacer arm for coupling antibodies or other large proteins containing -NH groups to Sepharose media by the cyanogen bromide method. In some embodiments, CNBr-activated Sepharose is used to immobilize antibodies or other large proteins containing -NH groups by coupling them to the CNBr-activated Sepharose matrix. In some embodiments, CNBr-activated Sepharose 4 Fast Flow is a beaded, highly cross-linked, pre-activated matrix produced by reacting Sepharose 4 Fast Flow with cyanogen bromide (CNBr). In some embodiments, CNBr-activated Sepharose 4 Fast Flow is based on an antigen-antibody reaction with an immobilized monoclonal antibody as a ligand.

[0086] In some embodiments, the kit comprises any one of the affinity chromatography resins described herein.

[0087] Antibody (e.g., monoclonal or polyclonal)-based immunoaffinity chromatography is a widely used method / process for purifying specific antigens or antigen-containing proteins (e.g., human tissue factor-containing proteins) using affinity chromatography resins containing antibodies or antigen-binding fragments bound to the base resin. In immunoaffinity methods, antibodies (e.g., monoclonal) or antibody mixtures (e.g., polyclonal) are covalently immobilized (e.g., coupled) onto a solid support (e.g., base resin) such as CNBr-activated Sepharose, NHS-Sepharose, and other related beads / resins suitable for protein coupling or immobilization. In some embodiments, the base resin is any material to which a biospecific ligand can be covalently attached. In some embodiments, the base resin is any material to which a biospecific ligand can be noncovalently attached. In some embodiments, examples of base resins used in affinity chromatography include agarose, Capto, cellulose, ceramic, dextran, polystyrene, polyacrylamide, silica, latex, controlled pore glass, and synthetic / organic polymers. Useful base resins can have properties such as a high surface area to volume ratio, chemical groups that are easily modified for covalent attachment of ligands, minimal nonspecific binding properties, good flow properties, and mechanical and chemical stability.

[0088] In some embodiments, the base resin is a sulfhydryl-coupled resin, and the antibody or antigen-binding fragment thereof is covalently attached to the base resin via free sulfhydryl and iodoacetyl groups on the base resin. In some embodiments, the base resin is an amine-coupled resin, which has reactive aldehyde groups that react with primary amines to form Schiff bases and are subsequently reduced with a suitable reducing agent (e.g., sodium cyanoborohydride) to form covalent bonds between the antibody or antigen-binding fragment and the base resin. In some embodiments, the base resin is an NHS-activated resin, and the antibody or antigen-binding fragment is attached to the resin via the reactive NHS group and primary amine that form covalent bonds. In some embodiments, the base resin is immobilized Protein A, Protein B, or Protein A / G, and the base resin binds to the constant domain of the antibody or antigen-binding fragment, ensuring that the antigen-binding domain faces outward from the base resin for optimal binding.

[0089] In some embodiments, the chromatography column is used for affinity chromatography, and the chromatography column comprises an affinity chromatography resin selected from the group consisting of CNBr-activated Sepharose, NHS-Sepharose, agarose, Capto, cellulose, ceramic, dextran, polystyrene, polyacrylamide, silica, latex, controlled pore glass, and synthetic / organic polymers.

[0090] Method for purifying tissue factor-containing proteins Provided herein are methods for purifying tissue factor-containing proteins, comprising the use of any of the affinity chromatography resins described herein. In some embodiments, the method comprises loading a liquid containing tissue factor-containing proteins onto the affinity chromatography resin, washing the affinity chromatography resin with one or more wash buffers, and eluting the tissue factor-containing proteins with an elution buffer. In some embodiments, the liquid containing tissue factor-containing proteins is a clarified liquid culture medium. In some embodiments, the liquid containing tissue factor-containing proteins comprises a cell lysate.

[0091] In some embodiments, the one or more wash buffers are (i) a first wash buffer comprising phosphate-buffered saline, and (ii) a second wash buffer comprising about 0.01 M to about 0.2 M citrate and having a pH of about 4.5 to about 5.5. In some embodiments, the first wash buffer is phosphate-buffered saline, and the second wash buffer is 0.1 M citrate, pH 5.0. In some embodiments, the elution buffer comprises 0.01 M to about 0.2 M acetate and has a pH of about 2.5 to about 3.5. In some embodiments, the elution buffer comprises 0.1 M acetate and has a pH of about 2.9.

[0092] In some embodiments, the second wash buffer has a concentration of about 0.01 M to about 0.2 M (e.g., about 0.01 M to about 0.18 M, about 0.01 M to about 0.16 M, about 0.01 M to about 0.14 M, about 0.01 M to about 0.12 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.08 M, about 0.01 M to about 0.06 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.04 M, about 0.01 M to about 0.03 M, about 0.01 M to about 0.02 M, about 0.02 M to about 0.2 M, about 0.02 M to about 0.18 M, about 0.02 M to about 0.16 M, about 0.02 M to about 0.14 M, about 0.02M~Approx. 0.12M, Approx. 0.02M~Approx. 0.1M, Approx. 0.02M~Approx. 0.08M, Approx. 0.02M~Approx. 0.06M, Approx. 0.02M~Approx. 0.05M, about 0.02M to about 0.04M, about 0.02M to about 0.03M, about 0.03M to about 0.2M, about 0.03M to about 0.18M, ​​about 0.03M~Approx. 0.16M, Approx. 0.03M~Approx. 0.14M, Approx. 0.03M~Approx. 0.12M, Approx. 0.03M~Approx. 0.1M, Approx. 0.03M~Approx. 0.08M, about 0.03M to about 0.06M, about 0.03M to about 0.05M, about 0.03M to about 0.04M, about 0.04M to about 0.2M, about 0 0.04M to approximately 0.18M, ​​approximately 0.04M to approximately 0.16M, approximately 0.04M to approximately 0.14M, approximately 0.04M to approximately 0.12M, approximately 0.04M to approximately 0.1M, approximately 0.04M to approximately 0.08M, approximately 0.04M to approximately 0.06M, approximately 0.04M to approximately 0.05M, approximately 0.05M to approximately 0.2M, approximately 0 .05M~approx. 0.18M, ​​approx. 0.05M~approx. 0.16M, approx. 0.05M~approx. 0.14M, approx. 0.05M~approx. 0.12M, approx. 0.05M~approx. 0.1M, about 0.05M to about 0.08M, about 0.05M to about 0.06M, about 0.06M to about 0.2M, about 0.06M to about 0.18M, ​​about 0. 06M~about 0.16M, about 0.06M~about 0.14M, about 0.06M~about 0.12M, about 0.06M~about 0.1M, about 0.06M~about 0. 08M, about 0.08M to about 0.2M, about 0.08M to about 0.18M, ​​about 0.08M to about 0.16M, about 0.08M to about 0.14M, about 0. 08M~about 0.12M, about 0.08M~about 0.1M, about 0.1M~about 0.2M, about 0.1M~about 0.18M, ​​about 0.1M~about 0.16M, Approx. 0.1M~Approx. 0.14M, Approx. 0.1M~Approx. 0.12M, Approx. 0.12M~Approx. 0.2M, Approx. 0.12M~Approx. 0.18M, ​​Approx. 0.12M~Approx. 0.The solution contains citric acid at a concentration of about 16M, about 0.12M to about 0.14M, about 0.14M to about 0.2M, about 0.14M to about 0.18M, ​​about 0.14M to about 0.16M, about 0.16M to about 0.2M, about 0.16M to about 0.18M, ​​or about 0.18M to about 0.2M.

[0093] In some embodiments, the second wash buffer has a pH of about 4.5 to about 5.5 (e.g., about 4.5 to about 5.4, about 4.5 to about 5.3, about 4.5 to about 5.2, about 4.5 to about 5.1, about 4.5 to about 5.0, about 4.5 to about 4.9, about 4.5 to about 4.8, about 4.5 to about 4.7, about 4.5 to about 4.6, about 4.6 to about 5.5, about 4.6 to about 5.4, About 4.6 to about 5.3, about 4.6 to about 5.2, about 4.6 to about 5.1, about 4.6 to about 5.0, about 4.6 to about 4.9, about 4.6 to about 4.8, about 4.6 to about 4.7, about 4.7 to about 5.5, about 4.7 to about 5.4, about 4.7 to about 5.3, about 4.7 to about 5.2, about 4.7 to about 5.1, about 4.7 to about 5.0, about 4.7 to about 4.9, about 4.7 to about 4 .8, about 4.8 to about 5.5, about 4.8 to about 5.4, about 4.8 to about 5.3, about 4.8 to about 5.2, about 4.8 to about 5.1, about 4.8 to about 5.0, about 4.8 to about 4.9, about 4.9 to about 5.5, about 4.9 to about 5.4, about 4.9 to about 5.3, about 4.9 to about 5.2, about 4.9 to about 5.1, about 4.9 to about 5.0, about 5.0 to about 5.5, about 5.0 about 5.4, about 5.0 to about 5.3, about 5.0 to about 5.2, about 5.0 to about 5.1, about 5.1 to about 5.5, about 5.1 to about 5.4, about 5.1 to about 5.3, about 5.1 to about 5.2, about 5.2 to about 5.5, about 5.2 to about 5.4, about 5.2 to about 5.3, about 5.3 to about 5.5, about 5.3 to about 5.4, or about 5.4 to about 5.5).

[0094] In some embodiments, the elution buffer contains about 0.01 M to about 0.2 M (e.g., about 0.01 M to about 0.18 M, about 0.01 M to about 0.16 M, about 0.01 M to about 0.14 M, about 0.01 M to about 0.12 M, about 0.01 M to about 0.1 M, about 0.01 M to about 0.08 M, about 0.01 M to about 0.06 M, about 0.01 M to about 0.05 M, about 0.01 M to about 0.04 M, about 0.01 M to about 0.03 M, about 0.01 M to about 0.02 M, about 0.02 M to about 0.2 M, about 0.02 M to about 0.18 M, about 0.02 M to about 0.16 M, about 0.02 M to about 0.14 ... M ~ about 0.12M, about 0.02M - about 0.1M, about 0.02M - about 0.08M, about 0.02M - about 0.06M, about 0.02M - about 0.05 M, about 0.02M to about 0.04M, about 0.02M to about 0.03M, about 0.03M to about 0.2M, about 0.03M to about 0.18M, ​​about 0.03M ~0.16M, approx. 0.03M~0.14M, approx. 0.03M~0.12M, approx. 0.03M~0.1M, approx. 0.03M~0.08M , about 0.03M~about 0.06M, about 0.03M~about 0.05M, about 0.03M~about 0.04M, about 0.04M~about 0.2M, about 0.04M~ Approximately 0.18M, ​​approximately 0.04M to approximately 0.16M, approximately 0.04M to approximately 0.14M, approximately 0.04M to approximately 0.12M, approximately 0.04M to approximately 0.1M, approximately 0.04M to approximately 0.08M, approximately 0.04M to approximately 0.06M, approximately 0.04M to approximately 0.05M, approximately 0.05M to approximately 0.2M, approximately 0.05M to approximately 0.18M, ​​about 0.05M to about 0.16M, about 0.05M to about 0.14M, about 0.05M to about 0.12M, about 0.05M to about 0.1M, about 0.05M~about 0.08M, about 0.05M~about 0.06M, about 0.06M~about 0.2M, about 0.06M~about 0.18M, ​​about 0.06M~about 0 .16M, about 0.06M to about 0.14M, about 0.06M to about 0.12M, about 0.06M to about 0.1M, about 0.06M to about 0.08M, about 0 .08M~approx. 0.2M, approx. 0.08M~approx. 0.18M, ​​approx. 0.08M~approx. 0.16M, approx. 0.08M~approx. 0.14M, approx. 0.08M~approx. 12M, about 0.08M to about 0.1M, about 0.1M to about 0.2M, about 0.1M to about 0.18M, ​​about 0.1M to about 0.16M, about 0.1M to about 0.14M, about 0.1M to about 0.12M, about 0.12M to about 0.2M, about 0.12M to about 0.18M, ​​about 0.12M to about 0.16M, about 0.The solution contains acetic acid at a concentration of 12M to about 0.14M, about 0.14M to about 0.2M, about 0.14M to about 0.18M, ​​about 0.14M to about 0.16M, about 0.16M to about 0.2M, about 0.16M to about 0.18M, ​​or about 0.18M to about 0.2M.

