Pancreatic cancer treatment methods

Abstract

Provided herein are methods of treating unresectable advanced / metastatic pancreatic cancer in a subject. TIFF2025508939000090.tif127170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 315,820, filed March 2, 2022, and U.S. Provisional Patent Application No. 63 / 315,840, filed March 2, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named 47039-0030WO1.xml. The XML file, created on February 23, 2023, is 96,770 bytes in size. The material in the XML file is incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure relates to the field of biotechnology, and more particularly to methods for treating pancreatic cancer in a subject using multi-chain chimeric polypeptides. [Background technology]

[0004] background 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 coagulation pathway, is the primary initiator of coagulation in vivo. Normally absent from circulating blood, tissue factor 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 the rapid degradation of FX to FXa and FIX to FIXa. The resulting FXa and activated membrane surface proteolytic activity then inefficiently converts small amounts of prothrombin to thrombin. Thrombin generated by FXa initiates platelet activation, activating trace amounts of pro-cofactors 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. 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.

[0005] In addition to the tissue factor-FVIIa complex, recent studies have shown that the tissue factor-FVIIa-FXa complex can activate FVIII, providing additional levels of FVIIIa during the initiation phase. The extrinsic pathway is most important for initiating coagulation through activation of limited amounts of thrombin, whereas the intrinsic pathway sustains coagulation by dramatically amplifying the initial signal.

[0006] The majority of tissue factor expressed on the cell surface is "encoded," and it must be "decoded" for full participation in coagulation. The mechanism of "decoding" cell surface tissue factor is currently unknown, but exposure of anionic phospholipids plays a key role in this process. Healthy cells actively sequester anionic phospholipids, such as phosphatidylserine (PS), in the inner leaflet of the cell membrane. Upon cell damage, activation, or cytosolic Ca release, the tissue factor becomes encoded and needs to be "decoded." 2+ This bilayer asymmetry is lost after increased levels of PS, resulting in increased PS exposure at the outer leaflet, which increases the specific activity of the cell surface tissue factor-FVIIa complex. Although PS exposure is known to decrease 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.

[0007] More than 62,000 Americans are expected to be diagnosed with pancreatic cancer in 2022 (see the Pancreatic Cancer Action Network website). Effective treatments for pancreatic cancer are desirable. Summary of the Invention [Means for solving the problem]

[0008] overview The present invention is based on the discovery that 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 through the binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII, can provide a treatment for pancreatic cancer in a subject.

[0009] Provided herein is a method for treating unresectable, advanced / metastatic pancreatic cancer in a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0010] Also provided herein is a method for improving the objective response rate in a subject with unresectable, advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0011] Also provided herein is a method for increasing progression-free survival or progression-free survival rate in a subject or population of subjects with unresectable, advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of 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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0012] Also provided herein is a method for increasing time to progression in a subject or population of subjects with unresectable advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0013] Also provided herein is a method for increasing duration of response in a subject or population of subjects with unresectable advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0014] Also provided herein is a method of increasing overall survival in a population of subjects with unresectable, advanced / metastatic pancreatic cancer, comprising administering to each subject in the population a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0015] In some embodiments of any of the methods described herein, the subject is 18 years of age or older. In some embodiments of any of the methods described herein, the subject has previously been treated with a standard first-line systemic therapy for pancreatic cancer, and the subject's pancreatic cancer has progressed on the previous treatment and / or has been intolerant to the previous treatment. In some embodiments of any of the methods described herein, the subject has previously been treated with a standard first-line systemic therapy for pancreatic cancer, and the subject has been intolerant to the first-line systemic therapy. In some embodiments of any of the methods described herein, the standard first-line systemic therapy comprises one or more of FOLFIRINOX, modified FOLFINIROX, gemcitabine, albumin-bound paclitaxel, cisplatin, erlotinib, capecitabine, docetaxel, fluoropyrimidines, and oxaliplatin. In some embodiments of any of the methods described herein, the first-line systemic therapy comprises one of (i) FOLFIRINOX, (ii) modified FOLFIRINOX, (iii) gemcitabine and albumin-bound paclitaxel, (iv) gemcitabine and erlotinib, (v) gemcitabine, (vi) gemcitabine and capecitabine, (vii) gemcitabine, docetaxel, and capecitabine, and (viii) a fluoropyrimidine and oxaliplatin. In some embodiments of any of the methods described herein, the subject has previously been identified as having a BRCA1, BRCA2, or PALB2 mutation, and thus the first-line systemic therapy comprises one of (i) FOLFIRINOX, (ii) modified FOLFIRINOX, and (iii) gemcitabine and cisplatin.

[0016] In some embodiments of any of the methods described herein, the subject has previously been treated with a second-line or post-line systemic therapy for pancreatic cancer, and the subject's pancreatic cancer has progressed on the previous treatment and / or has been intolerant to the previous treatment. In some embodiments of any of the methods described herein, the second-line or post-line systemic therapy includes one or more of another first-line systemic therapy, 5-fluorouracil, leucovorin, liposomal irinotecan, irinotecan, FOLFIRINOX, modified FOLFIRINOX, oxaliplatin, FOLFOX, capecitabine, gemcitabine, albumin-bound paclitaxel, cisplatin, erlotinib, pembrolizumab, larotrectinib, and entrectinib. In some embodiments of any of the methods described herein, the second-line or post-line systemic therapy is another first-line systemic therapy. In some embodiments of any of the methods described herein, the second-line or back-line systemic therapy comprises one of: (i) 5-fluorouracil, leucovorin, and liposomal irinotecan, (ii) 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI), (iii) FOLFIRINOX or modified FOLFIRINOX, (iv) oxaliplatin, 5-fluorouracil, and leucovorin (OFF), (v) FOLFOX, (vi) capecitabine and oxaliplatin, (vii) capecitabine, and (viii) 5-fluorouracil continuous infusion. In some embodiments of any of the methods described herein, the subject has been previously treated with a fluoropyrimidine-based therapy, such that the second-line or post-line systemic therapy comprises one of: (i) gemcitabine, (ii) gemcitabine and albumin-bound paclitaxel, and (iii) gemcitabine with erlotinib. In some embodiments of any of the methods described herein, the subject has been previously treated with a fluoropyrimidine-based therapy and has previously been identified as having a BRCA1, BRCA2, or PALB2 mutation, such that the second-line or post-line systemic therapy comprises gemcitabine and cisplatin.In some embodiments of any of the methods described herein, the subject has previously been treated with a fluoropyrimidine-based therapy and has not received prior treatment with irinotecan, so the second-line or rear-line systemic therapy comprises 5-fluorouracil, leucovorin, and liposomal irinotecan. In some embodiments of any of the methods described herein, the subject has previously been identified as having an MSI-H or dMMR tumor, so the second-line or rear-line systemic therapy comprises pembrolizumab. In some embodiments of any of the methods described herein, the subject has previously been identified as having an NTRK gene fusion, so the second-line or rear-line systemic therapy comprises larotrectinib or entrectinib.

[0017] In some embodiments of any of the methods described herein, the subject has distant metastatic disease. In some embodiments of any of the methods described herein, the subject has adequate cardiac, pulmonary, hepatic, and renal function. In some embodiments of any of the methods described herein, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, or 2. In some embodiments of any of the methods described herein, the subject has a life expectancy of at least 12 weeks prior to the administering step.

[0018] In some embodiments of any of the methods described herein, the subject is determined to have measurable disease as assessed by imaging tests before the administering step. In some embodiments of any of the methods described herein, the subject has received prior radiation therapy at least 4 weeks before the administering step. In some embodiments of any of the methods described herein, any acute effects of any prior therapy in the subject have been reduced to baseline or to a grade of 1 or less on the NCI CTCAE v5.0 before the administering step.

[0019] In some embodiments of any of the methods described herein, the subject has an absolute neutrophil count of 1,500 or more per microliter; a platelet count of 100,000 or more per microliter; a hemoglobin level of 9 g or more per deciliter; a glomerular filtration rate (GFR) of more than 40 mL / min or a serum creatinine level of 1.5 x the upper limit of normal (ULN) or less; a total bilirubin level of 2.0 x the ULN or less, or, if the subject has Gilbert's syndrome, 3.0 x the ULN or less; and aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels of 2.5 x the ULN or less, or, if liver metastases are present, 5.0 x the ULN or less. In some embodiments of any of the methods described herein, the subject has a pulmonary function test (PFT) value of greater than 50% forced expiratory volume (FEV1) if they have symptomatic or previously known functional impairment.

[0020] In some embodiments of any of the methods described herein, the subject is a female and the female has a negative pregnancy test within 14 days prior to the administering step. In some embodiments of any of the methods described herein, the female is using contraception or is surgically sterilized for at least 14 days prior to and during the administering step.

[0021] In some embodiments of any of the methods described herein, the subject is male, and the subject uses a barrier method of contraception during and for at least 28 days after the administering step.

[0022] In some embodiments of any of the methods described herein, the subject does not have a history of clinically significant vascular disease. In some embodiments of any of the methods described herein, the subject does not have a corrected QT interval (QTc) of 470 milliseconds or greater by Fridericia's correction. In some embodiments of any of the methods described herein, the subject does not have untreated CNS metastases. In some embodiments of any of the methods described herein, the subject has not received prior treatment for CNS metastases, and the subject is neurologically stable for at least two weeks prior to the administering step. In some embodiments of any of the methods described herein, the subject does not receive a corticosteroid during the administering step. In some embodiments of any of the methods described herein, the subject receives a stable or tapering dose of a corticosteroid of 10 mg or less daily during the administering step.

[0023] In some embodiments of any of the methods described herein, the subject has not undergone surgery, radiation therapy, chemotherapy, other immunotherapy, or investigational therapy within 14 days prior to the administering step. In some embodiments of any of the methods described herein, the subject has no other prior malignancies, except for adequately treated basal cell or squamous cell skin cancer, cervical intraepithelial neoplasia, adequately treated stage I or II cancer in which the subject is currently in complete remission, or any other cancer in which the subject is disease-free for three years after surgical treatment.

[0024] In some embodiments of any of the methods described herein, the subject has no known hypersensitivity or history of allergic reaction caused by compounds of similar chemical or biological composition to the multi-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the subject has not received prior treatment with a TGF-beta antagonist or IL-15 or an analog thereof.

[0025] In some embodiments of any of the methods described herein, the subject is not receiving concurrent herbal or unconventional therapy. In some embodiments of any of the methods described herein, the subject is not receiving an autoimmune disease requiring active treatment. In some embodiments of any of the methods described herein, the subject is not receiving a condition requiring systemic corticosteroid therapy or immunosuppressive therapy within 14 days of the administering step. In some embodiments of any of the methods described herein, the subject is not receiving an active autoimmune disease and is receiving a daily inhaled or topical steroid dose of 10 mg or less of prednisone equivalent, or an adrenal replacement steroid dose.

[0026] In some embodiments of any of the methods described herein, the subject does not have an active systemic infection requiring parenteral antibiotic therapy. In some embodiments of any of the methods described herein, the subject has not previously received an organ allograft or allogeneic transplant. In some embodiments of any of the methods described herein, the subject has not been identified or diagnosed as HIV-positive or having AIDS. In some embodiments of any of the methods described herein, the subject is a female, and the female is not pregnant or breastfeeding.

[0027] In some embodiments of any of the methods described herein, the subject does not have any ongoing toxicity from prior treatment. In some embodiments of any of the methods described herein, the ongoing toxicity is greater than Grade 1 using NCI CTCAE v5.0 or greater than baseline. In some embodiments of any of the methods described herein, the ongoing toxicity excludes peripheral neuropathy, alopecia, and fatigue. In some embodiments of any of the methods described herein, the subject does not have psychosis.

[0028] In some embodiments of any of the methods described herein, the first target binding domain and the soluble tissue factor domain are directly 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 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 directly 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.

[0029] 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 directly 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.

[0030] 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 are antigen binding domains. 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 are soluble interleukin or cytokine receptors. 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: 1. In some embodiments of any of the methods described herein, the pair of affinity domains is a sushi domain derived from the alpha chain of the human IL-15 receptor (IL-15Rα) and soluble IL-15.

[0032] In some embodiments of any of the methods described herein, the first target binding domain comprises a soluble TGF-βRII. In some embodiments of any of the methods described herein, the first target binding domain comprises a first sequence at least 80% identical to SEQ ID NO:66 and a second sequence at least 80% identical to SEQ ID NO:66, wherein the first and second sequences are separated by a linker. In some embodiments of any of the methods described herein, the first target binding domain comprises a first sequence at least 90% identical to SEQ ID NO:66 and a second sequence at least 90% identical to SEQ ID NO:66. In some embodiments of any of the methods described herein, the first target binding domain comprises a first sequence of SEQ ID NO:66 and a second sequence of SEQ ID NO:66.

[0033] In some embodiments of any of the methods described herein, the linker comprises the sequence of SEQ ID NO:7.

[0034] In some embodiments of any of the methods described herein, the first target binding domain comprises a sequence that is at least 80% identical to SEQ ID NO: 69. In some embodiments of any of the methods described herein, the first target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO: 69. In some embodiments of any of the methods described herein, the first target binding domain comprises the sequence of SEQ ID NO: 69.

[0035] In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 70. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 70. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 70. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 72.