[0095] In some embodiments, the elution buffer has a pH of about 2.5 to about 3.5 (e.g., about 2.5 to about 3.4, about 2.5 to about 3.3, about 2.5 to about 3.2, about 2.5 to about 3.1, about 2.5 to about 3.0, about 2.5 to about 2.9, about 2.5 to about 2.8, about 2.5 to about 2.7, about 2.5 to about 2.6, about 2.6 to about 3.5, about 2.6 to about 3.4, about 2. 6 to about 3.3, about 2.6 to about 3.2, about 2.6 to about 3.1, about 2.6 to about 3.0, about 2.6 to about 2.9, about 2.6 to about 2.8, about 2.6 to about 2.7, about 2.7 to about 3.5, about 2.7 to about 3.4, about 2.7 to about 3.3, about 2.7 to about 3.2, about 2.7 to about 3.1, about 2.7 to about 3.0, about 2.7 to about 2.9, about 2.7 to about 2.8 , about 2.8 to about 3.5, about 2.8 to about 3.4, about 2.8 to about 3.3, about 2.8 to about 3.2, about 2.8 to about 3.1, about 2.8 to about 3.0, about 2.8 to about 2.9, about 2.9 to about 3.5, about 2.9 to about 3.4, about 2.9 to about 3.3, about 2.9 to about 3.2, about 2.9 to about 3.1, about 2.9 to about 3.0, about 3.0 to about 3.5, about 3.0 to The pH is about 3.4, about 3.0 to about 3.3, about 3.0 to about 3.2, about 3.0 to about 3.1, about 3.1 to about 3.5, about 3.1 to about 3.4, about 3.1 to about 3.3, about 3.1 to about 3.2, about 3.2 to about 3.5, about 3.2 to about 3.4, about 3.2 to about 3.3, about 3.3 to about 3.5, about 3.3 to about 3.4, or about 3.4 to about 3.5.

[0096] Method for producing tissue factor-containing proteins Provided herein are methods for producing tissue factor-containing proteins, comprising: (i) purifying the tissue factor-containing protein using any of the methods described herein; and (ii) performing one or more additional unit operations on the eluate obtained from step (i). Non-limiting examples of unit operations that can be performed in a process for producing tissue factor-containing proteins include protein capture, protein purification, protein polishing, viral inactivation, adjusting the ionic concentration and / or pH of a fluid containing the protein, and filtering a liquid containing the protein. A non-limiting method for producing tissue factor-containing proteins is shown in Figure 1.

[0097] In some embodiments, the one or more additional unit operations comprise, in sequential order, performing low pH viral inactivation, performing depth filtration, performing polishing chromatography, performing nanofiltration, and performing ultrafiltration and diafiltration (UF / DF).

[0098] Also provided herein is a tissue factor-containing protein produced by any of the methods described herein. In some embodiments, a pharmaceutical composition comprises the produced tissue factor-containing protein.

[0099] Single-chain chimeric polypeptides A non-limiting example of a tissue factor-containing protein is a single-chain chimeric polypeptide comprising: (i) a first target binding domain (e.g., any of the target binding domains described herein or known in the art), (ii) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art), and (iii) a second target binding domain (e.g., any of the target binding domains described herein or known in the art).

[0100] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are immediately adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments of any of the single-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are immediately adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0101] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are immediately adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art). In some embodiments of any of the single-chain chimeric polypeptides described herein, the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are immediately adjacent to each other. In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art).

[0102] Some embodiments of any of the single-chain chimeric polypeptides described herein may further comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at their N-terminus and / or C-terminus.

[0103] In some embodiments, a single-chain chimeric polypeptide can comprise one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at its N-terminus. In some embodiments, one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide can be immediately adjacent to a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein).

[0104] In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at its C-terminus. In some embodiments, one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the at least one additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus of the single-chain chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein).

[0105] In some embodiments of any of the single-chain chimeric polypeptides described herein, the single-chain chimeric polypeptide comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at its N-terminus and its C-terminus. In some embodiments, one of the one or more additional antigen binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus of the single-chain chimeric polypeptide is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) at the N-terminus and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains).In some embodiments, one of the one or more additional antigen-binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains). In some embodiments, the single-chain chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between one of the one or more additional antigen-binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the C-terminus and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), or the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein).

[0106] In some embodiments of any of the single-chain chimeric polypeptides described herein, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) specifically bind to the same antigen. In some embodiments, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) specifically bind to the same epitope. In some embodiments, two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) comprise the same amino acid sequence.

[0107] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) each specifically bind to the same antigen. In some embodiments, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and the one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains each comprise the same amino acid sequence.

[0108] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) specifically bind to different antigens.

[0109] In some embodiments of any of the single-chain chimeric polypeptides, one or more of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen-binding domains (e.g., any of the exemplary target binding domains described herein or known in the art). In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and one or more additional target binding domains are each antigen-binding domains (e.g., any of the exemplary target binding domains described herein or known in the art). In some embodiments, the antigen-binding domain may comprise an scFv or a single-domain antibody.

[0110] In some embodiments of any of the single-chain chimeric polypeptides described herein, one of a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) include CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGI T, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HE R2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCII ligand, scTCR ligand, I The antibody specifically binds to a target selected from the group consisting of IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

[0111] In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. Non-limiting examples of soluble interleukin and soluble cytokine proteins include IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

[0112] In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine receptors. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKP30, soluble NKp44, soluble NKp46, soluble DNAM-1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.

[0113] In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the target binding domains described herein), the second target binding domain (e.g., any of the target binding domains described herein), and the one or more target binding domains (e.g., any of the target binding domains described herein) are each independently selected from the group consisting of CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, The antibody can specifically bind to a target selected from the group consisting of CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor.

[0114] In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is selected from the group of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

[0115] In some embodiments of any of the single-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine receptors. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble receptor is soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble TNFα receptor, soluble IL-4 receptor, or soluble IL-10 receptor.

[0116] In some embodiments, the single-chain chimeric polypeptide has a nucleic acid or amino acid sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to SEQ ID NOs: 22-29.

[0117] Soluble tissue factor domain In some embodiments of any of the polypeptides, compositions, or methods described herein, the soluble tissue factor domain can be a wild-type tissue factor polypeptide lacking the signal sequence, transmembrane domain, and intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor mutant, where the wild-type tissue factor polypeptide lacks the signal sequence, transmembrane domain, and intracellular domain and is further modified with selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, soluble mouse tissue factor domains, soluble rat tissue factor domains, and mutant soluble tissue factor domains are provided below. Exemplary soluble human tissue factor domain (SEQ ID NO: 10) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary nucleic acids encoding soluble human tissue factor domains (SEQ ID NO: 11) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGAC AGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG Exemplary soluble mouse tissue factor domain (SEQ ID NO: 12) agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnap kflpyrdtnlgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge Exemplary soluble rat tissue factor domain (SEQ ID NO: 13) agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 14) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 15) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0118] In some embodiments, the soluble tissue factor domain can comprise a sequence that is at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO: 10, 12, 13, 14, or 15. In some embodiments, the soluble tissue factor domain can comprise the sequence of SEQ ID NO: 10, 12, 13, 14, or 15 with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its N-terminus and / or 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its C-terminus.

[0119] As can be understood in the art, one of skill in the art will understand that mutations in amino acids that are conserved among different mammalian species are likely to reduce the activity and / or structural stability of a protein, while mutations in amino acids that are not conserved among different mammalian species are unlikely to reduce the activity and / or structural stability of a protein.

[0120] In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain cannot bind to factor VIIa. In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

[0121] In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain.

[0122] In some examples, the soluble tissue factor domain does not include one or more (e.g., 2, 3, 4, 5, 6, or 7) of the following: a lysine at the amino acid position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein, an isoleucine at the amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein, a tryptophan at the amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, an aspartic acid at the amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, a tyrosine at the amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, an arginine at the amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and a phenylalanine at the amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the mutant soluble tissue factor has the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0123] In some examples, the soluble tissue factor domain can be encoded by a nucleic acid comprising a sequence at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to SEQ ID NO:11.

[0124] In some embodiments, the soluble tissue factor domain is from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 160 amino acids, from about 20 amino acids to about 155 amino acids, from about 20 amino acids to about 150 amino acids, from about 20 amino acids to about 145 amino acids, from about 20 amino acids to about 140 amino acids, The total length may be about 20 to about 135 amino acids, about 20 to about 130 amino acids, about 20 to about 125 amino acids, about 20 to about 120 amino acids, about 20 to about 115 amino acids, about 20 to about 110 amino acids, about 20 to about 105 amino acids, about 20 to about 100 amino acids, about 20 to about 95 amino acids, about 20 to about 90 amino acids, about 20 to about 85 amino acids, about 20 to about 80 amino acids, about 20 to about 75 amino acids, about 20 to about 70 amino acids, about 20 to about 60 amino acids, about 20 to about 50 amino acids, about 20 to about 40 amino acids, or about 20 to about 30 amino acids.

[0125] Linker sequence In some embodiments, the linker sequence may be a flexible linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et al., Protein Engineering, Design & Selection 27(10):325-330, 2014, and Priyanka et al., Protein Sci. 22(2):153-167, 2013. In some examples, the linker sequence is a synthetic linker sequence.

[0126] Some embodiments of any of the single-chain chimeric polypeptides described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). Some embodiments of any of the single-chain chimeric polypeptides described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

[0127] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

[0128] In some embodiments, the linker sequence may have a total length of 1 to about 100 amino acids, 1 to about 90 amino acids, 1 to about 80 amino acids, 1 to about 70 amino acids, 1 to about 60 amino acids, 1 to about 50 amino acids, 1 to about 45 amino acids, 1 to about 40 amino acids, 1 to about 35 amino acids, 1 to about 30 amino acids, 1 to about 25 amino acids, 1 to about 24 amino acids, 1 to about 22 amino acids, 1 to about 20 amino acids, 1 to about 18 amino acids, 1 to about 16 amino acids, 1 to about 14 amino acids, 1 to about 12 amino acids, 1 to about 10 amino acids, 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids.

[0129] In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) (SEQ ID NO: 16) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGGS (SEQ ID NO: 16) sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) (SEQ ID NO: 17) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGGGS (SEQ ID NO: 17) sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) (SEQ ID NO: 18) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGSG (SEQ ID NO: 18) sequences.

[0130] In some embodiments, the linker sequence may comprise or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 19). In some embodiments, the linker sequence may be encoded by a nucleic acid comprising or consisting of GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 20). In some embodiments, the linker sequence may comprise or consist of GGGSGGGS (SEQ ID NO: 21).

[0131] Target Binding Domain In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the additional target binding domain or domains can be an antigen binding domain (e.g., any of the exemplary antigen binding domains described herein or known in the art), a soluble interleukin or cytokine protein (e.g., any of the exemplary soluble interleukin or soluble cytokine proteins described herein), and a soluble interleukin or cytokine receptor (e.g., any of the exemplary soluble interleukin or soluble cytokine receptors described herein).

[0132] In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the one or more additional target binding domains are each independently from about 5 amino acids to about 1000 amino acids, from about 5 amino acids to about 950 amino acids, from about 5 amino acids to about 900 amino acids, from about 5 amino acids to about 850 amino acids, from about 5 amino acids to about 800 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 650 amino acids, from about 5 amino acids to about 6 ...700 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 650 amino acids, from about 5 amino acids to about 600 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 650 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 about 5 to about 550 amino acids, about 5 to about 500 amino acids, about 5 to about 450 amino acids, about 5 to about 400 amino acids, about 5 to about 350 amino acids, about 5 to about 300 amino acids, about 5 to about 280 amino acids, about 5 to about 260 amino acids, about 5 to about 240 amino acids, about 5 to about 220 amino acids, about 5 to about 200 amino acids, about 5 to about 195 amino acids, about 5 to about 190 amino acids, about 5 to about 185 amino acids, about 5 to about 180 amino acids, about 5 to about 175 amino acids amino acids, about 5 to about 170 amino acids, about 5 to about 165 amino acids, about 5 to about 160 amino acids, about 5 to about 155 amino acids, about 5 to about 150 amino acids, about 5 to about 145 amino acids, about 5 to about 140 amino acids, about 5 to about 135 amino acids, about 5 to about 130 amino acids, about 5 to about 125 amino acids, about 5 to about 120 amino acids, about 5 to about 115 amino acids, about 5 to about 110 amino acids, about 5 to about 105 amino acids, about 5 to about 100 amino acids, about 5 amino acids about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids,It may have a total amino acid number of about 5 to about 10 amino acids.

[0133] Any of the target binding domains described herein may be used in combination with 1×10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M or 1 x 10 -13 The dissociation equilibrium constant (K D In some embodiments, the antigen binding protein constructs provided herein can bind to their targets at about 1 x 10 -3 M ~ approx. 1×10 -5 M, about 1 x 10 -4 M ~ approx. 1×10 -6 M, about 1 x 10 -5 M ~ approx. 1×10 -7 M, about 1 x 10 -6 M ~ approx. 1×10 -8 M, about 1 x 10 -7 M ~ approx. 1×10 -9 M, about 1 x 10 -8 M ~ approx. 1×10 -10 M, or approximately 1 x 10 -9 M ~ approx. 1×10 -11 K of M (including their upper and lower bounds) D It can bind to a specific antigen.

[0134] Any of the target-binding domains described in this specification can bind to its target with a K of about 1 pM to about 30 nM (e.g., about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about 650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM, about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM). D and can bind to its target.

[0135] Any of the target binding domains described herein may be administered at a concentration of about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about ~about 6nM, about 3nM to about 5nM, about 3nM to about 4nM, about 4nM to about 10nM, about 4nM to about 9nM, about 4nM to about 8nM, about 4nM ~about 7nM, about 4nM to about 6nM, about 4nM to about 5nM, about 5nM to about 10nM, about 5nM to about 9nM, about 5nM to about 8nM, about 5nM Approximately 7nM, approximately 5nM to approximately 6nM, approximately 6nM to approximately 10nM, approximately 6nM to approximately 9nM, approximately 6nM to approximately 8nM, approximately 6nM to approximately 7nM, approximately 7nM to approximately K of 10nM, about 7nM to about 9nM, about 7nM to about 8nM, about 8nM to about 10nM, about 8nM to about 9nM, and about 9nM to about 10nM) D can bind to its target.

[0136] A variety of different methods known in the art can be used to quantify any of the antigen binding protein constructs described herein. D Values ​​can also be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecular binding kinetic assays, etc.).

[0137] antigen-binding domain In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of these single-chain or multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these single-chain or multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0138] In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to different antigens.

[0139] In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are antigen-binding domains. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each antigen-binding domains. In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is an scFv or single-domain antibody (e.g., a VHH or VNAR domain).

[0140] In some examples, the antigen binding domain (e.g., any of the antigen binding domains described herein) is selected from the group consisting of CD16a (see, e.g., as described in U.S. Pat. No. 9,035,026), CD28 (see, e.g., as described in U.S. Pat. No. 7,723,482), CD3 (see, e.g., as described in U.S. Pat. No. 9,226,962), CD33 (see, e.g., as described in U.S. Pat. No. 8,759,494), CD20 (see, e.g., as described in WO2014 / 026054), CD19 (see, e.g., as described in U.S. Pat. No. 9,701,758), CD22 (see, e.g., as described in WO2003 / 104425), and the like. No. 2012 / 0171197), IL-10 (see, e.g., U.S. Patent Application Publication No. 2016 / 0340413), PDL-1 (see, e.g., Drees, et al., Protein Express. Purif. 94:60-66, 2014), TIGIT (see, e.g., as described in U.S. Patent Application Publication No. 2017 / 0198042), PD-1 (see, e.g., as described in U.S. Patent No. 7,488,802), TIM3 (see, e.g., as described in U.S. Patent No. 8,552,156), CTLA4 (see, e.g., as described in WO2012 / 120125), MICA (see, e.g., as described in WO2016 / 154585), MICB (see, e.g., as described in U.S. Patent No. 8,753,640), IL-6 (see, e.g., as described in Gejima et al., Human Antibodies 11(4):121-129,2002), IL-8 (see, e.g., U.S. Pat. No. 6,117,980), TNFα (see, e.g., U.S. Pat. No. 6,117,980), CD26 (see, e.g., WO2017 / 189526), ​​CD36 (see, e.g., U.S. Pat. App. Pub. No. 2015 / 0259429), ULBP2 (see, e.g., U.S. Pat. No. 9,273,136), CD30 (see, e.g., Homach et al., al., Scand. J. Immunol. 48(5):497-501, 1998), CD200 (see, e.g., U.S. Pat. No. 9,085,623), IGF-1R (see, e.g., U.S. Patent Application Publication No. 2017 / 0051063), MUC4AC (see, e.g., WO2012 / 170470), MUC5AC (see, e.g., U.S. Pat. No. 9,238,084), Trop-2 (see, e.g., WO2013 / 068946), CMET (see, e.g., Edwardraja et al., Biotechnol. Bioeng. 106(3):367-375, 2010), EGFR (see, e.g., Akbari et al., Protein Expr. Purif. 127:8-15, 2016), HER1 (see, e.g., as described in U.S. Patent Application Publication No. 2013 / 0274446), HER2 (see, e.g., as described in Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3 (see, e.g., as described in U.S. Patent No. 9,505,843), PSMA (see, e.g., as described in Parker et al., Protein Expr. Purif.89(2):136-145,2013), CEA (see, e.g., WO1995 / 015341), B7H3 (see, e.g., U.S. Pat. No. 9,371,395), EPCAM (see, e.g., WO2014 / 159531), BCMA (see, e.g., Smith et al., Mol. Ther. 26(6):1447-1456,2018), P-cadherin (see, e.g., U.S. Pat. No. 7,452,537), CEACAM5 (see, e.g., U.S. Pat. No. 9,617,345), UL16 binding protein (see, e.g., WO2017 / 083612), HLA-DR (see, e.g., Pistillo et al., Mol. Ther. 26(6):1447-1456,2018). al., Exp. Clin. Immunogenet. 14(2):123-130, 1997), DLL4 (see, e.g., those described in WO2014 / 007513), TYRO3 (see, e.g., those described in WO2016 / 166348), AXL (see, e.g., those described in WO2012 / 175692), MER (see, e.g., those described in WO2016 / 106221), CD122 (see, e.g., those described in U.S. Patent Application Publication No. 2016 / 0367664), CD155 (see, e.g., those described in WO2017 / 149538), or PDGF-DD (see, e.g., those described in U.S. Patent No. 9,441,034).

[0141] Each antigen-binding domain present in any of the single-chain or multi-chain chimeric polypeptides described herein is independently selected from the group consisting of a VHH domain, a VNAR domain, and an scFv. In some embodiments, any of the antigen-binding domains described herein is a BiTe, (scFv)2, a nanobody, a nanobody-HSA, a DART, a TandAb, a scDiabody, a scDiabody-CH3, a scFv-CH-CL-scFv, an HSAbody, a scDiabody-HAS, or a tandem-scFv. Additional examples of antigen-binding domains that can be used in either the single-chain or multi-chain chimeric polypeptides are known in the art.

[0142] VHH domains are single monomeric variable antibody domains that can be found in camelids. VNAR domains are single monomeric variable antibody domains that can be found in cartilaginous fish. H H domain and V NARNon-limiting examples of domains are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016, De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006, De Meyer et al., Trends Biotechnol. 32:263-270, 2014, Kijanka et al., Nanomedicine 10:161-174, 2015, Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014, Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016, Mujic-Delic et al., Trends Pharmacol.Sci.35:247-255,2014, Muyldermans,J.Biotechnol.74:277-302,2001, Muyldermans et al.,Trends Biochem.Sci.26:230-235,2001, Muyldermans,Ann.Rev.Biochem.82:775-797,2013, Rahbarizadeh et al.,Immunol.Invest.40:299-338,2011, Van Audenhove et al.,EBioMedicine 8:40-48,2016, Van Bockstaele et al.,Curr.Opin.Investig.Drugs 10:1212-1224,2009, Vincke et al.,Methods Mol. Biol. 911:15-26, 2012, and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0143] In some embodiments, each of the antigen-binding domains in a single-chain or multi-chain chimeric polypeptide described herein are both VHH domains, or at least one antigen-binding domain is a VHH domain. In some embodiments, each of the antigen-binding domains in a single-chain or multi-chain chimeric polypeptide described herein are both VNAR domains, or at least one antigen-binding domain is a VNAR domain. In some embodiments, each of the antigen-binding domains in a single-chain or multi-chain chimeric polypeptide described herein are both scFv domains, or at least one antigen-binding domain is an scFv domain.

[0144] In some embodiments, two or more polypeptides present in a single-chain or multi-chain chimeric polypeptide are assembled (e.g., non-covalently assembled) to form any of the antigen-binding domains described herein, e.g., an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), VHH-scAb, VHH-Fab, double-scFab, F(ab'), diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-hole common light chain, knobs-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, diabody-CH3, triplebody, minibody, minibody, TriBi minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock-and-lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv1-PEG-scFv2 can be formed. For a description of these components, see, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, incorporated herein in its entirety. Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments.Additional examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4), antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA, e.g., human or humanized IgA1 or IgA2), antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD), antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE), or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0145] An "Fv" fragment comprises a non-covalent dimer of one heavy-chain and one light-chain variable domain.

[0146] A "Fab" fragment contains the heavy and light chain variable domains of an Fv fragment as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ) is included.

[0147] An "F(ab')2" fragment contains two Fab fragments linked by a disulfide bond near the hinge region.

[0148] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to a multivalent and multispecific binding protein as described, for example, in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Pat. Nos. 7,612,181, 8,258,268, 8,586,714, 8,716,450, 8,722,855, 8,735,546, and 8,822,645, each of which is incorporated by reference in its entirety.