[0036] In some embodiments of any of the methods described herein, the second target binding domain comprises a soluble TGF-βRII. In some embodiments of any of the methods described herein, the second target binding domain comprises a first sequence at least 80% identical to SEQ ID NO:66 and a second sequence at least 80% identical to SEQ ID NO:66, wherein the first and second sequences are separated by a linker. In some embodiments of any of the methods described herein, the second target binding domain comprises a first sequence at least 90% identical to SEQ ID NO:66 and a second sequence at least 90% identical to SEQ ID NO:66. In some embodiments of any of the methods described herein, the second target binding domain comprises a first sequence of SEQ ID NO:66 and a second sequence of SEQ ID NO:66.

[0037] In some embodiments of any of the methods described herein, the linker comprises the sequence of SEQ ID NO:7.

[0038] In some embodiments of any of the methods described herein, the second target binding domain comprises a sequence that is at least 80% identical to SEQ ID NO: 69. In some embodiments of any of the methods described herein, the second target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO: 69. In some embodiments of any of the methods described herein, the second target binding domain comprises the sequence of SEQ ID NO: 69.

[0039] In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:74. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:70. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:74. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO:70. In some embodiments of any of the methods described herein, the second chimeric polypeptide comprises the sequence of SEQ ID NO:74. In some embodiments of any of the methods described herein, the first chimeric polypeptide comprises the sequence of SEQ ID NO:70.

[0040] In some embodiments of any of the methods described herein, the multi-chain chimeric polypeptide is administered subcutaneously to the subject. In other embodiments of any of the methods described herein, the multi-chain chimeric polypeptide is administered intravenously, intraperitoneally, intramuscularly, intratumorally, or subdermally to the subject. In some embodiments of any of the methods described herein, the subject receives a single administration of the multi-chain chimeric polypeptide. In some embodiments of any of the methods described herein, the single administration is 0.1 mg of the multi-chain chimeric polypeptide per kg of the subject's body weight (mg / kg). In some embodiments of any of the methods described herein, the single administration is 0.25 mg / kg. In some embodiments of any of the methods described herein, the single administration is 0.5 mg / kg. In some embodiments of any of the methods described herein, the single administration is 0.8 mg / kg. In some embodiments of any of the methods described herein, the single administration is 1.2 mg / kg.

[0041] In some embodiments of any of the methods described herein, the subject is administered the multi-chain chimeric polypeptide two or more times over the course of treatment. In some embodiments of any of the methods described herein, at least one of the two or more administrations is 0.1 mg of the multi-chain chimeric polypeptide per kg of subject body weight (mg / kg). In some embodiments of any of the methods described herein, at least one of the two or more administrations is 0.25 mg / kg. In some embodiments of any of the methods described herein, at least one of the two or more administrations is 0.5 mg / kg. In some embodiments of any of the methods described herein, at least one of the two or more administrations is 0.8 mg / kg. In some embodiments of any of the methods described herein, at least one of the two or more administrations is 1.2 mg / kg. In some embodiments of any of the methods described herein, the treatment period is about 4 weeks.

[0042] As used herein, the term "chimeric" refers to a polypeptide that includes amino acid sequences (e.g., domains) 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 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, at least two different domains can be synthetic sequences (e.g., two different scFvs).

[0043] 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 specificity and affinity similar to that of a naturally occurring antibody. In some aspects, the antigen-binding domain may be an antibody or fragment thereof. In some aspects, 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.

[0044] 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) to 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.

[0045] The term "soluble interleukin receptor" is used broadly herein to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) and 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 interleukin receptor can comprise 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 maintains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0046] The term "soluble cytokine receptor" is used broadly herein to refer to a polypeptide 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) and lacks a transmembrane domain (and optionally an intracellular domain). For example, a soluble cytokine receptor can comprise 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 maintains its ability to specifically bind to one or more of its natural ligands, but lacks its transmembrane domain (and optionally further lacks its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0047] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that comprises 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.

[0048] "Affinity" refers to the strength of the sum total 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 the antigen-binding domain and the antigen or epitope. The affinity of a molecule X for its partner Y is determined by the equilibrium dissociation constant (K D ) The kinetic components that contribute to the equilibrium dissociation 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 to its corresponding antigen or epitope are known in the art.

[0049] As used herein, a "multi-chain polypeptide" refers to a polypeptide (e.g., at least a first chimeric polypeptide and a second polypeptide) that comprises two or more (e.g., three, four, five, six, seven, eight, nine, or ten) protein chains, wherein the two or more protein chains associate through non-covalent bonds to form a quaternary structure.

[0050] The term "pair of affinity domains" refers to 1 × 10 -7Less 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 Less than M,) K 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.

[0051] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen-binding domain. An epitope can consist of, for example, surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former can be lost in the presence of denaturing solvents, while the binding to the latter is not lost in the presence of denaturing solvents. 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 to which an antigen-binding domain binds are known in the art.

[0052] The term "treatment" means 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, for example, reducing aberrant proliferation, gene expression, signaling, translation, and / or factor secretion. In some embodiments, treating cancer includes reducing the rate of cancer progression in a subject and / or the incidence of metastasis in a subject (e.g., compared to the rate of cancer progression and / or the incidence of metastasis in a similar subject not receiving treatment or receiving a different treatment). Generally, methods of treatment involve administering a therapeutically effective amount of a composition that reduces at least one symptom of a disorder to a subject in need of, or determined to be in need of, such treatment.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; however, other suitable methods and materials known in the art can also be used. The 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 case of conflict, the present specification, including definitions, will control.