[0149] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0150] In some embodiments of any of the antigen-binding domains described herein, they are capable of binding to an antigen selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

[0151] Additional examples and embodiments of antigen binding domains are known in the art.

[0152] Soluble interleukin or cytokine proteins In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain can be a soluble interleukin protein or a soluble cytokine protein. In some embodiments, the soluble interleukin or soluble cytokine protein is selected from the group of IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L are provided below. Human soluble IL-2 (SEQ ID NO: 104) TIFF0007758680000004.tif25170 Human soluble IL-3 (SEQ ID NO: 105) TIFF0007758680000005.tif27170 Human soluble IL-7 (SEQ ID NO: 106) TIFF0007758680000006.tif25170 Human soluble IL-8 (SEQ ID NO: 107) TIFF0007758680000007.tif17170 Human soluble IL-10 (SEQ ID NO: 108) TIFF0007758680000008.tif34170 Human soluble IL-15 (SEQ ID NO: 109) TIFF0007758680000009.tif25170 Human soluble IL-17 (SEQ ID NO: 110) TIFF0007758680000010.tif26170 Human soluble IL-18 (SEQ ID NO: 111) TIFF0007758680000011.tif26170 Human soluble PDGF-DD (SEQ ID NO: 112) TIFF0007758680000012.tif59170 Human soluble SCF (SEQ ID NO: 113) TIFF0007758680000013.tif40170 Human soluble FLT3L (SEQ ID NO: 114) TIFF0007758680000014.tif40170

[0153] Non-limiting examples of soluble MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 are provided below. Human soluble MICA (SEQ ID NO: 115) TIFF0007758680000015.tif70170 Human soluble MICB (SEQ ID NO: 116) TIFF0007758680000016.tif70170 Human soluble ULBP1 (SEQ ID NO: 117) TIFF0007758680000017.tif32170 Human soluble ULBP2 (SEQ ID NO: 118) TIFF0007758680000018.tif31170 Human soluble ULBP3 (SEQ ID NO: 119) TIFF0007758680000019.tif34170 Human soluble ULBP4 (SEQ ID NO: 120) TIFF0007758680000020.tif43170 Human soluble ULBP5 (SEQ ID NO: 121) TIFF0007758680000021.tif34170 Human soluble ULBP6 (SEQ ID NO: 122) TIFF0007758680000022.tif35170

[0154] Additional examples of soluble interleukin and cytokine proteins are known in the art.

[0155] Soluble receptors In some embodiments of any of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin receptor or a soluble cytokine receptor. In some embodiments, the soluble receptor is soluble TGF-β receptor II (TGF-β RII) (see, e.g., as described in Yung et al., Am. J. Resp. Crit. Care Med. 194(9):1140-1151, 2016), soluble TGF-βRIII (see, e.g., as described in Heng et al., Placenta 57:320, 2017), soluble NKG2D (see, e.g., Cosman et al., Immunity 14(2):123-133, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp30 (see, e.g., as described in Costa et al., al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp44 (see, e.g., Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp46 (see, e.g., Mandelboim et al., Nature 409:1055-1060, 2001; Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble DNAM1 (see, for example, those described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), scMHCI (see, for example, Washburn et al., PLoS One 6(3):e18439, 2011), scMHCII (see, e.g., Bishwajit et al., Cellular Immunol. 170(1):25-33, 1996), scTCR (see, e.g., Weber et al., Nature 356(6372):793-796, 1992), soluble CD155 (see, e.g., Tahara-Hanaoka et al., Int. Immunol. 16(4):533-538, 2004), or soluble CD28 (see, e.g., Hebbar et al., Clin. Exp. Immunol. 136:388-392, 2004). Additional examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0156] Exemplary Embodiments of Single-Chain Chimeric Polypeptides—Type A In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and / or the second target binding domain can independently specifically bind to CD3 (e.g., human CD3) or CD28 (e.g., human CD28). In some embodiments, the first target binding domain specifically binds to CD3 (e.g., human CD3) and the second target binding domain specifically binds to CD28 (e.g., human CD28). In some embodiments, the first target binding domain specifically binds to CD28 (e.g., human CD28) and the second target binding domain specifically binds to CD3 (e.g., human CD3).

[0157] Exemplary Embodiments of Single-Chain Chimeric Polypeptides—Type B In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and / or the second target binding domain can independently specifically bind to an IL-2 receptor (e.g., a human IL-2 receptor).

[0158] Exemplary Embodiments of Single-Chain Chimeric Polypeptides—Type C In some embodiments of any of the single-chain chimeric polypeptides described herein, the first target binding domain and / or the second target binding domain can independently specifically bind to an IL-15 receptor (e.g., a human IL-15 receptor).

[0159] Exemplary Single-Chain Chimeric Polypeptide Sequences The nucleic acid encoding the αCD3scFv / TF / αCD28scFv single chain polypeptide is as follows (SEQ ID NO: 22): (signal peptide) ATGAAGTGGGTGACCTTCATCAGCTTATTATTTTTATTCAGCTCCGCCTATTCC (αCD3 light chain variable region) CAGATCGTGCTGACCCAAAGCCCCGCCATCATGAGCGCTAGCCCCGGTGAGAAGGTGACCATGACATGCTCCGCTTCCAGCTCCGTGTCCTACATGAACTGGTATCAGCAGAAAAGCGGAACCAGCCCCAAAAGGTGGATCTACGACACCAGCAAGCTGG CCTCCGGAGTGCCCGCTCATTTCCGGGGCTCTGGATCCGGCACCAGCTACTCTTTAACCATTTCCGGCATGGAAGCTGAAGACGCTGCCACCTACTATTGCCAGCAATGGAGCAGCAACCCCTTCACATTCGGATCTGGCACCAAGCTCGAAATCAATCGT (Linker) GGAGGAGGTGGCAGCGGCGGCGGTGGATCCGGCGGAGGAGGAAGC (αCD3 heavy chain variable region) CAAGTTCAACTCCAGCAGAGCGGCGCTGAACTGGCCCGGCCCGGCGCCTCCGTCAAGATGAGCTGCAAGGCTTCCGCTATACATTTACTCGTTACACAATGCATGGGTCAAGCAGAGGCCCGGTCAAGGTTTAGAGTGGATCGGATATATCAACCCTTCCCGGGGCTACACCAACT ATAACCAAAGTTCAAGGATAAAGCCACTTTAACCACTGACAAAGAGCTCCTCCACCGCCTACATGCAGCTGTCCTCTTTAACCAGCGAGGACTCCGCTGTTTACTACTGCGCTAGGTATTACGACGACCACTACTGTTTAGACTATTGGGGACAAGGTACCACTTTAACCGTCAGCAGC (ヤーションション219 フェック) TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (αCD28 light chain variable region) GTCCAGCTGCAGCAGAGCGGACCCGAACTCGTGAAACCCGGTGCTTCCGTGAAAATGTCTTGTAAGGCCAGCGGATACACCTTCACCTCCTATGTGATCCAGTGGGTCAAACAGAAGCCCGGACAAGGTCTCGAGTGGATCGGCAGCATCAACCCTTACAACGACTATACC AAATACAACGAGAAGTTTAAGGGAAAGGCTACTTTAACCTCCGACAAAAGCTCCATCACAGCCTACATGGAGTTCAGCTCTTTAACATCCGAGGACAGCGCTCTGTACTATTGCGCCCGGTGGGGCGACGGCAATTACTGGGGACGGGGCACAACACTGACCGTGAGCAGC (Linker) GGAGGCGGAGGCTCCGGCGGAGGCGGATCTGGCGGTGGCGGCTCC (αCD28 light chain variable region) GACATCGAGATGACCCAGTCCCCCGCTATCATGTCCGCCTTTTAGGCGAGCGGGTCACAATGACTTGTACAGCCTCCTCCAGCGTCTCCTCCTCCTACTTCCATTGGTACCAACAGAAACCCGGAAGCTCCCCTAAACTGTGCATCTACAGCACCAGCA ATCTCGCCAGCGGCGTGCCCCCTAGGTTTTCCGGAAGCGGAAGCACCAGCTACTCTTTAACCATCTCCTCCATGGAGGCTGAGGATGCCGCCACCTACTTTTGTCACCAGTACCACCGGTCCCCCACCTTCGGAGGCGGCACCAAACTGGAGACAAAGAGG

[0160] The amino acid sequence of the αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide is as follows (SEQ ID NO: 23): (signal peptide) MKWVTFISLLFLFSSAYS (αCD3 light chain variable region) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (Linker) GGGGSGGGGSGGGGS (αCD3 heavy chain variable region) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (αCD28 light chain variable region) VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSS (Linker) GGGGSGGGGSGGGGS (αCD28 heavy chain variable region) DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR

[0161] The nucleic acid sequence encoding the αCD28scFv / TF / αCD3scFv single-chain polypeptide is as follows (SEQ ID NO: 24): (signal peptide) ATGAAATGGGTCACCTTCATCTCTTTACTGTTTTTATTTAGCAGCGCCTACAGC (αCD28 light chain variable region) GTGCAGCTGCAGCAGTCCGGACCCGAACTGGTCAAGCCCGGTGCCTCCGTGAAAATGTCTTGTAAGGCTTCTGGCTACACCTTTACCTCCTACGTCATCCAATGGGTGAAGCAGAAGCCCGGTCAAGGTCTCGAGTGGATCGGCAGCATCAATCCCTACAACGATTACACC AAGTATAACGAAAAGTTTAAGGGCAAGGCCACTCTGACAAGCGACAAGAGCTCCATTACCGCCTACATGGAGTTTTCCTCTTTAACTTCTGAGGACTCCGCTTTATACTATTGCGCTCGTTGGGGCGATGGCAATTATTGGGGCCGGGAACTACTTTAACAGTGAGCTCC (Linker) GGCGGCGGCGGAAGCGGAGGTGGAGGATCTGGCGGTGGAGGCAGC (αCD28 heavy chain variable region) GACATCGAGATGACACAGTCCCCCGCTATCATGAGCGCCTTTTAGGAGAACGTGTGACCATGACTTGTACAGCTTCCTCCAGCGTGAGCAGCTCCTATTTCCACTGGTACCAGCAGAAACCCGGCTCCTCCCTAAACTGTGTATCTACTCCACAAGCA ATTTAGCTAGCGGCGTGCCTCCTCGTTTTAGCGGCTCCGGCAGCACCTCTTACTCTTTAACCATTAGCTCTATGGAGGCCGAAGATGCCGCCACATACTTTTGCCATCAGTACCACCGGTCCCCTACCTTTGGCGGAGGCACAAAGCTGGAGACCAAGCGG (human tissue factor 219 form) AGCGGCACCACCAACACAGTGGCCGCCTACAATCTGACTTGGAAATCCACCAACTTCAAGACCATCCTCGAGTGGGAGCCCAAGCCCGTTAATCAAGTTTATACCGTGCAGATTTCCACCAAGAGCGGCGACTGGAAATCCAAGTGCTTCTATACCACAGACACCGAGTGCGATCTCACCGACGAGATCGTCAAAGACGTGAAGCAGACATATTTAGCTAGGGTGTTCTCCTACCCCGCTGGAAACGTGGAGAGCACCGGATCCGCTGGAGAGCCTTTATACGAGAACTCCCCCGAATTCACCCCCTATCTGGAAACCAATTTAGGCCAGCCCACCATCCAGAGCTTCGAACAAGTTGGCACAAAGGTGAACGTCACCGTCGAAGATGAGAGGACTTTAGTGCGGAGGAACAATACATTTTTATCCTTACGTGACGTCTTCGGCAAGGATTTAATCTACACACTGTATTACTGGAAGTCTAGCTCCTCCGGCAAGAAGACCGCCAAGACCAATACCAACGAATTTTTAATTGACGTGGACAAGGGCGAGAACTACTGCTTCTCCGTGCAAGCTGTGATCCCCTCCCGGACAGTGAACCGGAAGTCCACCGACTCCCCCGTGGAGTGCATGGGCCAAGAGAAGGGAGAGTTTCGTGAG (αCD3 light chain variable region) CAGATCGTGCTGACCCAGTCCCCCGCTATTATGAGCGCTAGCCCCGGTGAAAAGGTGACTATGACATGCAGCGCCAGCTCTTCCGTGAGCTACATGAACTGGTATCAGCAGAAGTCCGGCACCAGCCCTAAAAGGTGGATCTACGACACCAGCAAGCTGG CCAGCGGCGTCCCCGCTCACTTTCGGGGCTCCGGCTCCGGAACAAGCTACTCTCTGACCATCAGCGGCATGGAAGCCGAGGATGCCGCTACCTATTACTGTCAGCAGTGGAGCTCCAACCCCTTCACCTTTGGATCCGGCACCAAGCTCGAGATTAATCGT (Linker) GGAGGCGGAGGTAGCGGAGGAGGCGGATCCGGCGGTGGAGGTAGC (αCD3 heavy chain variable region) CAAGTTCAGCTCCAGCAAAGCGGCGCCGAACTCGCTCGGCCCGGCGCTTCCGTGAAGATGTCTTGTAAGGCCTCCGGCTATACCTTCACCCGGTACACAATGCACTGGGTCAAGCAACGGCCCGGTCAAGGTTTAGAGTGGATTGGCTATATCAACCCCTCCCGGGGCTATACCAACT ACAACCAGAAGTTCAAGGACAAAGCCACCCTCACCACCGACAAGTCCAGCAGCACCGCTTACATGCAGCTGAGCTCTTTAACATCCGAGGATTCCGCCGTGTACTACTGCGCTCGGTACTACGACGATCATTACTGCCTCGATTACTGGGGCCAAGGTACCACCTTAACAGTCTCCTCC

[0162] The amino acid sequence of the αCD28scFv / TF / αCD3scFv single-chain chimeric polypeptide is as follows (SEQ ID NO: 25): (signal peptide) MKWVTFISLLFLFSSAYS (αCD28 light chain variable region) VQLQQSGPELVKPGASVKMSCKASGYTFTSYVIQWVKQKPGQGLEWIGSINPYNDYTKYNEKFKGKATLTSDKSSITAYMEFSSLTSEDSALYYCARWGDGNYWGRGTTLTVSS (Linker) GGGGSGGGGSGGGGS (αCD28 heavy chain variable region) DIEMTQSPAIMSASLGERVTMTCTASSSVSSSYFHWYQQKPGSSPKLCIYSTSNLASGVPPRFSGSGSTSYSLTISSMEAEDAATYFCHQYHRSPTFGGGTKLETKR (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (αCD3 light chain variable region) QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (Linker) GGGGSGGGGSGGGGS (αCD3 heavy chain variable region) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS

[0163] The nucleic acid sequence encoding the IL-2 / TF / IL-2 single-chain chimeric polypeptide is as follows (SEQ ID NO: 26): (signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (First IL-2 fragment) GCCCCCACCTCCTCTCCACCAAGAAGACCCAGCTGCAGCTGGAGCATTTACTGCTGGATTTACAGATGATTTTAAACGGCATCAACAACTACAAGAACCCCAAGCTGACTCGTATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAGCATTTACAGTGTTTAGAGGAGGAGCTGAAGCCCTCG AGGAGGTGCTGAATTTAGCCCAGTCCAAGAATTTCCATTTAAGGCCCCGGGATTTAACAGCAACATCAACGTGATCGTTTTAGAGCTGAAGGGCTCCGAGACCACCTTCATGTGCGAGTACGCCGACGAGACCGCCACCATCGTGGAGTTTTTAAATCGTTGGATCACCTTCTGCCAGTCCATCATCTCCACTTTAACC (human tissue factor 219 form) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Second IL-2 fragment) GCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGG AGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTAACT

[0164] The amino acid sequence of the IL-2 / TF / IL-2 single-chain chimeric polypeptide is as follows (SEQ ID NO: 27): (signal peptide) MKWVTFISLLFLFSSAYS (human IL-2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (human IL-2) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0165] The nucleic acid sequence encoding the IL-15 / TF / IL-15 single-chain chimeric polypeptide is as follows (SEQ ID NO: 28): (signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (First IL-15 fragment) AACTGGGTGAACGTGATCAGCGATTTAAAGAAGATCGAGGATTTAATCCAGAGCATGCACATCGACGCCACTCTGTACACTGAGAGCGACGGTGCACCCTAGCTGCAAGGTGACTGCCATGAAGTGCTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGCGATGCC AGCATCCACGACACTGTGGAGAATTTAATCATTTTAGCCAACAACTCTTTAAGCAGCAACGGCAACGTGACAGAGAGCGGCTGCAAGGAGTGCGAGGAGCTGGAGGAGAAGAACATCAAGGAGTTTTTACAGAGCTTCGTGCACATCGTGCAGATGTTCATCAACACTAGC (human tissue factor 219 form) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (Second IL-15 fragment) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

[0166] The amino acid sequence of the IL-15 / TF / IL-15 single-chain chimeric polypeptide is as follows (SEQ ID NO: 29): (signal peptide) MKWVTFISLLFLFSSAYS (human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0167] Multi-chain chimeric polypeptides A non-limiting example of a tissue factor-containing protein is a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising (i) a first target binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains; and (b) a second chimeric polypeptide comprising (i) a second domain of the pair of affinity domains and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate via binding between the first domain and the second domain of the pair of affinity domains.

[0168] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are immediately adjacent to each other within the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) within the first chimeric polypeptide.

[0169] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the paired affinity domain (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) are immediately adjacent to each other within the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the paired affinity domain (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) within the first chimeric polypeptide.

[0170] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) in the second chimeric polypeptide.

[0171] In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art), wherein at least one of the one or more additional antigen binding domains is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide may further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art), and / or a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).

[0172] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0173] In some embodiments of any of the multi-chain chimeric polypeptides described herein, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is disposed at the N-terminus and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is positioned between a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the N-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the C-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the first domains described herein or any of the exemplary pair of affinity domains described herein) is immediately adjacent to the soluble tissue factor domain and / or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide (i) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein); at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein) and the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein); Between and / or (ii) a first domain of a pair of affinity domains; at least one of one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains; Between The amino acid sequence further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed at

[0174] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus and / or C-terminus of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the second domain of a pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second domain of the pair of affinity domains (e.g., any of the exemplary second domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the second target binding domain (e.g., any of the target binding domains described herein or known in the art) in the second chimeric polypeptide.In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0175] In some embodiments of any of the multi-chain chimeric polypeptides described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

[0176] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to different antigens. In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and one or more additional target binding domains are antigen binding domains. In some embodiments, the first target binding domain, the second target binding domain, and one or more additional target binding domains are each antigen binding domains (e.g., scFvs or single-domain antibodies).

[0177] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one of a first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), a second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) include CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGI T, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HE R2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKP30 ligand, scMHCII ligand, scTCR ligand, I The antibody specifically binds to a target selected from the group consisting of IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

[0178] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. Non-limiting examples of soluble interleukin and soluble cytokine proteins include IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

[0179] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine receptors. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKP30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.

[0180] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the target binding domains described herein), the second target binding domain (e.g., any of the target binding domains described herein), and the one or more additional target binding domains (e.g., any of the target binding domains described herein) are each independently selected from the group consisting of CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA , CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD ligand, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor.

[0181] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art), and one or more additional binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) are soluble interleukin or cytokine proteins. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is selected from the group of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

[0182] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble receptor is a soluble TGF-β receptor II (TGF-βRII), a soluble TGF-βRIII, a soluble TNFα receptor, a soluble IL-4 receptor, or a soluble IL-10 receptor.

[0183] A non-limiting example of a cell activator is a multi-chain chimeric polypeptide comprising: (a) a first and a second chimeric polypeptide, each comprising (i) a first target binding domain, (ii) an Fc domain, and (iii) a first domain of a pair of affinity domains; and (b) a third and a fourth chimeric polypeptide, each comprising (i) a second domain of the pair of affinity domains and (ii) a second target binding domain, wherein the first and second chimeric polypeptides and the third and fourth chimeric polypeptides associate via the binding between the first and second domains of the pair of affinity domains, and the first and second chimeric polypeptides associate via their Fc domains.

[0184] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the Fc domain (e.g., any of the exemplary Fc domains described herein) are immediately adjacent to each other in the first and second chimeric polypeptides. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first and second chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the Fc domain (e.g., any of the exemplary Fc domains described herein) in the first and second chimeric polypeptides.

[0185] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the Fc domain (e.g., any of the exemplary Fc domains described herein) and the first domain of the paired affinity domain (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) are immediately adjacent to each other in the first and second chimeric polypeptides. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first and second chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the Fc domain (e.g., any of the exemplary Fc domains described herein) and the first domain of the paired affinity domain (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first and second chimeric polypeptides.

[0186] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are immediately adjacent to each other in the third and fourth chimeric polypeptides. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the third and fourth chimeric polypeptides further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of the pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) in the third and fourth chimeric polypeptides.

[0187] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain comprise the same amino acid sequence. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to different antigens. In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are antigen binding domains (e.g., any of the exemplary second target binding domains described herein). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each antigen binding domains (e.g., any of the exemplary second target binding domains described herein). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen binding domain (e.g., any of the exemplary second target binding domains described herein) comprises an scFv or a single domain antibody.

[0188] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) are selected from the group consisting of CD16a, CD28, CD3, CD33, CD20, CD19, CD22, CD1 23, IL-1R, IL-1, VEGF, IL-6R, IL-4, IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP 2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACA The antibody specifically binds to a target selected from the group consisting of M5, UL16 binding protein, HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

[0189] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) is a soluble interleukin or cytokine protein. Non-limiting examples of soluble interleukin and cytokine proteins include IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

[0190] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) are soluble interleukin or cytokine receptors. Non-limiting examples of soluble interleukin receptors and soluble cytokine receptors include soluble TGF-β receptor II (TGF-βRII), soluble TGF-βRIII, soluble NKG2D, soluble NKp30, soluble NKp44, soluble NKp46, soluble DNAM1, scMHCI, scMHCII, scTCR, soluble CD155, soluble CD122, soluble CD3, or soluble CD28.

[0191] In some embodiments of any of the methods described herein, the first target binding domain and the second target binding domain are each independently selected from the group consisting of CD16a, CD33, CD20, CD19, CD22, CD123, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26, CD36, ULBP2, CD30, CD200, IGF-1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding protein, HLA-DR, The antibody can specifically bind to a target selected from the group consisting of DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NKp30 ligand, scMHCI ligand, scMHCII ligand, scTCR ligand, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16 binding protein receptor, CD155 receptor, and CD122 receptor.