[0054] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0055] [Figure 1] A schematic diagram of the TGFRt15-TGFRs construct is shown. [Figure 2] An additional schematic of the TGFRt15-TGFRs construct is shown. [Figure 3] 1 shows the results of inhibition of TGFβ1 by TGFRt15-TGFRs and TGFR-Fc. [Figure 4] 1 shows the results of a 32D β cell proliferation assay using TGFRt15-TGFRs or recombinant IL-15. [Figure 5] 5A and 5B show the results of detecting IL-15 and TGF RII in TGFRt15-TGFRs with the corresponding antibodies using ELISA. [Figure 6] 1 is a line graph showing the chromatographic profile of the culture supernatant of cells containing TGFRt15-TGFRs protein after binding to and elution from an anti-TF antibody resin. [Figure 7] 1 shows the analytical SEC profile of TGFRt15-TGFRs. [Figure 8]Shown are TGFRt15-TGFRs before and after deglycosylation as analyzed by reducing SDS-PAGE. [Figure 9] Figures 9A and 9B show spleen weights and percentages of immune cell types in TGFRt15-TGFRs-treated and control-treated mice. Figure 9A shows spleen weights in mice treated with TGFRt15-TGFRs compared to PBS controls. Figure 9B shows the percentages of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with TGFRt15-TGFRs compared to PBS controls. [Figure 10] Figures 10A and 10B show spleen weight and immune stimulation over 92 hours in mice treated with TGFRt15-TGFRs. Figure 10A shows the spleen weight of mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. Figure 10B shows the percentage of immune cells in mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. [Figure 11] 11A and 11B show the expression of Ki67 and Granzyme B over time in mice treated with TGFRt15-TGFRs. [Figure 12] 1 shows enhanced cytotoxicity of splenocytes by TGFRt15-TGFRs in C57BL / 6 mice. [Figure 13] 1 shows the change in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or the combination of chemotherapy and TGFRt15-TGFRs in a mouse model of pancreatic cancer. [Figure 14] 1 shows the cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFRs. [Figure 15]Figures 15A-15B show the immune stimulation results of an exemplary multi-chain polypeptide in C57BL / 6 mice. Figure 15A shows the spleen weight of mice treated with increasing doses of an exemplary multi-chain polypeptide compared to mice treated with a control solution. Figure 15B shows the percentage of immune cell types present in the spleens of mice treated with increasing doses of an exemplary multi-chain polypeptide compared to mice treated with a control solution. [Figure 16] Figures 16A-16B show the duration of immune stimulation of an exemplary multi-chain polypeptide in C57BL / 6 mice. Figure 16A shows spleen weight over 92 hours in mice treated with 3 mg / kg of an exemplary multi-chain polypeptide. Figure 16B shows the percentage of immune cell types present in the spleen over 92 hours in mice treated with 3 mg / kg of an exemplary multi-chain polypeptide. [Figure 17] Figures 17A-17B show the expression of Ki67 and granzyme B in immune cells induced by an exemplary multi-chain polypeptide. Figure 17A shows the expression of Ki67 in CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and CD19+ B cells at various time points after treatment with the multi-chain polypeptide. Figure 17B shows the expression of granzyme B in CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and CD19+ B cells at various time points after treatment with the multi-chain polypeptide. [Figure 18] 1 shows the effect of tumor inhibition by splenocytes prepared from mice treated with an exemplary multi-chain polypeptide at various time points after treatment. [Figure 19A]Figures 19A and 19B show the percentages and proliferation rates of CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and CD19+ B cells in the blood of untreated B6.129P2-ApoEtm1Unc / J mice (purchased from Jackson Laboratory) fed a control diet, a high-fat diet, and mice fed a high-fat diet and treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFR. Figure 19A shows the percentages of various cell types in each control and experimental group. Figure 19B shows the proliferation rates of various cell types in each control and experimental group. [Figure 19B] See legend to Figure 19A. [Figure 20] 20A-20E show exemplary physical appearances of mice fed either a control diet or a high-fat diet and either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFR. [Figure 21] Fasting body weights of mice fed either a control or high-fat diet and either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFR are shown. [Figure 22] 1 shows fasting blood glucose levels in mice fed either a control diet or a high-fat diet and either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFR. [Figure 23] Figures 23A-23F show chemotherapy-induced senescent B16F10 cells and the expression of senescence genes. Figure 23A shows chemotherapy induction of senescent B16F10 cells visualized using SA β-gal staining. Figures 23B-23F show the expression of p21CIP1, IL6, DPP4, RATE1E, and ULBP1 over time in chemotherapy-induced senescent B16F10 cells. [Figure 24-1]Figures 24A-24F show colony formation and expression of stem cell markers by chemotherapy-induced senescent B16F10 cells. Figure 24A shows colony formation by chemotherapy-induced senescent B16F10 cells. Figures 24B and 24C show expression of Oct4 mRNA and Notch4 mRNA by chemotherapy-induced senescent B16F10 cells compared to control B16F10 cells. Figures 24D-24F show the percentage of chemotherapy-induced senescent B16F10 cells that are double-positive for two of three stem cell markers, including CD44, CD24, and CD133. [Figure 24-2] See description of Figure 24-1. [Figure 25] Figures 25A-25C show the migration and invasion properties of chemotherapy-induced senescent B16F10 cells. Figure 25A shows the results of a migration assay comparing chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) with control B16F10 cells. Figures 25B and 25C show the results of an invasion assay comparing chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) with control B16F10 cells. [Figure 26] Figures 26A and 26B show in vitro expanded NK cells and their cytotoxicity against chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) or control B16F10 cells. Figure 26A shows an exemplary schematic diagram of the process for obtaining in vitro expanded NK cells. Figure 26B shows the cytotoxicity of expanded NK cells against chemotherapy-induced senescent cells with stem cell properties (B16F10-SNC-CSC) or control B16F10 cells. [Figure 27] Figures 27A-27C show the results of combination treatment using a mouse melanoma model. Figure 27A shows an exemplary schematic for treating melanoma in a mouse model. Figures 27B and 27C show the change in tumor volume over time with combination treatment including TGFRt15-TGFRs compared to chemotherapy or TA99 treatment alone. [Figure 28] 28A-28C are a series of graphs showing immune stimulation in C57BL / 6 mice following treatment with 2t2. [Figure 29] Figures 29A and 29B are a series of graphs showing immune stimulation in C57BL / 6 mice following treatment with TGFRt15-TGFRs. [Figure 30-1] 30A-30C are a series of graphs showing in vivo stimulation of Tregs, NK cells, and CD8+ T cells in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs or 2t2. [Figure 30-2] See description of Figure 30-1. [Figure 31] Figures 31A and 31B are a series of graphs showing induction of splenocyte proliferation by 2t2 in C57BL / 6 mice. [Figure 32] 32A-32C are a series of graphs depicting immune stimulation in C57BL / 6 mice following treatment with TGFRt15-TGFRs. [Figure 33] Figures 33A and 33B are a series of graphs showing in vivo induction of NK cell and CD8+ T cell proliferation in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs or 2t2. [Figure 34] 1 is a schematic diagram and series of graphs showing the persistence of 7t15-21 and anti-TF antibody expanded NK cells in NSG mice after treatment with 7t15-21, TGFRt15-TGFRs or 2t2. [Figure 35] Figures 35A and 35B are a series of graphs showing enhanced NK cell cytotoxicity following treatment of NK cells with TGFRt15-TGFRs. [Figure 36] Figures 36A and 36B are a series of graphs showing enhanced ADCC activity of NK cells following treatment of the NK cells with TGFRt15-TGFRs. [Figure 37] 1 is a graph of in vitro killing of senescent B16F10 melanoma cells by TGFRt15-TGFRs / 2t2 activated mouse NK cells. [Figure 38-1]Figures 38A-38H are a series of graphs showing the anti-tumor activity of TGFRt15-TGFRs plus anti-TRP1 antibody (TA99) in combination with chemotherapy in a melanoma mouse model. [Figure 38-2] See description of Figure 38-1. [Figure 38-3] See description of Figure 38-1. [Figure 38-4] See description of Figure 38-1. [Figure 39] 39A-39C are a series of graphs showing amelioration of Western diet-induced hyperglycemia in ApoE− / − mice by 2t2. [Figure 40] The upper panel shows the upregulation of CD44 memory T cells upon treatment with TGFRt15-TGFRs. The lower panel shows the upregulation of CD44 memory T cells upon treatment with 2t2. [Figure 41] 1 is a series of graphs showing immunophenotype and cell proliferation after treatment with IL-15 based agents at 3 days post-treatment. [Figure 42] Figures 42A-42C are graphs showing that TGFRt15-TGFRs treatment reduces senescence-associated gene expression in C57BL / 6 mice. These graphs show the expression of p21CIP1p21 and CD26 in lung (42A and 42B) and p21CIP1p21 in liver tissue (42C), respectively. [Figure 43] 1 is a series of graphs showing the percentage and proliferation of CD4+, CD8+, and Treg cells. [Figure 44] 1 is a series of graphs showing the percentage and proliferation of NK, CD19+, and monocytic cells. [Figure 45] Figures 45A-45C are graphs showing the assessment of senescence markers p21CIP1p21 and CD26 in lung and liver tissues. Figures 208A and 208B show the lung p21CIP1p21 (45A) and lung CD26 (45B) senescence markers. Figure 45C shows the liver p21CIP1p21 senescence marker. [Figure 46]1 is a series of graphs showing immunophenotypes from peripheral blood analysis 4 days after a single dose treatment with TGFRt15-TGFRs. [Figure 47] 1 is a series of graphs showing immunophenotypes from peripheral blood analysis 4 days after a single dose treatment with TGFRt15-TGFRs. [Figure 48] 10 is a series of graphs showing β-Gal staining analysis by FACS at day 7 after the second dose with TGFRt15-TGFRs. [Figure 49] 10 is a series of graphs showing levels of senescence markers in liver tissue determined using qPCR at day 7 after the second dose with TGFRt15-TGFRs. [Figure 50] 10 is a series of graphs showing levels of senescence markers in kidney tissue as determined using qPCR at day 7 after the second dose with TGFRt15-TGFRs. [Figure 51] 10 is a series of graphs showing levels of aging markers in skin tissue as determined using qPCR at day 7 after the second dose with TGFRt15-TGFRs. [Figure 52] 10 is a series of graphs showing levels of senescence markers in lung tissue determined using qPCR at day 7 after the second dose with TGFRt15-TGFRs. [Figure 53] 1 is a series of histological images showing β-Gal staining of kidney tissues 7 days after the second administration of TGFRt15-TGFRs. [Figure 54] FIG. 1 shows a schematic diagram of the interaction between exemplary TGFβRII / IL-15RαSu and TGFβRII / TF / IL-15Mut proteins resulting in a TGFRt15*-TGFR complex. [Figure 55] FIG. 1 shows a schematic diagram of the interaction between exemplary TGFβRII / IL-15RαSu and TGFβRII / TF / IL-15Mut proteins. [Figure 56]Figure 56A is a graph showing the binding activity of TGFRt15-TGFRs to TGF-β1 and LAP. Figure 56B is a graph showing the binding activity of TGFRII / Fc to TGF-β1 and LAP. Figure 56C is a graph showing the binding activity of TGFRt15-TGFRs to TGF-β1 and LAP. Figure 56D is a graph showing the binding activity of TGFRt15*-TGFR to TGF-β1 and LAP. Figure 56E is a graph showing the binding activity of TGFRt15-TGFRs, TGFRt15*-TGFR, and 7t15-21 to CTLL-2 cells. [Figure 57] Figure 57A is a graph of the TGF-β1 blocking activity of TGFRt15-TGFRs and TGFRt15*-TGFR. Figure 57B is a graph of the IL-15 biological activity of TGFRt15-TGFRs and TGFRt15*-TGFR. Figure 57C is a graph showing that TGF-β1, TGF-β2, and TGF-β3 each similarly inhibit IL-4-induced CTLL-2 proliferation in the absence of TGFRt15*-TGFR. Figure 57D is a graph showing that TGFRt15*-TGFR significantly reversed TGF-β1 and TGF-β3 inhibition of IL-4-induced CTLL-2 cell proliferation. [Figure 58] Figure 58A is a graph showing no significant damage to the IL-15 domain of TGFRt15-TGFRs after 10 days of incubation at 4°C, 25°C, or 37°C. Figure 58B is a graph showing no significant damage to the TGFβ-RII domain of TGFRt15-TGFRs after 10 days of incubation at 4°C, 25°C, or 37°C. Figure 58C is a graph showing the TGF-β1 neutralizing activity of TGFRt15-TGFRs after 10 days of incubation in human serum at 4°C, 25°C, or 37°C. Figure 58D is a graph showing the IL-15 activity of TGFRt15-TGFRs after 10 days of incubation in human serum at 4°C, 25°C, or 37°C. [Figure 59]Figure 59A is a graph showing cell-mediated cytotoxicity in an assay using NK cells and the indicated constructs. Figure 59B is a graph showing cell-mediated cytotoxicity in an assay using PBMCs and the indicated constructs. Figure 59C is a graph showing intracellular granzyme B production in an assay using NK cells and the indicated constructs. Figure 59D is a graph showing intracellular granzyme B production in an assay using PBMCs and the indicated constructs. Figure 59E is a graph showing interferon-gamma production in an assay using NK cells and the indicated constructs. Figure 59F is a graph showing interferon-gamma production in an assay using PBMCs and the indicated constructs. [Figure 60] 1 is a graph showing the pharmacokinetics (half-life, t1 / 2) of TGFRt15-TGFRs evaluated in female C57BL / 6 mice. [Figure 61] 1 is a graph showing the toxicity of TGFRt15-TGFRs in C57BL / 6 mice. [Figure 62] 1 is a graph showing the antitumor activity of TGFRt15-TGFRs in a C57BL / 6 mouse melanoma model. [Figure 63] The activity of TGFRt15-TGFRs in 9-week-old C57BL6 / j male mice was shown, in which the mice were given 50 μl of bleomycin (2.5 mg / kg, single dose) orally via the pharynx, and then TGFRt15-TGFRs (3 mg / kg) was given subcutaneously 17 days after bleomycin treatment. [Figure 64] 1 shows fasting plasma glucose concentrations in db / db mice 4 days after treatment with TGFRt15-TGFRs or TGFRt15*-TGFR. [Figure 65] Figures 65A-65C show the levels of TGFβ1-3 in db / db mice 4 days after treatment with TGFRt15-TGFRs or TGFRt15*-TGFR: TGFβ1 (Figure 65A), TGFβ2 (Figure 65B), and TGFβ3 (Figure 65C). [Figure 66]Figures 66A-66E show lymphocyte subsets in db / db mice 4 days after treatment with TGFRt15-TGFRs or TGFRt15*-TGFR: blood NK cells (Figure 66A), blood Ki67+ NK cells (Figure 66B), blood granzyme B+ (GzmB+) (Figure 66C), blood CD8+ (Figure 66D), and blood CD8+Ki67+ T cells (Figure 66E). [Figure 67] Figure 67A shows the interaction of TGFRt15*-TGFRs or TGFRt15-TGFRs with latent TGFβ1 (SLC) or with CD39 (control), and Figure 67B shows the interaction of TGFRt15*-TGFRs and TGFRII-Fc with latent TGFβ1. [Figure 68] 1 is a graph showing the clotting time of Innovin in a PT assay. [Figure 69] 1 is a graph showing the clotting time of TGFRt15-TGFRs in a PT assay. [Figure 70] 1 is a series of graphs showing gene expression of senescence markers PAI-1, IL-1α, IL6, and IL-1β in the kidney, comparing young mice with PBS or aged mice treated with TGFRt15-TGFRs at short-term versus long-term follow-up. [Figure 71] 1 is a series of graphs showing gene expression of senescence markers IL-1α and IL6 in the liver. [Figure 72] 1 shows the protein expression of the aging marker PAI-1 in the kidney. [Figure 73] 1 is a series of graphs showing that IL15SA (positive control) or TGFRt15*-TGFR+IL15SA mediated an increase in the percentage of CD3+CD8+, CD3-NK1.1+, and CD3+CD45+ immune cells in the blood, while treatment with TGFRt15*-TGFR had little or no effect on the percentage of these cell populations. [Figure 74]1 is a series of graphs showing that IL15SA (positive control) or TGFRt15*-TGFR+IL15SA mediated an increase in the percentage of CD3+CD8+, CD3-NK1.1+, and CD3+CD45+ immune cells in the spleen, while treatment with TGFRt15*-TGFR had little or no effect on the percentage of these cell populations. [Figure 75A] 1 shows gene expression of the senescence marker p21 in kidney and liver tissues after treatment with the test substance. [Figure 75B] 1 shows gene expression of the senescence marker PAI1 in kidney and liver tissues after test treatments. [Figure 75C] 1 shows gene expression of the senescence marker IL-1α in kidney and liver tissues after test treatments. [Figure 75D] 1 shows gene expression of the senescence marker IL-6 in kidney and liver tissues after test treatments. [Figure 76A] The percentages and proliferation of CD4+, CD8+, and Treg cells after treatment with the indicated agents are shown. [Figure 76B] The percentage and proliferation of NK, CD19+, and monocytic cells after treatment with the indicated agents are shown. [Figure 77] Figure 77A shows the evaluation of gene expression of the senescence marker p21 in lung tissue of mice after chemotherapy and treatment with the indicated agents. Figure 77B shows the evaluation of gene expression of the senescence marker CD26 in lung tissue of mice after chemotherapy and treatment with the indicated agents. Figure 77C shows the evaluation of gene expression of the senescence marker p21 in kidney tissue of mice after chemotherapy and treatment with the indicated agents. [Figure 78A] Figures 78A and 78B are graphs showing that TGFRt15-TGFRs treatment enhances immune cell proliferation, expansion and activation in the peripheral blood of B16F10 tumor-bearing mice. [Figure 78B] See legend to Figure 78A. [Figure 79]1 is a series of graphs showing that TGFRt15-TGFRs treatment reduces levels of TGFβ in the plasma of B16F10 tumor-bearing mice. [Figure 80] 1 is a series of graphs showing that TGFRt15-TGFRs treatment reduces the levels of pro-inflammatory cytokines in the plasma of B16F10 tumor-bearing mice. [Figure 81] 1 shows that TGFRt15-TGFRs treatment enhances NK and CD8 expansion in the spleens of B16F10 tumor-bearing mice. [Figure 82A] Figures 82A and 82B show that treatment with TGFRt15-TGFRs enhances glycolytic activity of splenocytes in B16F10 tumor-bearing mice. [Figure 82B] See legend to Figure 82A. [Figure 83A] Figures 83A and 83B show that treatment with TGFRt15-TGFRs enhances mitochondrial respiration of splenocytes in B16F10 tumor-bearing mice. [Figure 83B] See legend to Figure 83A. [Figure 84A] Figures 84A and 84B show that treatment with TGFRt15-TGFRs enhances NK and CD8 immune cell infiltration (TIL) into tumors in B16F10 tumor-bearing mice. [Figure 84B] See legend to Figure 84A. [Figure 85] Histopathological analysis of tumors after TGFRt15-TGFRs treatment shows that tumors after TGFRt15-TGFRs + TA99 antibody treatment showed little mitotic or necrotic activity. The mitotic index correlates with dividing cells, and the presence of necrosis is a measure of more aggressive features and a poor prognosis. [Figure 86] Figure 10 is a graph showing anti-PD-L1 antibody in combination with TGFRt15-TGFRs+TA99 antibody and chemotherapy in a B16F10 melanoma mouse model. [Figure 87]1 is a graph showing that the anti-tumor effect of TGFRt15-TGFRs in a B16F10 melanoma mouse model is dependent on NK and CD8 T cells. [Figure 88A] Figures 88A and 88B are graphs showing gene expression of senescence markers p21, IL-1α and IL6 in liver and lung tissues of tumor-bearing mice after chemotherapy. [Figure 88B] See legend to Figure 88A. [Figure 89] 1 is a graph showing induction of gene expression of senescence markers p21, IL6, H2AX, and NK cell ligands, Rae1e and ULBP1, by docetaxel treatment of B16F10 GFP cells. [Figure 90] Tumor-infiltrating lymphocytes per day 4 days after treatment in tumor-bearing mice are shown. [Figure 91A] Figures 91A and 91B show flow cytometry analysis suggesting that mice treated with immunotherapy exhibited lower numbers of GFP-positive senescent tumor cells 4 and 10 days after treatment compared to the PBS control group (Figure 91A), and tumor cells plated in 24-well plates were assessed by fluorescence microscopy (Figure 91B). [Figure 91B] See legend to Figure 91A. [Figure 92] 1 shows the levels of TGFβ in the kidneys of mice after cisplatin-induced renal injury and treatment with TGFRt15-TGFRs. [Figure 93] Figures 93A-93C show the toxic effects of repeated subcutaneous administration of TGFRt15-TGFRs in C57BL / 6 mice. Body weight changes are shown through SD21 (Figure 93A). Spleen weight (Figure 93B) and blood counts and differentials (Figure 93C) are shown for mice on SD7 after one dose of TGFRt15-TGFRs and on SD21 after two doses. [Figure 94] 1 shows plasma levels of TGF-β isoforms in mice after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg / kg) or TGFRt15*-TGFR (3 mg / kg). [Figure 95] Figures 95A and 95B show the changes in the rate of glycolytic capacity (ECAR) (Figure 95A) and the rate of mitochondrial respiratory capacity (OCR) (Figure 95B) in mouse splenocytes after in vivo treatment with PBS, TGFRt15-TGFRs, TGFRt15*-TGFR, or IL15SA. [Figure 96] Figures 96A and 96B show the changes in the rate of glycolytic capacity (ECAR) (Figure 96A) and the rate of mitochondrial respiratory capacity (OCR) (Figure 96B) in mouse splenocytes after in vitro treatment with PBS, TGFRt15-TGFRs, or TGFRt15*-TGFR. [Figure 97-1] Figures 97A-97E show changes in tumor growth and survival of B16F10 melanoma tumors in C57BL / 6 mice after in vitro treatment with PBS, TGFRt15-TGFRs, or TGFRt15*-TGFR. Tumor volume (Figure 97A) and mouse survival (based on tumor volumes of <4000 mm3) (Figure 97B) were assessed. As shown in Figure 97A, mice were intraperitoneally treated with anti-CD8, anti-NK, or anti-CD8 and anti-NK Abs for 1 week to deplete immune cells prior to injection with tumor cells of B16F10 melanoma tumors. Tumor-bearing mice were then treated with PBS or 20 mg / kg TGFRt15-TGFRs on days 1 and 4 after tumor cell inoculation. Animal tumor volume (Figure 97C) and mouse survival (Figure 97D) were assessed. B16F10 tumor-bearing mice were treated with PBS or 20 mg / kg TGFRt15-TGFRs on days 1 and 7 after tumor inoculation (Figure 97E). Tumors were harvested on day 11 after tumor inoculation, and tumor-infiltrating NK1.1+ cells and CD8+ T cells were quantified by flow cytometry. [Figure 97-2] See description of Figure 97-1. [Figure 98-1] FIG. 98A shows the fold change in the level of gene expression in the pancreas of db / db mice receiving TGFRt15-TGFRs compared to PBS controls. [Figure 98-2]Figures 98B-98D show the mean fold change in pancreatic expression levels for SASP, aging, and beta cell index genes, respectively, for db / db mice receiving TGFRt15-TGFRs compared to PBS controls. [Figure 99-1] Figures 99A and 99B show multispectral imaging of pancreatic tissue sections from db / db mice treated with PBS (control) (Figure 99A) or TGFRt15-TGFRs (Figure 99B). Representative islet cells are shown, with insulin+ islet beta cells (such as OPAL-520) and insulin+ p21+ beta cells (such as OPAL-570) (seen as white cells in the grayscale image) reduced in the TGFRt15-TGFRs-treated group (Figure 99B) compared to the PBS-treated group (Figure 99A). [Figure 99-2] Figures 99C and 99D show the levels of islet insulin+ cells (Figure 99C) and islet insulin+p21+ cells (Figure 99D) in pancreatic tissue sections from db / db mice treated with PBS (control) or TGFRt15-TGFRs. [Figure 100] 100A-100C show the effect of treatment on the percentages of blood immune cell subsets in db / db mice receiving PBS (control) or TGFRt15-TGFRs. [Figure 101] 1 shows the percentage of Ki67-positive immune cells induced in the blood after subcutaneous treatment of cynomolgus monkeys with TGFRt15-TGFRs compared to PBS (vehicle). [Figure 102] Figure 1 shows the extracellular acidification rate (ECAR), which indicates the glycolytic function of splenocytes isolated from young (6-week-old) and aged (72-week-old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg / kg), or TGFRt15*-TGFR (3 mg / kg). [Figure 103] Oxygen consumption rate (OCR), representing mitochondrial respiration, of splenocytes isolated from young (6-week-old) and aged (72-week-old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg / kg), or TGFRt15*-TGFR (3 mg / kg) is shown. [Figure 104] Shown are the percentages of immune cell subsets in the blood of young (6-week-old) and aged (72-week-old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs (3 mg / kg), or TGFRt15*-TGFR (3 mg / kg). [Figure 105] Shown are the percentages of immune cell subsets in the spleens of young (6-week-old) and aged (72-week-old) mice 4 days after in vivo treatment with PBS, TGFRt15-TGFRs, or TGFRt15*-TGFR. [Figure 106-1] FIG. 106 shows gene expression levels of IL1-α, IL1-β, IL-6, p21, and PAI-1 in the liver of aged mice after one or two doses of TGFRt15-TGFRs treatment. [Figure 106-2] See description of Figure 106-1. [Figure 107] 1 shows inflammation scores of liver tissue from aged mice after one or two doses of TGFRt15-TGFRs treatment. [Figure 108] 1 shows the expression levels of IL1-α, IL1-β, IL-6, IL-8, TGF-β, PAI-1, collagen, and fibronectin proteins in the liver of aged mice after one or two doses of TGFRt15-TGFRs treatment. [Figure 109] 1 shows the levels of β-galactosidase in liver tissue of aged mice 4 days after in vivo treatment with PBS or TGFRt15-TGFRs. [Figure 110] 1 shows survival curves of 72-week-old C57BL / 6 mice after a single subcutaneous treatment with PBS or TGFRt15-TGFRs (3 mg / kg). [Figure 111] 1 shows protein levels of SASP factors in the liver of B16F10 tumor-bearing mice after chemotherapy and TGFRt15-TGFRs+TA99 therapy. [Figure 112]Figures 112A and 112B show the effect of CD8+ T cell (dpCD8) and NK cell (dpNK) antibody depletion on the levels of TIS B16F10-GFP cells (Figure 112A) and NK and CD8+ T cell levels (Figure 112B) in tumors in mice after chemotherapy and TGFRt15-TGFRs+TA99 therapy. [Figure 113-1] Figures 113A-113E show the anti-tumor activity and mechanism of action of TGFRt15-TGFRs+TA99 in combination with immune checkpoint inhibitors in B16F10 tumor-bearing mice. Figure 113A shows an exemplary schematic for treating B16F10 melanoma in a mouse model. Figure 113B shows the change in tumor volume over time and at day 18 after combination treatment including TGFRt15-TGFRs+TA99+anti-PD-L1 antibody followed by docetaxel, compared with PBS or chemotherapy treatment alone. Figures 113C and 113D show the effect of treatment on the percentage of tumor-infiltrating CD28+CD8+ T cells and splenic IFNγ+CD8+ T cells at day 18. Figure 113E shows the effect of treatment on the level of NKG2D (MFI) of tumor-infiltrating CD8+ and CD8+CD44hi T cells at day 18. [Figure 113-2] See description of Figure 113-1. [Figure 113-3] See description of Figure 113-1. [Figure 114-1] Figures 114A-114D show the changes in tumor growth and survival of SW1990 human pancreatic tumors in C57BL / 6 scid mice after in vitro treatment with PBS, gemcitabine and nab-paclitaxel chemotherapy, TGFRt15-TGFRs, or TGFRt15-TGFRs plus chemotherapy. Figure 114A shows an exemplary schematic for treating SW1990 human pancreatic tumors in a xenograft mouse model. Figures 114B and 114C show the changes in tumor volume over time and at day 38, respectively, after combination treatment including TGFRt15-TGFRs plus chemotherapy compared with PBS or chemotherapy treatment alone. Figure 114D shows the effect of treatment on survival of mice bearing SW1990 human pancreatic tumors. [Figure 114-2] See description of Figure 114-1. [Figure 115-1] Figures 115A-115C are a series of graphs showing levels of gene expression of senescence markers (IL-1α, IL-6, and PAI-1, respectively) in tissues of aged mice after treatment with PBS, TGFRt15-TGFRs, 2t2, one dose of TGFRt15-TGFRs on day 0 with a second dose of 2t2 on day 60, or one dose of 2t2 on day 0 with a second dose of TGFRt15-TGFRs on day 60. [Figure 115-2] See description of Figure 115-1. [Figure 116] FIG. 1 is an exemplary schematic diagram of an experimental design using a melanoma mouse model. [Figure 117A] 117A-117H are graphs showing the effect of administration of TGFRt15-TGFRs on the proliferation, expansion, and activation of NK / T cells in the blood of a melanoma mouse model. [Figure 117B] See legend to Figure 117A. [Figure 117C] See legend to Figure 117A. [Figure 117D] See legend to Figure 117A. [Figure 117E] See legend to Figure 117A. [Figure 117F] See legend to Figure 117A. [Figure 117G] See legend to Figure 117A. [Figure 117H] See legend to Figure 117A. [Figure 118A] 118A-118C are graphs showing the effect of TGFRt15-TGFRs treatment on plasma TGF-β1, TGF-β2, and TGF-β3 levels in a melanoma mouse model. [Figure 118B] See legend to Figure 118A. [Figure 118C] See legend to Figure 118A. [Figure 119A]119A-119E are graphs showing the effect of treatment with dexamethasone or a combination of TGFRt15-TGFRs and dexamethasone on plasma levels of IL-2, IL-1β, IL-6, and GM-CSF in a melanoma mouse model. [Figure 119B] See legend to Figure 119A. [Figure 119C] See legend to Figure 119A. [Figure 119D] See legend to Figure 119A. [Figure 119E] See legend to Figure 119A. [Figure 120] Figures 120A and 120B are graphs showing the effect of treatment with dexamethasone or a combination of TGFRt15-TGFRs and dexamethasone on NK cells and CD8+ T cells in the spleen of a melanoma mouse model. [Figure 121A] 121A-121C are a series of graphs showing the effect of treatment with saline (black line), dexamethasone (dark gray line), or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 (light gray line) on the glycolytic activity of splenocytes. [Figure 121B] See legend to Figure 121A. [Figure 121C] See legend to Figure 121A. [Figure 122-1] Figures 122A-122L are a series of graphs showing the effect of treatment with saline, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on glycolytic activity (glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification) of splenocytes from a melanoma mouse model. [Figure 122-2] See description of Figure 122-1. [Figure 122-3] See description of Figure 122-1. [Figure 122-4] See description of Figure 122-1. [Figure 122-5] See description of Figure 122-1. [Figure 122-6] See description of Figure 122-1. [Figure 123A] 123A-123C are a series of graphs showing the effect of treatment with PBS, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on mitochondrial respiration of splenocytes from a melanoma mouse model. [Figure 123B] See legend to Figure 123A. [Figure 123C] See legend to Figure 123A. [Figure 124-1] Figures 124A-124L are a series of graphs showing the effect of treatment with PBS, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on mitochondrial respiration (basal respiration, maximal respiration, spare respiratory capacity, and ATP production) of splenocytes from a melanoma mouse model. [Figure 124-2] See description of Figure 124-1. [Figure 124-3] See description of Figure 124-1. [Figure 124-4] See description of Figure 124-1. [Figure 124-5] See description of Figure 124-1. [Figure 124-6] See description of Figure 124-1. [Figure 125-1] Figures 125A-125H are a series of graphs showing the effect of treatment with PBS, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on the infiltration of NK / Ki67 cells, CD8 / Ki67 cells, NK cells, CD8 cells, NK / CD25 cells, NK / granzyme B cells, CD8 / CD25 cells, and CD8 / granzyme B cells into melanoma tumors in a melanoma mouse model. [Figure 125-2] See description of Figure 125-1. [Figure 125-3] See description of Figure 125-1. [Figure 125-4] See description of Figure 125-1. [Figure 126-1]FIG. 126A is a schematic diagram of the experimental design for therapy-induced senescence in B16F10 tumors in a melanoma mouse model. [Figure 126-2] Figures 126B-126E are a series of graphs showing the effect of DTX treatment on senescence-associated gene expression (DPP4, IL-6, p16, and p21, respectively) in B16F10 tumor cells in mice. [Figure 126-3] See description of Figure 126-2. [Figure 127-1] FIG. 127A is a schematic diagram of the experimental design for therapy-induced senescence in B16F10 tumors in a melanoma mouse model. [Figure 127-2] Figures 127B and 127C are a series of graphs showing the effect of treatment with saline, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on the expression of p21 and IL-6, respectively, in a B16F10 melanoma tumor model. [Figure 128-1] Figures 128A-128D are a series of graphs showing the levels of protein expression of senescence markers (PAI1, IL-1α, CXCL1, and IL-2, respectively) in the plasma of aged mice after treatment with PBS, TGFRt15-TGFRs, 2t2, one dose of TGFRt15-TGFRs on day 0 with a second dose of 2t2 on day 60, or one dose of 2t2 on day 0 with a second dose of TGFRt15-TGFRs on day 60. [Figure 128-2] See description of Figure 128-1. [Figure 129] RNA-seq analysis of differentially expressed genes between PBS (control group) and TGFRt15-TGFRs (TGFRt15-TGFRs group) in the liver of db / db mice. [Figure 130] 1 shows RNA-seq analysis of differentially expressed genes between PBS (control group) and TGFRt15-TGFRs (TGFRt15-TGFRs group) in the livers of aging mice. DETAILED DESCRIPTION OF THE INVENTION