[0192] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine protein. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble interleukin or cytokine protein is selected from the group consisting of IL-1, IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, and SCF.

[0193] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain is a soluble interleukin or cytokine receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble receptor is a soluble TGF-β receptor II (TGF-βRII), a soluble TGF-βRIII, a soluble TNFα receptor, a soluble IL-4 receptor, or a soluble IL-10 receptor.

[0194] In some embodiments, the multi-chain chimeric polypeptide has a nucleic acid sequence or amino acid sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to SEQ ID NOs: 30-101.

[0195] Additional antigen-binding domains In some embodiments of any of the single-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides, at least one of the one or more additional antigen-binding domains may be positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide may further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art), and / or a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).

[0196] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first domain of the pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0197] In some embodiments of any of the multi-chain chimeric polypeptides described herein, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is disposed at the N-terminus and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is positioned between a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the N-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the C-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the pair of affinity domains (e.g., any of the first domains described herein or any of the exemplary pair of affinity domains described herein) is immediately adjacent to the soluble tissue factor domain and / or the first domain of the pair of affinity domains. In some embodiments, the first chimeric polypeptide (i) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein); at least one of one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein) and the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein); Between and / or (ii) a first domain of a pair of affinity domains; at least one of one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains; Between The amino acid sequence further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed at

[0198] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) at the N-terminus and / or C-terminus of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the second domain of a pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second domain of the pair of affinity domains (e.g., any of the exemplary second domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the second target binding domain (e.g., any of the target binding domains described herein or known in the art) in the second chimeric polypeptide.In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0199] In some embodiments of any of the multi-chain chimeric polypeptides described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

[0200] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to different antigens. In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) of the first target binding domain, the second target binding domain, and one or more additional target binding domains are antigen binding domains. In some embodiments, the first target binding domain, the second target binding domain, and one or more additional target binding domains are each antigen binding domains (e.g., scFvs or single-domain antibodies).

[0201] Paired affinity domains In some embodiments, a multi-chain chimeric polypeptide comprises 1) a first chimeric polypeptide comprising a first domain of a pair of affinity domains and 2) a second chimeric polypeptide comprising a second domain of the pair of affinity domains, such that the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first and second domains of the pair of affinity domains. In some embodiments, the pair of affinity domains is a sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. The sushi domain, also known as the short consensus repeat or type 1 glycoprotein motif, is a common motif for protein-protein interactions. Sushi domains have been identified in several protein-binding molecules, including complement components C1r, C1s, factor H, and C2m, as well as the non-immunological molecules factor XIII and β2-glycoprotein. A typical sushi domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp. Biocomput. 2000:155-67). The first cysteine ​​can form a disulfide bond with the third cysteine, and the second cysteine ​​can form a disulfide bridge with the fourth cysteine. In some embodiments where one member of the paired affinity domains is soluble IL-15, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments where one member of the paired affinity domains is human IL-15 receptor alpha chain (IL15Rα), the human IL15Rα is mature full-length IL15Rα. In some embodiments, the paired affinity domains are barnase and barnstar. In some embodiments, the paired affinity domains are PKA and AKAP.In some embodiments, the pair of affinity domains is an adapter / docking tag module based on a mutant RNase I fragment (Rossi, Proc Natl Acad Sci USA. 103:6841-6846, 2006; Sharkey et al., Cancer Res. 68:5282-5290, 2008; Rossi et al., Trends Pharmacol Sci. 33:474-481, 2012), or a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492, 2003).

[0202] In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first domain of a pair of affinity domains, a second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a second domain of the pair of affinity domains, and the first domain of the pair of affinity domains and the second domain of the pair of affinity domains are at least 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M or 1 x 10 -13 The dissociation equilibrium constant (K D In some embodiments, the first domain of the pair of affinity domains and the second domain of the pair of affinity domains bind to each other at about 1×10 -4 M ~ approx. 1×10 -6 M, about 1 x 10 -5 M ~ approx. 1×10 -7 M, about 1 x 10 -6 M ~ approx. 1×10 -8 M, about 1 x 10 -7 M ~ approx. 1×10 -9 M, about 1 x 10 -8 M ~ approx. 1×10 -10 M, about 1 x 10 -9 M ~ approx. 1×10 -11 M, about 1 x 10 -10 M ~ approx. 1×10-12 M, about 1 x 10 -11 M ~ approx. 1×10 -13 M, about 1 x 10 -4 M ~ approx. 1×10 -5 M, about 1 x 10 -5 M ~ approx. 1×10 -6 M, about 1 x 10 -6 M ~ approx. 1×10 -7 M, about 1 x 10 -7 M ~ approx. 1×10 -8 M, about 1 x 10 -8 M ~ approx. 1×10 -9 M, about 1 x 10 -9 M ~ approx. 1×10 -10 M, about 1 x 10 -10 M ~ approx. 1×10 -11 M, about 1 x 10 -11 M ~ approx. 1×10 -12 M, or approximately 1 x 10 -12 M ~ approx. 1×10 -13 K of M (including their upper and lower bounds) D The K of binding of the first domain of the pair of affinity domains and the second domain of the pair of affinity domains can be determined using any of a variety of different methods known in the art. D Values ​​can be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, biomolecular binding kinetic assays, etc.).

[0203] In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first domain of a pair of affinity domains, and a second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a second domain of the pair of affinity domains, wherein the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both, are about 10 to 100 amino acids in length. For example, the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both may be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 ... The length may be about 0 amino acids, about 10 to 75 amino acids, about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both, are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

[0204] In some embodiments, any of the first and / or second domains of a pair of affinity domains disclosed herein may contain one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and / or C-terminus, so long as the function of the first and / or second domain of the pair of affinity domains remains intact. For example, the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα) may contain one or more additional amino acids at the N-terminus and / or C-terminus while still retaining the ability to bind to soluble IL-15. Additionally or alternatively, soluble IL-15 may contain one or more additional amino acids at the N-terminus and / or C-terminus while still retaining the ability to bind to the sushi domain derived from the human IL-15 receptor alpha chain (IL15Rα).

[0205] Non-limiting examples of sushi domains from the IL-15 receptor alpha chain (IL15Rα) can include sequences at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 123). In some embodiments, the sushi domain from the IL15Rα alpha chain can be encoded by a nucleic acid comprising ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 124).

[0206] In some embodiments, the soluble IL-15 may comprise a sequence at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 125). In some embodiments, soluble IL-15 can be encoded by a nucleic acid comprising the sequence AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 126).

[0207] Exemplary Multi-Chain Chimeric Polypeptides—Type A In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to an IL-18 receptor or an IL-12 receptor. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0208] In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain are soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the IL-18 receptor or the IL-12 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0209] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-12 receptor and the second target binding domain specifically binds to the IL-18 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-18 receptor and the second target binding domain specifically binds to the IL-12 receptor.

[0210] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-18 (eg, soluble human IL-18).

[0211] In some embodiments of these multi-chain chimeric polypeptides, the second target-binding domain comprises soluble IL-12 (e.g., soluble human IL-12). In some embodiments of these multi-chain chimeric polypeptides, the soluble human IL-15 comprises a soluble human IL-12β(p40) sequence and a soluble human IL-12α(p35) sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble IL-15 human IL-15 further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein) between the soluble IL-12β(p40) sequence and the soluble human IL-12α(p35) sequence.

[0212] Exemplary Multi-Chain Chimeric Polypeptides—Type B In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to IL-21 receptor or TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0213] In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain is soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or a soluble TGF-β receptor (e.g., a soluble TGFRβRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-21 or a soluble TGF-β receptor (e.g., a soluble TGFRβRII receptor). In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the IL-21 receptor or TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0214] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-21 receptor and the second target binding domain specifically binds to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF-β and the second target binding domain specifically binds to the IL-21 receptor.

[0215] In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises a soluble TGF-β receptor (e.g., a soluble TGFRβRII receptor (e.g., a soluble human TGFRβRII receptor)). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0216] Exemplary Multi-Chain Chimeric Polypeptides—Type C In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to an IL-7 receptor or an IL-21 receptor. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0217] In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain are soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-21 or soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the IL-21 receptor or the IL-7 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0218] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-21 receptor and the second target binding domain specifically binds to the IL-7 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-7 receptor and the second target binding domain specifically binds to the IL-21 receptor.

[0219] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-21 (eg, soluble human IL-21).

[0220] Exemplary Multi-Chain Chimeric Polypeptides—Type D In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to an IL-7 receptor or an IL-21 receptor. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0221] In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain are soluble IL-21 (e.g., a soluble human IL-21 polypeptide) or soluble IL-7 (e.g., a soluble human IL-7 polypeptide). In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-21 or soluble IL-7. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the IL-21 receptor or the IL-7 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0222] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-21 receptor and the second target binding domain specifically binds to the IL-7 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-7 receptor and the second target binding domain specifically binds to the IL-21 receptor.

[0223] Exemplary Multi-Chain Chimeric Polypeptides—Type E In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to an IL-18 (e.g., soluble human IL-18) receptor, an IL-12 (e.g., soluble human IL-12) receptor, or CD16 (e.g., an anti-CD16 scFv). In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to CD16 or the IL-12 receptor.

[0224] In some embodiments of these multi-chain chimeric polypeptides, one or both of the first target binding domain and the second target binding domain are soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are each independently soluble IL-15 or soluble IL-18. In some embodiments of these multi-chain chimeric polypeptides, both the first target binding domain and the second target binding domain specifically bind to the IL-18 receptor or the IL-12 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0225] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-12 receptor and the second target binding domain specifically binds to the IL-18 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-18 receptor and the second target binding domain specifically binds to the IL-12 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to CD16 and the second target binding domain specifically binds to the IL-18 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to the IL-18 receptor and the second target binding domain specifically binds to CD16.

[0226] In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

[0227] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-18 (eg, soluble human IL-18).

[0228] Exemplary Multi-Chain Chimeric Polypeptides—Type F In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to an IL-7 (e.g., soluble human IL-7) receptor, CD16 (e.g., anti-CD16 scFv), or IL-21 (e.g., soluble human IL-21) receptor. In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a CD16 or IL-21 receptor.

[0229] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain specifically binds to the IL-7 receptor and the second target binding domain specifically binds to the CD16 or IL-21 receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain comprises a soluble IL-7 protein. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-7 protein is soluble human IL-7. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain comprises a target binding domain that specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain comprises an scFv that specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain specifically binds to the IL-21 receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to the IL-21 receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to CD16.

[0230] In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

[0231] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises soluble IL-7 (eg, soluble human IL-7).

[0232] Exemplary Multi-Chain Chimeric Polypeptides—Type G In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a TGFβ (e.g., human TGFβRII receptor), CD16 (e.g., anti-CD16 scFv), or IL-21 (e.g., soluble human IL-21) receptor. In some embodiments of these multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a CD16 or IL-21 receptor.

[0233] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β, CD16, or IL-21 receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain specifically binds to TGF-β, and the second target binding domain specifically binds to CD16 or IL-21 receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain is a soluble TGF-β receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble TGF-β receptor is a soluble TGFβRII receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target binding domain specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain comprises an antigen-binding domain that specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second antigen-binding domain comprises an scFv that specifically binds to CD16. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain specifically binds to the IL-21 receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second target-binding domain comprises soluble human IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target-binding domain that specifically binds to the IL-21 receptor. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the additional target-binding domain comprises soluble IL-21. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble IL-21 is soluble human IL-21.In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain that specifically binds to CD16.