[0056] Detailed Description Provided herein is a method for treating unresectable, advanced / metastatic pancreatic cancer in a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII.

[0057] Also provided herein are methods for improving objective response rates in subjects with unresectable advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides described herein.

[0058] Also provided herein are methods for increasing progression-free survival or progression-free survival rate in a subject or population of subjects with unresectable, advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides described herein.

[0059] Also provided herein are methods for increasing the progression-free interval in a subject or population of subjects with unresectable, advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides described herein.

[0060] Also provided herein are methods for increasing the duration of response in a subject or population of subjects with unresectable advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides described herein.

[0061] Also provided herein are methods for increasing overall survival in a population of subjects with unresectable, advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of any of the multi-chain chimeric polypeptides described herein.

[0062] In some examples of any of the multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide is each independently between about 50 amino acids and about 3000 amino acids, between about 50 amino acids and about 2500 amino acids, between about 50 amino acids and about 2000 amino acids, between about 50 amino acids and about 1500 amino acids, between about 50 amino acids and about 1000 amino acids, between about 50 amino acids and about 800 amino acids, between about 50 amino acids and about 600 amino acids, between about 50 amino acids and about 50 0 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 250 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, About 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 450 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 350 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 250 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 150 amino acids, about 150 amino acids to about 3000 amino acids, about 150 amino acids to about 2500 amino acids, about 150 amino acids to about 2000 amino acids, about 150 amino acids to about 1500 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids,About 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 250 amino acids, about 150 amino acids to about 200 amino acids, about 200 amino acids to about 3000 amino acids, about 200 amino acids to about 2500 amino acids, about 200 amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 600 amino acids , about 200 amino acids to about 500 amino acids, about 200 amino acids to about 450 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 350 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 250 amino acids, about 250 amino acids to about 3000 amino acids, about 250 amino acids to about 2500 amino acids, about 250 amino acids to about 2000 amino acids, about 250 amino acids to about 1500 amino acids, about 250 amino acids to about 1000 amino acids , about 250 amino acids to about 800 amino acids, about 250 amino acids to about 600 amino acids, about 250 amino acids to about 500 amino acids, about 250 amino acids to about 450 amino acids, about 250 amino acids to about 400 amino acids, about 250 amino acids to about 350 amino acids, about 250 amino acids to about 300 amino acids, about 300 amino acids to about 3000 amino acids, about 300 amino acids to about 2500 amino acids, about 300 amino acids to about 2000 amino acids, about 300 amino acids to about 1500 amino acids, About 300 amino acids to about 1000 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 450 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 350 amino acids, about 350 amino acids to about 3000 amino acids, about 350 amino acids to about 2500 amino acids, about 350 amino acids to about 2000 amino acids, about 350 amino acids to about 1500 amino acids,About 350 amino acids to about 1000 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 600 amino acids, about 350 amino acids to about 500 amino acids, about 350 amino acids to about 450 amino acids, about 350 amino acids to about 400 amino acids, about 400 amino acids to about 3000 amino acids, about 400 amino acids to about 2500 amino acids, about 400 amino acids to about 2000 amino acids, about 400 amino acids to about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 4 00 amino acids to about 800 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 450 amino acids, about 450 amino acids to about 3000 amino acids, about 450 amino acids to about 2500 amino acids, about 450 amino acids to about 2000 amino acids, about 450 amino acids to about 1500 amino acids, about 450 amino acids to about 1000 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 500 amino acids, about 500 amino acids to about 3000 amino acids, about 500 amino acids to about 2500 amino acids, about 500 amino acids to about 2000 amino acids, about 500 amino acids to about 1500 amino acids, about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 600 amino acids, about 600 amino acids to about 3000 amino acids, about 600 amino acids to about 2500 amino acids, about 600 amino acids to about 2000 amino acids, about 600 to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 800 amino acids, about 800 amino acids to about 3000 amino acids, about 800 amino acids to about 2500 amino acids, about 800 amino acids to about 2000 amino acids, about 800 amino acids to about 1500 amino acids, about 800 amino acids to about 1000 amino acids, about 1000 amino acids to about 3000 amino acids, about 1000 amino acids to about 2500 amino acids, about 1000 amino acids to about 2000 amino acids,It may be about 1000 to about 1500 amino acids, about 1500 to about 3000 amino acids, about 1500 to about 2500 amino acids, about 1500 to about 2000 amino acids, about 2000 to about 3000 amino acids, about 2000 to about 2500 amino acids, or about 2500 to about 3000 amino acids.

[0063] 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 in 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) in the first chimeric polypeptide.

[0064] 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 pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein) are immediately adjacent to each other in 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 pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein) in the first chimeric polypeptide.

[0065] 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 the exemplary pair of affinity domains described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are directly 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 the exemplary pair 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.

[0066] Non-limiting aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are described below and can be used in any combination without limitation. Additional aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are known in the art.

[0067] 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 Identification Number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258 and one S-palmitoylation site at Cys245. No deletions or mutations in the cytoplasmic domain that affect tissue factor clotting activity have been identified. Tissue factor has one S-palmitoylation site in the intracellular domain of the protein at Cys245, which is located at the amino acid terminal of the intracellular domain and near the membrane surface. The transmembrane domain of tissue factor consists of a single-spanning α-helix.

[0068] The extracellular domain of tissue factor, consisting of two fibronectin type III domains, is connected to the transmembrane domain through a six-amino acid linker. This linker provides conformational flexibility, separating the extracellular domain of tissue factor from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping β-sheets, with an upper sheet domain containing three antiparallel β-strands and a lower sheet domain containing four β-strands. The β-strands are connected by β-loops between βA and βB, between βC and βD, and between βE and βF, all of which have conserved conformations in the two modules. There are three short α-helical segments connecting the β-strands. A unique feature of tissue factor is a 17-amino acid β-hairpin between the β10 and β11 strands, 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 that is absent in the C-terminal domain and is unique to tissue factor. Such fibronectin type III domain structures are characteristic of the immunoglobulin-like family of protein folds and are conserved among a wide variety of extracellular proteins.

[0069] Zymogen FVII is rapidly converted to FVIIa by limited proteolysis upon binding to tissues and forming 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 in the tissue factor-FVIIa complex significantly increases the enzymatic activity of FVIIa: the rate of hydrolysis of small chromogenic peptidyl substrates increases approximately 20- to 100-fold, and the rate of activation of the natural polymer substrates FIX and FX increases by nearly one million-fold. The formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface), in cooperation with the allosteric activation of the FVIIa activation site upon binding to tissue factor, significantly increases the rate of FIX or FX activation. 2+The nearly million-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complexes compared with free FVIIa is a key regulatory point for the coagulation cascade.

[0070] FVII is a single polypeptide chain of approximately 50 kDa consisting of 406 amino acid residues, which contains an N-terminal gamma-carboxyglutamic acid-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EGF2), and a C-terminal serine protease domain. FVII is a single polypeptide chain consisting of approximately 50 kDa, consisting of an Ile-Ile residue in the short linker region between the EGF2 and protease domains. 154 -Arg 152 It is activated to FVIIa by specific proteolytic cleavage at the Cys 135 and Cys 262 FVIIa binds to Ca through its N-terminal GLA domain. 2+ Immediately adjacent to the C-terminus to the GLA domain is an aromatic stack and two EGF domains. The aromatic stack binds GLA to a single Ca 2+ This connects to the EGF1 domain, which is bound to the Ca ion. 2+ Occupation of the binding site increases the amidolytic activity of FVIIa and the association of tissue factor. 193 , Asp 242 , and Ser 344 The catalytic triad and a single Ca in the FVIIa protease domain 2+ Ion binding is important for its catalytic activity. Proteolytic activation of FVII to FVIIa occurs via Ile 153 The newly formed amino terminus is then released and folded back into the activation pocket, where it is inserted into the Asp 343A salt bridge forms with the carboxylic acid of FVIIa, generating an oxyanion hole. This salt bridge formation is important 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.

[0071] Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is important for FVIIa to target substrates. Free FVIIa adopts a stable, extended conformation when bound to a membrane, with its active site positioned approximately 80 Å above the membrane surface. Upon binding of FVIIa to a tissue molecule, 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 on the membrane, demonstrating that tissue factor can fully support FVIIa active site positioning even in the absence of FVIIa-membrane interactions. Additional data showed that tissue factor supported full FVIIa proteolytic activity as long as the extracellular domain of tissue factor was somehow tethered to the membrane surface. However, elevating the FVIIa active site 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.

[0072] Alanine-scanning mutagenesis has been used to evaluate the role of specific amino acid side chains in the extracellular domain of tissue factor 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 replacement reduced the affinity for FVIIa binding by 5-10 fold. Most of the side chains of these residues were found to be well exposed to solvent in the crystal structure, consistent with a macromolecular ligand interaction. The FVIIa ligand-binding site is located over an extensive region at the interface between the two molecules. 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 It provides continuity for the main 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 extending to the concave side of the intermodular angle with the more distant base Val 207 The binding region extends from Asp58 to Lys 48 , Lys 46 , Gln 37 , Asp 44 , and Trp 45 It extends to the concave region formed by Trp 45 and Asp 44 does not interact independently with FVIIa, and Trp 45 The effect of mutation at position Asp 44 and Gln 37The interaction region is located between two surface-exposed aromatic residues, Phe and Phe, which may reflect the structural importance of this side chain for local packing adjacent to the side chain. 76 and Tyr 78 which form part of the hydrophobic cluster in the N-module.

[0073] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, which are specific proteinase-activated receptors. Mutational analysis has identified a number of residues that, when mutated, support full FVIIa amidolytic activity toward 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 loop region of tissue factor at residues 159-165, and residues within or adjacent to this flexible loop, have been shown to be important for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate-binding exosite region of tissue factor that is quite far from the FVIIa active site. Substitution of only slightly bulky glycine residues with alanine significantly impairs tissue factor-FVIIa proteolytic activity. This suggests that the flexibility conferred by glycine is important for the loop at residues 159-165 for recognition of tissue factor macromolecular substrates.

[0074] Residue Lys 165 and Lys 166 Lys has also been demonstrated to be important for substrate recognition and binding. Mutation of either of these residues to alanine results in a significant decrease in tissue factor cofactor function. 165 and Lys 166and have their backs to each other, Lys 165 points towards FVIIa in most tissue factor-FVIOIa structures, and Lys 166 The Lys of FVIIa is oriented toward the substrate-binding exosite region in the crystal structure. 165 and Gla 35 The putative salt bridge formation between tissue factor and the GLA domain of FVIIa would support the view 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 domain, a possible adjacent EGF1 domain, of FIX and FX, and that the presence of the GLA domain of FVIIa may regulate these interactions either directly or indirectly.

[0075] Soluble tissue factor domain In some aspects 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 variant in which the wild-type tissue factor polypeptide lacks the signal sequence, transmembrane domain, and intracellular domain and is further modified at 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, mouse soluble tissue factor domains, rat soluble tissue factor domains, and mutant soluble tissue factor domains are provided below.

[0076] Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO:1) TIFF2025508939000002.tif28170

[0077] Exemplary nucleic acids encoding soluble human tissue factor domains (SEQ ID NO:2) TIFF2025508939000003.tif86170

[0078] Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO:3) TIFF2025508939000004.tif35170

[0079] Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO:4) TIFF2025508939000005.tif33170

[0080] Exemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO:5) TIFF2025508939000006.tif30170

[0081] Exemplary Soluble Rat Tissue Factor Domain (SEQ ID NO:6) TIFF2025508939000007.tif28170

[0082] 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:1, 3, 4, 5, or 6. In some embodiments, soluble tissue factor can comprise the sequence of SEQ ID NO:1, 3, 4, 5, or 6, 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) 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) removed from its C-terminus.

[0083] As appreciated in the art, one skilled 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 the 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 the protein.

[0084] 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. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the human soluble tissue factor domain does not initiate blood clotting.

[0085] In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some aspects, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some aspects, the soluble tissue factor domain can be a soluble rat tissue factor domain.

[0086] In some examples, the soluble tissue factor domain does not include one or more (e.g., two, three, four, five, six, or seven) 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 possesses the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0087] In some examples, the soluble tissue factor domain can be encoded by a nucleic acid comprising 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:2.

[0088] 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 160 amino acids, from about 20 amino acids to about 1 40 amino acids, about 20 to about 120 amino acids, about 20 to about 100 amino acids, about 20 to about 80 amino acids, about 20 to about 60 amino acids, about 20 to about 40 amino acids, about 40 to about 220 amino acids, about 40 to about 215 amino acids, about 40 to about 210 amino acids, about 40 to about 205 amino acids, about 40 to about 200 amino acids, about 40 to about 195 amino acids, about 40 amino acids up to about 190 amino acids, about 40 amino acids to about 185 amino acids, about 40 amino acids to about 180 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 215 amino acids, about 60 amino acids to about 210 amino acids, about 60 Amino acids up to about 205 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 195 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids,About 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 100 amino acids amino acids, about 100 to about 220 amino acids, about 100 to about 215 amino acids, about 100 to about 210 amino acids, about 100 to about 205 amino acids, about 100 to about 200 amino acids, about 100 to about 195 amino acids, about 100 to about 190 amino acids, about 100 to about 185 amino acids, about 100 to about 180 amino acids, about 100 to about 160 amino acids, about 100 to about 140 amino acids, about 100 Amino acids up to about 120 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 215 amino acids, about 120 amino acids to about 210 amino acids, about 120 amino acids to about 205 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 195 amino acids, about 120 amino acids to about 190 amino acids, about 120 amino acids to about 185 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 14 0 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 215 amino acids, about 140 amino acids to about 210 amino acids, about 140 amino acids to about 205 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 160 amino acids, about 160 amino acids to about 220 amino acids,About 160 amino acids to about 215 amino acids, about 160 amino acids to about 210 amino acids, about 160 amino acids to about 205 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 195 amino acids, about 160 amino acids to about 190 amino acids, about 160 amino acids to about 185 amino acids, about 160 amino acids to about 180 amino acids, about 180 amino acids to about 220 amino acids, about 180 amino acids to about 215 amino acids, about 180 amino acids to about 210 amino acids, about 1 80 amino acids to about 205 amino acids, about 180 amino acids to about 200 amino acids, about 180 amino acids to about 195 amino acids, about 180 amino acids to about 190 amino acids, about 180 amino acids to about 185 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 215 amino acids, about 185 amino acids to about 210 amino acids, about 185 amino acids to about 205 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 from about 190 amino acids, from about 190 amino acids to about 220 amino acids, from about 190 amino acids to about 215 amino acids, from about 190 amino acids to about 210 amino acids, from about 190 amino acids to about 205 amino acids, from about 190 amino acids to about 200 amino acids, from about 190 amino acids to about 195 amino acids, from about 195 amino acids to about 220 amino acids, from about 195 amino acids to about 215 amino acids, from about 195 amino acids to about 210 amino acids, from about 195 amino acids to about 205 amino acids, The amino acid sequence may have a total length of about 200 to about 200 amino acids, about 200 to about 220 amino acids, about 200 to about 215 amino acids, about 200 to about 210 amino acids, about 200 to about 205 amino acids, about 205 to about 220 amino acids, about 205 to about 215 amino acids, about 205 to about 210 amino acids, about 210 to about 220 amino acids, about 210 to about 215 amino acids, or about 215 to about 220 amino acids.

[0089] 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; Priyanka et al., Protein Sci. 22(2):153-167, 2013. In some examples, the linker sequence is a synthetic linker sequence.

[0090] In some aspects 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 aspects 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).

[0091] In some embodiments, the linker sequence is 1 amino acid to about 100 amino acids, 1 amino acid to about 90 amino acids, 1 amino acid to about 80 amino acids, 1 amino acid to about 70 amino acids, 1 amino acid to about 60 amino acids, 1 amino acid to about 50 amino acids, 1 amino acid to about 40 amino acids, 1 amino acid to about 30 amino acids, 1 amino acid to about 25 amino acids, 1 amino acid to about 20 amino acids, 1 amino acid to about 15 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 8 amino acids, amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 4 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 15 amino acids amino acids, about 2 to about 10 amino acids, about 2 to about 8 amino acids, about 2 to about 6 amino acids, about 2 to about 4 amino acids, about 4 to about 100 amino acids, about 4 to about 90 amino acids, about 4 to about 80 amino acids, about 4 to about 70 amino acids, about 4 to about 60 amino acids, about 4 to about 50 amino acids, about 4 to about 40 amino acids, about 4 to about 30 amino acids, about 4 to about 25 amino acids , about 4 amino acids to about 20 amino acids, about 4 amino acids to about 15 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 90 amino acids, about 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 30 amino acids,About 6 amino acids to about 25 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 15 amino acids, about 6 amino acids to about 10 amino acids, about 6 amino acids to about 8 amino acids, about 8 amino acids to about 100 amino acids, about 8 amino acids to about 90 amino acids, about 8 amino acids to about 80 amino acids, about 8 amino acids to about 70 amino acids, about 8 amino acids to about 60 amino acids, about 8 amino acids to about 50 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 30 amino acids , about 8 amino acids to about 25 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 15 amino acids, about 8 amino acids to about 10 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids up to about 25 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 15 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 70 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 30 amino acids, about 15 amino acids to about 25 amino acids, about 15 amino acids to about 20 Amino acids, about 20 to about 100 amino acids, about 20 to about 90 amino acids, about 20 to about 80 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, about 20 to about 30 amino acids, about 20 to about 25 amino acids, about 25 to about 100 amino acids, about 25 to about 90 amino acids, about 25 to about 80 amino acids,About 25 to about 70 amino acids, about 25 to about 60 amino acids, about 25 to about 50 amino acids, about 25 to about 40 amino acids, about 25 to about 30 amino acids, about 30 to about 100 amino acids, about 30 to about 90 amino acids, about 30 to about 80 amino acids, about 30 to about 70 amino acids, about 30 to about 60 amino acids, about 30 to about 50 amino acids, about 30 to about 40 amino acids, about 40 to about 100 amino acids, about 40 to about 90 amino acids, about 40 to about 80 amino acids, about 40 to about 70 amino acids, about 40 to about 60 amino acids The amino acid sequence may be about 40 to about 50 amino acids, about 50 to about 100 amino acids, about 50 to about 90 amino acids, about 50 to about 80 amino acids, about 50 to about 70 amino acids, about 50 to about 60 amino acids, about 60 to about 100 amino acids, about 60 to about 90 amino acids, about 60 to about 80 amino acids, about 60 to about 70 amino acids, about 70 to about 100 amino acids, about 70 to about 90 amino acids, about 70 to about 80 amino acids, about 80 to about 100 amino acids, about 80 to about 90 amino acids, or about 90 to about 100 amino acids.

[0092] 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), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

[0093] In some embodiments, the linker sequence can comprise or consist of GGGGSGGGSGGGGS (SEQ ID NO:7). In some embodiments, the linker sequence can be encoded by a nucleic acid that comprises or consists of GCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO:8). In some embodiments, the linker sequence can comprise or consist of GGGSGGGS (SEQ ID NO:9).

[0094] Target Binding Domain In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the additional target binding domain(s) 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).

[0095] 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 an antigen binding domain. 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.

[0096] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises one or more additional target binding domains. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more additional target binding domains.

[0097] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the one or more additional target binding domains are each independently selected from the group consisting of CD16a, CD28, CD3 (e.g., one or more of CD3α, CD3β, CD3δ, CD3ε, and CD3γ), 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, CD26a, CD36, ULBP2, CD30, CD200, IGF-1R, MU C4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, UL16 binding proteins (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, ligand for TGF-β receptor II (TGF-βRII), ligand for TGF-βRIII, ligand for DNAM-1, ligand for NKp46, ligand for NKp44, ligand for NKG2D, NK P 30 ligand, a ligand for scMHCI, a ligand for scMHCII, a ligand for scTCR, a receptor for IL-1, a receptor for IL-2, a receptor for IL-3, a receptor for IL-7, a receptor for IL-8, a receptor for IL-10, a receptor for IL-12, a receptor for IL-15, a receptor for IL-17, a receptor for IL-18, a receptor for IL-21, a receptor for PDGF-DD, a receptor for stem cell factor (SCF), a receptor for stem cell-like tyrosine kinase 3 ligand (FLT3L), a receptor for MICA, a receptor for MICB, a receptor for ULP16 binding protein, a receptor for CD155, a receptor for CD122, and a receptor for CD28.

[0098] In some embodiments of any of the 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 900 amino acids, from about 5 amino acids to about 800 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 600 amino acids, from about 5 amino acids to about 500 amino acids, from about 5 amino acids to about 400 amino acids, from about 5 amino acids to about 300 amino acids, about 5 amino acids to about 280 amino acids, about 5 amino acids to about 260 amino acids, about 5 amino acids to about 240 amino acids, about 5 amino acids to about 220 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 60 amino acids, About 5 amino acids to about 40 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 10 amino acids, about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 300 amino acids, about 10 amino acids to about 280 amino acids, about 10 amino acids to about 260 amino acids, about 10 amino acids to about 240 amino acids, about 10 amino acids to about 220 amino acids, about 10 amino acids to about 200 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 40 amino acids,About 10 amino acids to about 20 amino acids, about 20 amino acids to about 1000 amino acids, about 20 amino acids to about 900 amino acids, about 20 amino acids to about 800 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 300 amino acids, about 20 amino acids to about 280 amino acids, about 20 amino acids to about 260 amino acids, about 20 amino acids to about 240 amino acids amino acids, about 20 to about 220 amino acids, about 20 to about 200 amino acids, about 20 to about 180 amino acids, about 20 to about 160 amino acids, about 20 to about 140 amino acids, about 20 to about 120 amino acids, about 20 to about 100 amino acids, about 20 to about 80 amino acids, about 20 to about 60 amino acids, about 20 to about 40 amino acids, about 40 to about 1000 amino acids, about 40 to about 900 amino acids, about 40 amino acids to about 800 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 400 amino acids, about 40 amino acids to about 300 amino acids, about 40 amino acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40 amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 200 amino acids, about 40 Amino acids to about 180 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 700 amino acids, about 60 amino acids to about 600 amino acids,About 60 amino acids to about 500 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 300 amino acids, about 60 amino acids to about 280 amino acids, about 60 amino acids to about 260 amino acids, about 60 amino acids to about 240 amino acids, about 60 amino acids to about 220 amino acids, about 60 amino acids to about 200 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 120 amino acids, about 60 to about 100 amino acids, about 60 to about 80 amino acids, about 80 to about 1000 amino acids, about 80 to about 900 amino acids, about 80 to about 800 amino acids, about 80 to about 700 amino acids, about 80 to about 600 amino acids, about 80 to about 500 amino acids, about 80 to about 400 amino acids, about 80 to about 300 amino acids, about 80 to about 280 amino acids, about 80 amino acids about 80 to about 260 amino acids, about 80 to about 240 amino acids, about 80 to about 220 amino acids, about 80 to about 200 amino acids, about 80 to about 180 amino acids, about 80 to about 160 amino acids, about 80 to about 140 amino acids, about 80 to about 120 amino acids, about 80 to about 100 amino acids, about 100 to about 1000 amino acids, about 100 to about 900 amino acids, about 100 to about 800 amino acids amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 300 amino acids, about 100 amino acids to about 280 amino acids, about 100 amino acids to about 260 amino acids, about 100 amino acids to about 240 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 180 amino acids,About 100 amino acids to about 160 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 800 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 600 amino acids, about 120 amino acids to about 500 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 300 amino acids, about 120 to about 280 amino acids, about 120 amino acids to about 260 amino acids, about 120 amino acids to about 240 amino acids, about 120 amino acids to about 220 amino acids, about 120 amino acids to about 200 amino acids, about 120 amino acids to about 180 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 140 amino acids, about 140 amino acids to about 1000 amino acids, about 140 amino acids to about 900 amino acids, about 140 amino acids to about 800 amino acids, about 140 amino acids up to about 700 amino acids, about 140 amino acids to about 600 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 300 amino acids, about 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 160 amino acids, about 160 to about 1000 amino acids, about 160 to about 900 amino acids, about 160 to about 800 amino acids, about 160 to about 700 amino acids, about 160 to about 600 amino acids, about 160 to about 500 amino acids, about 160 to about 400 amino acids, about 160 to about 300 amino acids, about 160 to about 280 amino acids, about 160 to about 260 amino acids, about 160 to about 240 amino acids,About 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids, about 180 amino acids to about 900 amino acids, about 180 amino acids to about 800 amino acids, about 180 amino acids to about 700 amino acids, about 180 amino acids to about 600 amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to about 400 amino acids, about 180 amino acids to about 300 amino acids, about 180 from about 280 amino acids, from about 180 amino acids to about 260 amino acids, from about 180 amino acids to about 240 amino acids, from about 180 amino acids to about 220 amino acids, from about 180 amino acids to about 200 amino acids, from about 200 amino acids to about 1000 amino acids, from about 200 amino acids to about 900 amino acids, from about 200 amino acids to about 800 amino acids, from about 200 amino acids to about 700 amino acids, from about 200 amino acids to about 600 amino acids, from about 200 amino acids to about 500 amino acids, from about 200 amino acids to about About 400 amino acids, about 200 to about 300 amino acids, about 200 to about 280 amino acids, about 200 to about 260 amino acids, about 200 to about 240 amino acids, about 200 to about 220 amino acids, about 220 to about 1000 amino acids, about 220 to about 900 amino acids, about 220 to about 800 amino acids, about 220 to about 700 amino acids, about 220 to about 600 amino acids, about 220 to about 500 amino acids amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 300 amino acids, about 220 amino acids to about 280 amino acids, about 220 amino acids to about 260 amino acids, about 220 amino acids to about 240 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 500 amino acids,About 240 amino acids to about 400 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 280 amino acids, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 700 amino acids, About 260 amino acids to about 600 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 300 amino acids, about 260 amino acids to about 280 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids up to about 500 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 300 amino acids, about 300 amino acids to about 1000 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 400 amino acids, about 400 amino acids to about 1000 amino acids amino acids, about 400 to about 900 amino acids, about 400 to about 800 amino acids, about 400 to about 700 amino acids, about 400 to about 600 amino acids, about 400 to about 500 amino acids, about 500 to about 1000 amino acids, about 500 to about 900 amino acids, about 500 to about 800 amino acids, about 500 to about 700 amino acids, about 500 to about 600 amino acids, about 600 amino acids The total number of amino acids may be from about 1,000 amino acids, from about 600 amino acids to about 900 amino acids, from about 600 amino acids to about 800 amino acids, from about 600 amino acids to about 700 amino acids, from about 700 amino acids to about 1,000 amino acids, from about 700 amino acids to about 900 amino acids, from about 700 amino acids to about 800 amino acids, from about 800 amino acids to about 1,000 amino acids, from about 800 amino acids to about 900 amino acids, or from about 900 amino acids to about 1,000 amino acids.