[0234] In some embodiments of these multi-chain chimeric polypeptides, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence.

[0235] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain comprises a TGFβRII receptor (e.g., a soluble human TGFβRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0236] Exemplary Multi-Chain Chimeric Polypeptides—Type H In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to an IL-7 receptor. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0237] Exemplary Multi-Chain Chimeric Polypeptides—Type I In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0238] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0239] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are soluble TGF-β receptors (e.g., soluble TGFβRII receptors, e.g., soluble human TGFβRII). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0240] Exemplary Multi-Chain Chimeric Polypeptides—Type J In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to an IL-7 receptor, an IL-21 receptor, or a CD137L receptor. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to an IL-21 (e.g., soluble IL-21, e.g., soluble human IL-21) receptor or a CD137L (e.g., soluble CD137L, e.g., soluble human CD137L) receptor.

[0241] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to an IL-7 receptor and the second target binding domain specifically binds to an IL-21 receptor or a CD137L receptor. In some embodiments, the additional target binding domain specifically binds to an IL-21 receptor or a CD137L receptor.

[0242] Exemplary Multi-Chain Chimeric Polypeptides—Type K In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to IL-7 receptor or TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0243] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to IL-7 receptor and the second target binding domain specifically binds to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF-β and the second target binding domain specifically binds to IL-7 receptor.

[0244] In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain comprises a target binding domain that specifically binds to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the second target binding domain is a soluble TGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., a soluble human TGFβRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0245] Exemplary Multi-Chain Chimeric Polypeptides—Type L In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β, an IL-21 receptor, or a CD137L receptor. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to an IL-21 (e.g., soluble IL-21, e.g., soluble human IL-21) receptor or a CD137L (e.g., soluble CD137L, e.g., soluble human CD137L) receptor.

[0246] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF-β and the second target binding domain specifically binds to the IL-21 receptor or the CD137L receptor.

[0247] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is a soluble TGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., a soluble human TGFβRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0248] Exemplary Multi-Chain Chimeric Polypeptides—Type M In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a TGF-β or IL-21 receptor. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to an IL-21 (e.g., soluble IL-21, e.g., soluble human IL-21) receptor or a TGF-β receptor (e.g., a soluble TGF-β receptor, e.g., a soluble TGFβRII receptor).

[0249] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF-β, and the second target binding domain specifically binds to a TGF-β or IL-21 receptor. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is a soluble TGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., a soluble human TGFβRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0250] Exemplary Multi-Chain Chimeric Polypeptides—Type N In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β or CD16. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to CD16 (e.g., an anti-CD16 scFv) or TGF-β (e.g., a soluble TGF-β receptor, e.g., a soluble TGFβRII receptor).

[0251] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds to TGF-β, and the second target binding domain specifically binds to TGF-β or CD16. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain is a soluble TGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., a soluble human TGFβRII receptor). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0252] Exemplary Multi-Chain Chimeric Polypeptides—Type O In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to a TGF-β or CD137L receptor. In some embodiments of these multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises an additional target binding domain. In some embodiments of these multi-chain chimeric polypeptides described herein, the additional target binding domain specifically binds to a TGF-β receptor (e.g., a soluble TGF-β receptor, e.g., a soluble TGFβRII receptor) or a CD137L receptor.

[0253] In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other within the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain within the first chimeric polypeptide.

[0254] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the pair of affinity domains are immediately adjacent to each other within the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the pair of affinity domains within the first chimeric polypeptide.

[0255] In some embodiments of these multi-chain chimeric polypeptides, the second domain of the pair of affinity domains and the second target binding domain are immediately adjacent to each other within the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the second target binding domain within the second chimeric polypeptide.

[0256] In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains at the N-terminus or C-terminus of the second chimeric polypeptide.

[0257] In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second domain of the pair of affinity domains are immediately adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second domain of the pair of affinity domains and the additional target binding domain in the second chimeric polypeptide.

[0258] In some embodiments of these multi-chain chimeric polypeptides, the additional target binding domain and the second target binding domain are immediately adjacent to each other within the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the second target binding domain and the additional target binding domain within the second chimeric polypeptide.

[0259] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pair of affinity domains described herein.

[0260] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain specifically binds TGF-β and the second target binding domain specifically binds CD137L. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain or the additional target binding domain is a soluble TGF-β receptor (e.g., a soluble TGFβRII receptor, e.g., a soluble human TGFβRII receptor).

[0261] In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first soluble human TGFRβRII sequence and a second soluble human TGFRβRII sequence. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first soluble human TGFRβRII sequence and the second soluble human TGFRβRII sequence.

[0262] Exemplary Multi-Chain Chimeric Polypeptide Sequences The nucleic acid sequence of the IL12 / IL-15RαSu construct (including the signal peptide sequence) is as follows (SEQ ID NO: 30): (signal peptide) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (Human IL-12 subunit beta (p40)) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCC ATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACGATTTAACCTTCTCCGGAAAAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTC AGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAATAGAGCGCTTGTCCCGCTCGAAGAATCTTTACCCATTGAGGTGATGGTGGACCCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTATCCGGGACATATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCCAAAATAGCCGGCAA TTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTC (linker) GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35)) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCAT GAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGG CTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTT AAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCTCGAGGAGCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC (human IL-15Rα sushi domain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

[0263] The amino acid sequence of the IL12 / IL-15RαSu fusion protein (including the signal peptide sequence) is as follows (SEQ ID NO: 31): (signal peptide) MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40)) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker) GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35)) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0264] The nucleic acid sequence of the IL-18 / TF / IL-15 construct is as follows (SEQ ID NO: 32): (signal peptide) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (human IL-18) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGGACAATGCCCCCCGGACCATCTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACA AGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCTCCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAAACGAGGAT (ヤーションション219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (ヒトIL-15) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

[0265] The amino acid sequence of the IL-18 / TF / IL-15 fusion protein (including the signal peptide sequence) is as follows (SEQ ID NO: 33): (signal peptide) MKWVTFISLLFLFSSAYS (human IL-18) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0266] The nucleic acid sequence of the IL-12 / IL-15RαSu / αCD16scFv construct is as follows (SEQ ID NO: 34): (signal peptide) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (Human IL-12 subunit beta (p40)) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCC ATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACGATTTAACCTTCTCCGGAAAAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTC AGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAATAGAGCGCTTGTCCCGCTCGAAGAATCTTTACCCATTGAGGTGATGGTGGACCCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTATCCGGGACATATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCCAAAATAGCCGGCAA TTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTC (linker) GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35)) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC (Human IL-15Rα sushi domain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (Anti-human CD16 light chain variable domain) TCCGAGCTGACCCAGGACCCTGCTGTGTCCGTGGCTCTGGGCCAGACCGTGAGGATCACCTGCCAGGGCGACTCCCTGAGGTCCTACTACGCCTCCTGGTACCAGCAGAAGCCCGGCCAGGCTCCTGTGCTGGTGATCTACGGCAAGAACAACAGGCCCTCC GGCATCCCTGACAGGTTCTCCGGATCCTCCTCCGGCAACACCGCCTCCTGACCATCACAGGCGCTCAGGCCGAGGACGAGGCTGACTACTACTGCAACTCCAGGGACTCCTCCGGCAACCATGTGGTGTTCGGCGGCGGCACCAAGCTGACCGTGGGCCAT (Linker) GGCGGCGGCGGCTCCGGAGGCGGCGGCAGCGGCGGAGGAGGATCC (anti-human CD16 heavy chain variable domain) GAGGTGCAGCTGGTGGAGTCCGGAGGAGGAGTGGTGAGGCCTGGAGGCTCCCTGAGGCTGAGCTGTGCTGCCTCCGGCTTCACCTTCGACGACTACGGCATGTCCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGTCCGGCATCAACTGGAACGGCGGATCCACCG GCTACGCCGATTCCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCCCTGAGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGGCAGGTCCCTGCTGTTCGACTACTGGGGACAGGGCACCCTGGTGACCGTGTCCAGG

[0267] The amino acid sequence of the IL-12 / IL-15RαSu / αCD16scFv fusion protein (including the signal peptide sequence) is as follows (SEQ ID NO: 35): (signal peptide) MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40)) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker) GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35)) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (anti-human CD16 light chain variable domain) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVGH (Linker) GGGGSGGGGSGGGGS (anti-human CD16 heavy chain variable domain) EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGRSLLFDYWGQGTLVTVSR

[0268] The nucleic acid sequence of the IL-18 / IL-15RαSu construct (including the signal peptide sequence) is as follows (SEQ ID NO: 36): (signal peptide) ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (human IL-18) TACTTCGGCAAACTGGAATCCAAGCTGAGCGTGATCCGGAATTTAAACGACCAAGTTCTGTTTATCGATCAAGGTAACCGGCCTCTGTTCGAGGACATGACCGACTCCGATTGCCGGACAATGCCCCCCGGACCATCTTCATTATCTCCATGTACAAGGACAGCCAGCCCCGGGGCATGGCTGTGACAATTAGCGTGAAGTGTGAGAAAATCAGCACTTTATCTTGTGAGAACA AGATCATCTCCTTTAAGGAAATGAACCCCCCCGATAACATCAAGGACACCAAGTCCGATATCATCTTCTTCCAGCGGTCCGTGCCCGGTCACGATAACAAGATGCAGTTCGAATCCTCCTCCTACGAGGGCTACTTTTTAGCTTGTGAAAAGGAGAGGGATTTATTCAAGCTGATCCTCAAGAAGGAGGACGAGCTGGGCGATCGTTCCATCATGTTCACCGTCCAAAACGAGGAT (human IL-15Rα sushi domain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