[0099] Any of the target binding domains described herein can be administered in a concentration of 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 equilibrium dissociation 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 (inclusive) D The antibody can bind to the antigen identified in

[0100] Any of the target binding domains described herein may be present in a concentration of from about 1 pM to about 30 nM (e.g., from about 1 pM to about 25 nM, from about 1 pM to about 20 nM, from about 1 pM to about 15 nM, from about 1 pM to about 10 nM, from about 1 pM to about 5 nM, from about 1 pM to about 2 nM, from about 1 pM to about 1 nM, from about 1 pM to about 950 pM, from about 1 pM to about 900 pM, from about 1 pM to about 850 pM, from about 1 pM to about 800 pM, from about 1 pM to about 750 pM, from about 1 pM to about 700 pM, from about 1 pM to about 650 pM, from about 1 pM to about 600 pM, from about 1 pM to about 550 pM, from about 1 pM to about 500 pM, from about 1 pM to about 450 pM, Approximately 1 pM to approximately 400 pM, approximately 1 pM to approximately 350 pM, approximately 1 pM to approximately 300 pM, approximately 1 pM to approximately 250 pM, approximately 1 pM to approximately 200 pM, approximately 1 pM~about 150pM, about 1pM~about 100pM, about 1pM~about 90pM, about 1pM~about 80pM, about 1pM~about 70pM, about 1pM~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 ~5pM, about 1pM to about 4pM, about 1pM to about 3pM, about 1pM to about 2pM, about 2pM to about 30nM, about 2pM to about 25nM, about 2pM Approximately 20nM, approximately 2pM to approximately 15nM, approximately 2pM to approximately 10nM, approximately 2pM to approximately 5nM, approximately 2pM to approximately 2nM, approximately 2pM to approximately 1nM, approximately 2pM ~950pM, approximately 2pM to approximately 900pM, approximately 2pM to approximately 850pM, approximately 2pM to approximately 800pM, approximately 2pM to approximately 750pM, approximately 2pM to approximately 7 00pM, about 2pM to about 650pM, about 2pM to about 600pM, about 2pM to about 550pM, about 2pM to about 500pM, about 2pM to about 450 pM, about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, Approx. 2 pM to approx. 150 pM, approx. 2 pM to approx. 100 pM, approx. 2 pM to approx. 90 pM, approx. 2 pM to approx. 80 pM, approx. 2 pM to approx. 70 pM, approx. 2 pM Approximately 60 pM, approximately 2 pM to approximately 50 pM, approximately 2 pM to approximately 40 pM, approximately 2 pM to approximately 30 pM, approximately 2 pM to approximately 20 pM, approximately 2 pM to approximately 10 pM, approximately 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5pM to about 15nM, about 5pM to about 10nM, about 5pM to about 5nM, about 5pM to about 2nM, about 5pM to about 1nM, about 5pM to about 950pM,about 5 pM to about 900 pM, about 5 pM to about 850 pM, about 5 pM to about 800 pM, about 5 pM to about 750 pM, about 5 pM to about 700 pM, about 5 pM to about 650 pM, about 5 pM to about 600 pM, about 5 pM to about 550 pM, about 5 pM to about 500 pM, about 5 pM to about 450 pM, about 5 pM to about about 400 pM, about 5 pM to about 350 pM, about 5 pM to about 300 pM, about 5 pM to about 250 pM, about 5 pM to about 200 pM, about 5 pM to about 150 pM, about 5 pM to about 100 pM, about 5 pM to about 90 pM, about 5 pM to about 80 pM, about 5 pM to about 70 pM, about 5 pM to about 60 pM, about 5 pM to about 50 pM, about 5 pM to about 40 pM, about 5 pM to about 30 pM, about 5 pM to about 20 pM, about 5 pM to about 10 pM, about 10 pM to about 30 nM, about 10 pM to about 25 nM, about 10 pM to about 20 nM, about 10 pM to about 15 nM, about 10 pM to about 10 nM, about 10 pM to about 5 nM, about 10 pM to about 2 nM, about 10 pM to about 1 nM, about 10 pM to about 950 pM, about 10 pM to about 900 pM, about 10 pM to about 850 pM, about 10 pM to about 800 pM, about 10 pM to about 750 pM, about 10 pM to about 700 pM, about 10 pM to about 650 pM, about 10 pM to about 600 pM, about 10 pM to about 550 pM, about 10 pM to about 500 pM, about 10 pM to about 450 pM, about 10 pM to about 400 pM, about 10 pM to about 350 pM, about 10 pM to about 300 pM, about 10 pM to about 250 pM, about 10 pM to about 200 pM, about 10 pM to about 150 pM, about 10 pM to about 1 00 pM, about 10 pM to about 90 pM, about 10 pM to about 80 pM, about 10 pM to about 70 pM, about 10 pM to about 60 pM, about 10 pM to about 50 pM, about 10 pM to about 40 pM, about 10 pM to about 30 pM, about 10 pM to about 20 pM, about 15 pM to about 30 nM, about 15 pM to about 25 nM, about 15 pM to about 20 nM, about 15 pM to about 15 nM, about 15 pM to about 10 nM, about 15 pM to about 5 nM, about 15 pM to about 2 nM, about 15 pM to about 1 nM, about 15 pM to about 950 pM, about 15 pM to about 900 pM, about 15 pM to about 850 pM, about 15 pM to about 800 p ... pM to about 750 pM, about 15 pM to about 700 pM, about 15 pM to about 650 pM, about 15 pM to about 600 pM, about 15 pM to about 550 pM, about 15 pM to about 500 pM, about 15 pM to about 450 pM, about 15 pM to about 400 pM, about 15 pM to about 350 pM, about 15 pM to about 300 pM,about 15 pM to about 250 pM, about 15 pM to about 200 pM, about 15 pM to about 150 pM, about 15 pM to about 100 pM, about 15 pM to about 90 pM, about 15 pM to about 80 pM, about 15 pM to about 70 pM, about 15 pM to about 60 pM, about 15 pM to about 50 pM, about 15 pM to about 40 pM, about 15 pM to about 30 pM, about 15 pM to about 20 pM, about 20 pM to about 30 nM, about 20 pM to about 25 nM, about 20 pM to about 20 nM, about 20 pM to about 15 nM, about 20 pM to about 10 nM, about 20 pM to about 5 nM, about 20 pM to about 2 nM, about 20 pM to about 1 nM, about 20 pM to about 950 pM, about 20 pM to about 900 pM, about 20 pM to about 850 pM, about 20 pM to about 800 pM, about 20 pM to about 750 pM, about 20 pM to about 700 pM, about 20 pM to about 650 pM, about 20 pM to about 600 pM, about 20 pM to about 550 pM, about 20 pM to about 500 pM, about 20 pM to about 450 pM, about 20 pM to about 400 pM, about 20 pM to about 350 pM, about 20 pM to about 300 pM, about 20 pM to about 250 pM, about 20 pM to about 20 pM, about 200 pM to about 150 pM, about 20 pM to about 100 pM, about 20 pM to about 90 pM, about 20 pM to about 80 pM M, about 20 pM to about 70 pM, about 20 pM to about 60 pM, about 20 pM to about 50 pM, about 20 pM to about 40 pM, about 20 pM to about 30 pM, about 30 pM to about 30 nM, about 30 pM to about 25 nM, about 30 pM to about 30 nM, about 30 pM to about 15 nM, about 30 pM to about 10 nM, about 3 0pM to about 5nM, about 30pM to about 2nM, about 30pM to about 1nM, about 30pM to about 950pM, about 30pM to about 900pM, about 30pM to about 850pM, about 30pM to about 800pM, about 30pM to about 750pM, about 30pM to about 700pM, about 30pM to about 650pM, about 3 0 pM to about 600 pM, about 30 pM to about 550 pM, about 30 pM to about 500 pM, about 30 pM to about 450 pM, about 30 pM to about 400 pM, about 30 pM to about 350 pM, about 30 pM to about 300 pM, about 30 pM to about 250 pM, about 30 pM to about 200 pM, about 30 pM to about 15 0 pM, about 30 pM to about 100 pM, about 30 pM to about 90 pM, about 30 pM to about 80 pM, about 30 pM to about 70 pM, about 30 pM to about 60 pM, about 30 pM to about 50 pM, about 30 pM to about 40 pM, about 40 pM to about 30 nM, about 40 pM to about 25 nM, about 40 pM to about 30 nM,about 40 pM to about 15 nM, about 40 pM to about 10 nM, about 40 pM to about 5 nM, about 40 pM to about 2 nM, about 40 pM to about 1 nM, about 40 pM to about 950 pM, about 40 pM to about 900 pM, about 40 pM to about 850 pM, about 40 pM to about 800 pM, about 40 pM to about 750 pM, about 40 pM to about 700 pM, about 40 pM to about 650 pM, about 40 pM to about 600 pM, about 40 pM to about 550 pM, about 40 pM to about 500 pM, about 40 pM to about 450 pM, about 40 pM to about 400 pM, about 40 pM to about 350 pM, about 40 pM to about 300 pM, about 40 pM to about 250 pM M, about 40 pM to about 200 pM, about 40 pM to about 150 pM, about 40 pM to about 100 pM, about 40 pM to about 90 pM, about 40 pM to about 80 pM, about 40 pM to about 70 pM, about 40 pM to about 60 pM, about 40 pM to about 50 pM, about 50 pM to about 30 nM, about 50 pM to about 25 nM, about 50 pM to about 30 nM, about 50 pM to about 15 nM, about 50 pM to about 10 nM, about 50 pM to about 5 nM, about 50 pM to about 2 nM, about 50 pM to about 1 nM, about 50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about 850 pM, about 50 pM to about 800 pM, about 50 pM to about 100 nM, about 50 pM to about 50 nM, about 50 pM to about 2 nM, about 50 pM to about 1 nM, about 50 pM to about 950 pM, about 50 pM to about 900 pM, about 50 pM to about 850 pM, about 50 pM to about 800 pM pM to about 750 pM, about 50 pM to about 700 pM, about 50 pM to about 650 pM, about 50 pM to about 600 pM, about 50 pM to about 550 pM, about 50 pM to about 500 pM, about 50 pM to about 450 pM, about 50 pM to about 400 pM, about 50 pM to about 350 pM, about 50 pM to about 300 pM , about 50 pM to about 250 pM, about 50 pM to about 200 pM, about 50 pM to about 150 pM, about 50 pM to about 100 pM, about 50 pM to about 90 pM, about 50 pM to about 80 pM, about 50 pM to about 70 pM, about 50 pM to about 60 pM, about 60 pM to about 30 nM, about 60 pM to about 25 nM, about 60 pM to about 30 nM, about 60 pM to about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM, about 60 pM to about 2 nM, about 60 pM to about 1 nM, about 60 pM to about 950 pM, about 60 pM to about 900 pM, about 60 pM to about 850 pM, about 60 pM to about 800 pM, about 60 pM to about 10 ... pM to about 750 pM, about 60 pM to about 700 pM, about 60 pM to about 650 pM, about 60 pM to about 600 pM, about 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM to about 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM, about 60 pM to about 300 pM,about 60 pM to about 250 pM, about 60 pM to about 200 pM, about 60 pM to about 150 pM, about 60 pM to about 100 pM, about 60 pM to about 90 pM, about 60 pM to about 80 pM, about 60 pM to about 70 pM, about 70 pM to about 30 nM, about 70 pM to about 25 nM, about 70 pM to about 30 nM, about 70 pM to about 15 nM, about 70 pM to about 10 nM, about 70 pM to about 5 nM, about 70 pM to about 2 nM, about 70 pM to about 1 nM, about 70 pM to about 950 pM, about 70 pM to about 900 pM, about 70 pM to about 850 pM, about 70 pM to about 800 pM, about 70 pM to about 750 pM, about 70 pM pM to about 700 pM, about 70 pM to about 650 pM, about 70 pM to about 600 pM, about 70 pM to about 550 pM, about 70 pM to about 500 pM, about 70 pM to about 450 pM, about 70 pM to about 400 pM, about 70 pM to about 350 pM, about 70 pM to about 300 pM, about 70 pM to about 250 pM , about 70 pM to about 200 pM, about 70 pM to about 150 pM, about 70 pM to about 100 pM, about 70 pM to about 90 pM, about 70 pM to about 80 pM, about 80 pM to about 30 nM, about 80 pM to about 25 nM, about 80 pM to about 30 nM, about 80 pM to about 15 nM, about 80 pM to about 10 nM, about 80 pM to about 5 nM, about 80 pM to about 2 nM, about 80 pM to about 1 nM, about 80 pM to about 950 pM, about 80 pM to about 900 pM, about 80 pM to about 850 pM, about 80 pM to about 800 pM, about 80 pM to about 750 pM, about 80 pM to about 700 pM, about 80 pM to about 650 pM, about 8 0 pM to about 600 pM, about 80 pM to about 550 pM, about 80 pM to about 500 pM, about 80 pM to about 450 pM, about 80 pM to about 400 pM, about 80 pM to about 350 pM, about 80 pM to about 300 pM, about 80 pM to about 250 pM, about 80 pM to about 200 pM, about 80 pM to about 150 pM, about 80 pM to about 100 pM, about 80 pM to about 90 pM, about 90 pM to about 30 nM, about 90 pM to about 25 nM, about 90 pM to about 30 nM, about 90 pM to about 15 nM, about 90 pM to about 10 nM, about 90 pM to about 5 nM, about 90 pM to about 2 nM, about 90 pM to about 1 nM, about 90 pM to about pM to about 950 pM, about 90 pM to about 900 pM, about 90 pM to about 850 pM, about 90 pM to about 800 pM, about 90 pM to about 750 pM, about 90 pM to about 700 pM, about 90 pM to about 650 pM, about 90 pM to about 600 pM, about 90 pM to about 550 pM, about 90 pM to about 500 pM,about 90 pM to about 450 pM, about 90 pM to about 400 pM, about 90 pM to about 350 pM, about 90 pM to about 300 pM, about 90 pM to about 250 pM, about 90 pM to about 200 pM, about 90 pM to about 150 pM, about 90 pM to about 100 pM, about 100 pM to about 30 nM, about 100 pM to, about 25nM, about 100pM to about 30nM, about 100pM to about 15nM, about 100pM to about 10nM, about 100pM to about 5nM, about 100pM to about 2nM, about 100pM to about 1nM, about 100pM to about 950pM, about 100pM to about 900pM, about 100pM to about 850pM, about 100 pM to about 800 pM, about 100 pM to about 750 pM, about 100 pM to about 700 pM, about 100 pM to about 650 pM, about 100 pM to about 600 pM, about 100 pM to about 550 pM, about 100 pM to about 500 pM, about 100 pM to about 450 pM, about 100 pM to about 400 pM , about 100 pM to about 350 pM, about 100 pM to about 300 pM, about 100 pM to about 250 pM, about 100 pM to about 200 pM, about 100 pM to about 150 pM, about 150 pM to about 30 nM, about 150 pM to about 25 nM, about 150 pM to about 30 nM, about 150 pM to about 15 nM, about 150 pM to about 10 nM, about 150 pM to about 5 nM, about 150 pM to about 2 nM, about 150 pM to about 1 nM, about 150 pM to about 950 pM, about 150 pM to about 900 pM, about 150 pM to about 850 pM, about 150 pM to about 800 pM, about 150 pM to about 750 pM, about 150 pM to about about 700 pM, about 150 pM to about 650 pM, about 150 pM to about 600 pM, about 150 pM to about 550 pM, about 150 pM to about 500 pM, about 150 pM to about 450 pM, about 150 pM to about 400 pM, about 150 pM to about 350 pM, about 150 pM to about 300 pM, about 150 pM to about 350 pM M to about 250 pM, about 150 pM to about 200 pM, about 200 pM to about 30 nM, about 200 pM to about 25 nM, about 200 pM to about 30 nM, about 200 pM to about 15 nM, about 200 pM to about 10 nM, about 200 pM to about 5 nM, about 200 pM to about 2 nM, about 200 pM to about 1 nM, about 200 pM to about 950 pM, about 200 pM to about 900 pM, about 200 pM to about 850 pM, about 200 pM to about 800 pM, about 200 pM to about 750 pM, about 200 pM to about 700 pM, about 200 pM to about 650 pM, about 200 pM to about 600 pM, about 200 pM to about 550 pM M, about 200 pM to about 500 pM, about 200 pM to about 450 pM, about 200 pM to about 400 pM, about 200 pM to about 350 pM, about 200 pM to about 300 pM, about 200 pM to about 250 pM, about 300 pM to about 30 nM, about 300 pM to about 25 nM, about 300 pM to about 30 nM,about 300 pM to about 15 nM, about 300 pM to about 10 nM, about 300 pM to about 5 nM, about 300 pM to about 2 nM, about 300 pM to about 1 nM, about 300 pM to about 950 pM, about 300 pM to about 900 pM, about 300 pM to about 850 pM, about 300 pM to about 800 pM, about 300 pM to about 10 ... about 750 pM, about 300 pM to about 700 pM, about 300 pM to about 650 pM, about 300 pM to about 600 pM, about 300 pM to about 550 pM, about 300 pM to about 500 pM, about 300 pM to about 450 pM, about 300 pM to about 400 pM, about 300 pM to about 350 pM, about 400 pM to about 30 nM, about 400 pM to about 25 nM, about 400 pM to about 30 nM, about 400 pM to about 15 nM, about 400 pM to about 10 nM, about 400 pM to about 5 nM, about 400 pM to about 2 nM, about 400 pM to about 1 nM, about 400 pM to about 950 pM, about 400 pM to about 900 pM, about 4 00 pM to about 850 pM, about 400 pM to about 800 pM, about 400 pM to about 750 pM, about 400 pM to about 700 pM, about 400 pM to about 650 pM, about 400 pM to about 600 pM, about 400 pM to about 550 pM, about 400 pM to about 500 pM, about 500 pM to about 30 nM, about 5 00 pM to about 25 nM, about 500 pM to about 30 nM, about 500 pM to about 15 nM, about 500 pM to about 10 nM, about 500 pM to about 5 nM, about 500 pM to about 2 nM, about 500 pM to about 1 nM, about 500 pM to about 950 pM, about 500 pM to about 900 pM, about 500 pM to about 850 pM, about 500 pM to about 800 pM, about 500 pM to about 750 pM, about 500 pM to about 700 pM, about 500 pM to about 650 pM, about 500 pM to about 600 pM, about 500 pM to about 550 pM, about 600 pM to about 30 nM, about 600 pM to about 25 nM, about 600 pM to about 30 nM , about 600 pM to about 15 nM, about 600 pM to about 10 nM, about 600 pM to about 5 nM, about 600 pM to about 2 nM, about 600 pM to about 1 nM, about 600 pM to about 950 pM, about 600 pM to about 900 pM, about 600 pM to about 850 pM, about 600 pM to about 800 pM, about 600 pM to about 750 pM, about 600 pM to about 700 pM, about 600 pM to about 650 pM, about 700 pM to about 30 nM, about 700 pM to about 25 nM, about 700 pM to about 30 nM, about 700 pM to about 15 nM, about 700 pM to about 10 nM, about 700 pM to about 5 nM, about 700 pM to about 2 nM,about 700 pM to about 1 nM, about 700 pM to about 950 pM, about 700 pM to about 900 pM, about 700 pM to about 850 pM, about 700 pM to about 800 pM, about 700 pM to about 750 pM, about 800 pM to about 30 nM, about 800 pM to about 25 nM, about 800 pM to about 30 nM, about 800 pM to about 15 nM, about 800 pM to about 10 nM, about 800 pM to about 5 nM, about 800 pM to about 2 nM, about 800 pM to about 1 nM, about 800 pM to about 950 pM, about 800 pM to about 900 pM, about 800 pM to about 850 pM, about 900 pM to about 30 nM, about 900 pM to about 25 nM, about 900 pM to about 30 nM, about 900 pM to about 15 nM, about 900 pM to about 10 nM, about 900 pM to about 5 nM, about 900 pM to about 2 nM, about 900 pM to about 1 nM, about 900 pM to about 950 pM, about 1 nM to about 30 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 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30 nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nM to about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM, about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nM to about 25 nM), D The combination of the でその standard and the することができる.