[0269] The amino acid sequence of the IL-18 / IL-15RαSu fusion protein (including the signal peptide sequence) is as follows (SEQ ID NO: 37): (signal peptide) MKWVTFISLLFLFSSAYS (human IL-18) YFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED (human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0270] The nucleic acid sequence of the IL-12 / TF / IL-15 construct (including the leader sequence) is as follows (SEQ ID NO: 38): (signal peptide) ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (Human IL-12 subunit beta (p40)) ATTTGGGAACTGAAGAAGGACGTCTACGTGGTCGAACTGGACTGGTATCCCGATGCTCCCGGCGAAATGGTGGTGCTCACTTGTGACACCCCCGAAGAAGACGGCATCACTTGGACCCTCGATCAGAGCAGCGAGGTGCTGGGCTCCGGAAGACCCTCACAATCCAAGTTAAGGAGTTCGGAGACGCTGGCCAATACACATGCCACAAGGGAGGCGAGGTGCTCAGCC ATTCCTTATTATTATTACACAAGAAGGAAGACGGAATCTGGTCCACCGACATTTTAAAAGATCAGAAGGAGCCCAAGAATAGACCTTTTTAAGGTGTGAGGCCAAAAACTACAGCGGTCGTTTCACTTGTTGGTGGCTGACCACCATTTCCACGATTTAACCTTCTCCGGAAAAGCCGGGGGAAGCTCCGACCCTCAAGGTGTGACATGTGGAGCCGCTACCCTC AGCGCTGAGAGGGTTCGTGGCGATAACAAGGAATACGAGTACAGCGTGGAGTGCCAAGAATAGAGCGCTTGTCCCGCTCGAAGAATCTTTACCCATTGAGGTGATGGTGGACCCCGTGCACAAACTCAAGTACGAGAACTACACCTCCTCCTTCTTATCCGGGACATATTAAGCCCGATCCTCCTAAGAATTTACAGCTGAAGCCTCTCCAAAATAGCCGGCAA TTGAGGTCTCTTGGGAATATCCCGACACTTGGAGCACACCCCACAGCTACTTCTCTTTAACCTTTTGTGTGCAAGTTCAAGGTAAAGCAAGCGGGAGAAGAAAGACCGGGTGTTTACCGACAAAACCAGCGCCACGTCATCTGTCGGAAGAACGCCTCCATCAGCGTGAGGGCTCCAAGATCGTTATTACTCCAGCAGCTGGTCCGAGTGGGCCAGCGTGCCTTGTTC (linker) GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGGCGGCGGAGGATCT (Human IL-12 subunit alpha (p35)) CGTAACCTCCCCGTGGCTACCCCCGATCCCGGAATGTTCCCTTGTTTACACCACAGCCAGAATTTACTGAGGGCCGTGAGCAACATGCTGCAGAAAGCTAGGCAGACTTTAGAATTTTACCCTTGCACCAGCGAGGAGATCGACCATGAAGATATCACCAAGGACAAGACATCCACCGTGGAGGCTTGTTTACCTCTGGAGCTGACAAAGAACGAGTCTTGTCTCAACTCTCGTGAAACCAGCTTCATCACAAATGGCTCTTGTTTAGCTTCCCGGAAGACCTCCTTTATGATGGCTTTATGCCTCAGCTCCATCTACGAGGATTTAAAGATGTACCAAGTGGAGTTCAAGACCATGAACGCCAAGCTGCTCATGGACCCTAAACGGCAGATCTTTTTAGACCAGAACATGCTGGCTGTGATTGATGAGCTGATGCAAGCTTTAAACTTCAACTCCGAGACCGTCCCTCAGAAGTCCTCCCTCGAGGAGCCCGATTTTTACAAGACAAAGATCAAACTGTGCATTTTACTCCACGCCTTTAGGATCCGGGCCGTGACCATTGACCGGGTCATGAGCTATTTAAACGCCAGC (Human tissue factor 219) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (ヒトIL-15) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

[0271] The amino acid sequence of the IL-12 / TF / IL-15 fusion protein (including the leader sequence) is as follows (SEQ ID NO: 39): (signal peptide) MKWVTFISLLFLFSSAYS (Human IL-12 subunit beta (p40)) IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS (Linker) GGGGSGGGGSGGGGS (Human IL-12 subunit alpha (p35)) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0272] The nucleic acid sequence of the TGFβRII / IL-15RαSu construct (including the signal sequence) is as follows (SEQ ID NO: 40): (signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (human TGFβRII-first fragment) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (Linker) GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT (Human TGFβRII - second fragment) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (Human IL - 15Rα sushi domain) ATCACGTGTCCTCCTCCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCAGGGAGCGGTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACGTCCAGCCTGACGGAGTGCGTGTTGAACAAGGCCACGAATGTCGCCCACTGGACAACCCCCAGTCTCAAATGTATTAGA

[0273] The amino acid sequence of the TGFβRII / IL-15RαSu construct (including the signal peptide sequence) is as follows (SEQ ID NO: 41): (signal peptide) MKWVTFISLLFLFSSAYS (human TGFβRII-first fragment) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker) GGGGSGGGGSGGGGS (human TGFβRII-second fragment) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0274] The nucleic acid sequence of the IL-21 / TF / IL-15 construct (including the leader sequence) is as follows (SEQ ID NO: 42): (signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (Human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCCGCCAACACCGGCAACAACGAGCGGATCATCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (Human Tissue Factor 219) TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (ヒトIL-15) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

[0275] The amino acid sequence of the mature IL-21 / TF / IL-15 fusion protein (including the signal peptide sequence) is as follows (SEQ ID NO: 43): (signal peptide) MKWVTFISLLFLFSSAYS (human IL-21) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE (human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0276] The nucleic acid sequence of the IL-21 / TF mutant / IL-15 construct (including the signal peptide sequence) is as follows (SEQ ID NO: 44, bolded nucleotides are mutants, mutant codons are underlined): (signal sequence) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCGGCCAACACCGGCAACAACGAGCGGATCA TCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (human tissue factor 219 mutant) TIFF0007758680000023.tif104170 (human IL-15) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

[0277] The amino acid sequence of the IL-21 / TF mutant / IL-15 construct (including the signal peptide sequence) is as follows (SEQ ID NO: 45, substituted residues are shown in bold and underlined): (signal peptide) MKWVTFISLLFLFSSAYS (human IL-21) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (human tissue factor 219) TIFF0007758680000024.tif40170 (Human IL-15) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0278] The nucleic acid sequence of the IL-21 / IL-15RαSu construct (including the signal sequence) is as follows (SEQ ID NO: 46): (signal sequence) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (human IL-21) CAGGGCCAGGACAGGCACATGATCCGGATGAGGCAGCTCATCGACATCGTCGACCAGCTGAAGAACTACGTGAACGACCTGGTGCCCGAGTTTCTGCCTGCCCCCGAGGACGTGGAGACCAACTGCGAGTGGTCCGCCTTCTCCTGCTTTCAGAAGGCCCAGCTGAAGTCGGCCAACACCGGCAACAACGAGCGGATCA TCAACGTGAGCATCAAGAAGCTGAAGCGGAAGCCTCCCTCCACAAACGCCGGCAGGAGGCAGAAGCACAGGCTGACCTGCCCCAGCTGTGACTCCTACGAGAAGAAGCCCCCCAAGGAGTTCCTGGAGAGGTTCAAGTCCCTGCTGCAGAAGATGATCCATCAGCACCTGTCCTCCAGGACCCACGGCTCCGAGGACTCC (human IL-15Rα sushi domain) ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG

[0279] The amino acid sequence of the IL-21 / IL-15RαSu construct (including the signal peptide sequence) is as follows (SEQ ID NO: 101): (signal peptide) MKWVTFISLLFLFSSAYS (human IL-21) QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0280] The nucleic acid sequence of the TGFβRII / TF / IL-15 construct (including the leader sequence) is as follows (SEQ ID NO: 47): (signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (human TGFβRII-first fragment) ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCACGATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAAT GACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT (Linker) GGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGT (human TGFβRII-second fragment) ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCCCGTGAAATTTCCCAGCTGTGCAAATTCTGCGATGAGGTTTTCCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCACAATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGCTGGCGGAAGAAT GACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC (ヤーションション219) TCCGGCACCACCAATACCGTGGCCGCTTATAACCTCACATGGAAGAGCACCAACTTCAAGACAATTCTGGAATGGGAACCCAAGCCCGTCAATCAAGTTTACACCGTGCAGATCTCCACCAAATCCGGAGACTGGAAGAGCAAGTGCTTCTACACAACAGACACCGAGTGTGATTTAACCGACGAAATCGTCAAGGACGTCAAGCAAACCTATCTGGCTCGGGTCTTTTCCTACCCCGCTGGCAATGTCGAGTCCACCGGCTCCGCTGGCGAGCCTCTCTACGAGAATTCCCCCGAATTCACCCCTTATTTAGAGACCAATTTAGGCCAGCCTACCATCCAGAGCTTCGAGCAAGTTGGCACCAAGGTGAACGTCACCGTCGAGGATGAAAGGACTTTAGTGCGGCGGAATAACACATTTTTATCCCTCCGGGATGTGTTCGGCAAAGACCTCATCTACACACTGTACTATTGGAAGTCCAGCTCCTCCGGCAAAAAGACCGCTAAGACCAACACCAACGAGTTTTTAATTGACGTGGACAAAGGCGAGAACTACTGCTTCAGCGTGCAAGCCGTGATCCCTTCTCGTACCGTCAACCGGAAGAGCACAGATTCCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG (ヒトIL-15) AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCT AGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC

[0281] The amino acid sequence of the TGFβRII / TF / IL-15 fusion protein (including the signal peptide) is as follows (SEQ ID NO: 48): (signal peptide) MKWVTFISLLFLFSSAYS (human TGFβRII-first fragment) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (Linker) GGGGSGGGGSGGGGS (human TGFβRII-second fragment) IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (human tissue factor 219) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENS...

Claims

1. 1. A method for purifying a tissue factor-containing multi-chain chimeric polypeptide, comprising: loading a liquid containing the multi-chain chimeric polypeptide onto the affinity chromatography resin; washing the affinity chromatography resin with one or more wash buffers; and eluting the multi-chain chimeric polypeptide with an elution buffer. Including, the affinity chromatography resin comprises an anti-tissue factor antibody or an antigen-binding fragment thereof, the antibody comprising a heavy chain variable domain comprising CDR1 of SEQ ID NO: 1, CDR2 of SEQ ID NO: 2 or 9, and CDR3 of SEQ ID NO: 3, and a light chain variable domain comprising CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6, bound to a base resin; The multi-chain chimeric polypeptide comprises: (a) (i), (ii) and (iii) below, namely: (i) a first target binding domain comprising a sequence having at least 90% identity to amino acids 19-305 of SEQ ID NO: 70; (ii) a soluble tissue factor domain comprising a sequence having at least 90% identity to SEQ ID NO: 10; and (iii) a first domain of a pair of affinity domains comprising a sequence having at least 90% identity to SEQ ID NO: 125; a first chimeric polypeptide comprising: (b) (i) and (ii) below, namely: (i) a second domain of the pair of affinity domains comprising a sequence having at least 90% identity to SEQ ID NO: 123; and (ii) a second target binding domain comprising a sequence having at least 90% identity to amino acids 19-305 of SEQ ID NO:

70. a second chimeric polypeptide comprising Including, the first chimeric polypeptide and the second chimeric polypeptide are associated with each other via binding between the first domain and the second domain of the pair of affinity domains; The method, wherein the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

2. loading a liquid containing the tissue factor-containing protein onto the affinity chromatography resin; washing the affinity chromatography resin with one or more wash buffers; eluting the tissue factor-containing protein using an elution buffer; The method of claim 1 , comprising:

3. 3. The method of claim 2, wherein the liquid containing the tissue factor-containing protein is a clarified liquid culture medium.

4. The method of claim 2 , wherein the liquid containing the tissue factor-containing protein comprises a cell lysate.

5. the one or more wash buffers (i) a first wash buffer comprising phosphate buffered saline; and (ii) a second wash buffer containing 0.01 M to 0.2 M citrate and having a pH of 4.5 to 5.5; The method according to any one of claims 2 to 4, wherein

6. (i) the first wash buffer is phosphate buffered saline; (ii) the second wash buffer is 0.1 M citrate, pH 5.0; The method of claim 5.

7. The method of any one of claims 2 to 6, wherein the elution buffer comprises 0.01 M to 0.2 M acetate and has a pH of 2.5 to 3.

5.

8. 8. The method of claim 7, wherein the elution buffer comprises 0.1 M acetate and has a pH of 2.

9.

9. 1. A method for producing a tissue factor-containing protein, comprising: (i) purifying a tissue factor-containing protein using the method of any one of claims 1 to 8; (ii) subjecting the eluate obtained from step (i) to one or more additional unit operations.

10. The one or more additional unit operations, in sequential order: performing low pH viral inactivation; Performing depth filtration, performing polishing chromatography; performing nanofiltration; and Performing ultrafiltration and diafiltration (UF / DF) 10. The method of claim 9, comprising:

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