[0101] Any of the target binding domains described herein may be present in 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 3 nM, nM to 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 M ~ about 7nM, about 4nM - about 6nM, about 4nM - about 5nM, about 5nM - about 10nM, about 5nM - about 9nM, about 5nM - about 8nM, about 5nM ~about 7nM, about 5nM to about 6nM, about 6nM to about 10nM, about 6nM to about 9nM, about 6nM to about 8nM, about 6nM to about 7nM, about 7nM about 10 nM, about 7 nM to about 9 nM, about 7 nM to about 8 nM, about 8 nM to about 10 nM, about 8 nM to about 9 nM, and about 9 nM to about 10 nM) D can bind to its target.

[0102] A variety of different methods known in the art can be used to quantify the K of any of the antigen binding protein constructs described herein. D Values ​​can be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecular binding kinetic rate assays, etc.).

[0103] antigen-binding domain 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 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.

[0104] 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.

[0105] 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. 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.

[0106] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is an scFv or a single-domain antibody (e.g., a VHH or VNAR domain).

[0107] In some examples, the first and / or second target-binding domain is an antigen-binding domain. Non-limiting examples of antigen-binding domains capable of specifically binding to a ligand of TGF-βRII include the antigen-binding domains of SAR-439459, NIS793, SRK-181, and GC1008 (fresolimumab).

[0108] In some examples, the one or more additional target binding domains can be antigen binding domains (e.g., any of the antigen binding domains described herein) that specifically bind to any one of the following: CD16a (see, e.g., those described in U.S. Pat. No. 9,035,026), CD28 (see, e.g., those described in U.S. Pat. No. 7,723,482), CD3 (see, e.g., those described in U.S. Pat. No. 9,226,962), CD33 (see, e.g., those described in U.S. Pat. No. 8,759,494), CD20 (see, e.g., those described in WO 2014 / 026054), CD19 (see, e.g., those described in U.S. Pat. No. 9,701,758), CD22 (see, e.g., those described in U.S. Pat. No. 9,701,758), CD33 (see, e.g., those described in U.S. Pat. No. 8,759,494), CD20 (see, e.g., those described in WO 2014 / 026054), CD19 (see, e.g., those described in U.S. Pat. No. 9,701,758), CD22 (see, e.g., No. 2003 / 104425), CD123 (see, e.g., as described in WO 2014 / 130635), IL-1R (see, e.g., as described in U.S. Pat. No. 8,741,604), IL-1 (see, e.g., as described in WO 2014 / 095808), VEGF (see, e.g., as described in U.S. Pat. No. 9,090,684), IL-6R (see, e.g., as described in U.S. Pat. No. 7,482,436), IL-4 (see, e.g., as described in U.S. Pat. App. No. 2012 / 0171197), IL-10 (see, e.g., as described in U.S. Pat. App. No. 2016 / 0340413), PDL-1 (see, e.g., as described in Drees et al., Protein Express. Purif. 94:60-66, 2014), TIGIT (see, for example, those described in U.S. Patent Application Publication No. 2017 / 0198042), PD-1 (see, for example, those described in U.S. Patent No. 7,488,802), TIM3 (see, for example, those described in U.S. Patent No. 8,552,156), CTLA4 (see, for example, those described in WO 2012 / 120125), MICA (see, for example, those described in WO 2016 / 154585), MICB (see, for example, those described in U.S. Patent No. 8,753,640), IL-6 (see, for example, those 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), CD26a (see, e.g., WO 2017 / 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., as described in U.S. Pat. No. 9,085,623), IGF-1R (see, e.g., as described in U.S. Patent Application Publication No. 2017 / 0051063), MUC4AC (see, e.g., as described in WO 2012 / 170470), MUC5AC (see, e.g., as described in U.S. Pat. No. 9,238,084), Trop-2 (see, e.g., as described in WO 2013 / 068946), CMET (see, e.g., as described in Edwardraja et al., Biotechnol. Bioeng. 106(3):367-375, 2010), EGFR (see, e.g., as described in Akbari et al., Protein Expr. Purif. 127:8-15, 2016), HER1 (see, for example, those described in U.S. Patent Application Publication No. 2013 / 0274446), HER2 (see, for example, those described in Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3 (see, for example, those described in U.S. Patent No. 9,505,843), PSMA (see, for example, those described in Parker et al., Protein Expr. Purif.89(2):136-145,2013), CEA (see, for example, those described in WO 1995 / 015341), B7H3 (see, for example, those described in U.S. Pat. No. 9,371,395), EPCAM (see, for example, those described in WO 2014 / 159531), BCMA (see, for example, those described in Smith et al., Mol. Ther. 26(6):1447-1456,2018), P-cadherin (see, for example, those described in U.S. Pat. No. 7,452,537), CEACAM5 (see, for example, those described in U.S. Pat. No. 9,617,345), UL16 binding protein (see, for example, those described in WO 2017 / 083612), HLA-DR (see, for example, Pistillo et al., al., Exp. Clin. Immunogenet. 14(2):123-130, 1997), DLL4 (see, for example, those described in WO 2014 / 007513), TYRO3 (see, for example, those described in WO 2016 / 166348), AXL (see, for example, those described in WO 2012 / 175692), MER (see, for example, those described in WO 2016 / 106221), CD122 (see, for example, those described in U.S. Patent Application Publication No. 2016 / 0367664), CD155 (see, for example, those described in WO 2017 / 149538), or PDGF-DD (see, for example, those described in U.S. Patent No. 9,441,034).

[0109] Each antigen-binding domain present in any of the 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 any of the multi-chain chimeric polypeptides are known in the art.

[0110] VHH domains are single monomeric variable antibody domains found in camelids. VNAR domains are single monomeric variable antibody domains found in cartilaginous fish. VHH domains and V NARNon-limiting aspects of the domain 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.

[0111] In some embodiments, each of the antigen-binding domains in the multi-chain chimeric polypeptides 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 the multi-chain chimeric polypeptides 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 the multi-chain chimeric polypeptides described herein are both scFv domains, or at least one antigen-binding domain is an scFv domain.

[0112] In some embodiments, two or more of the polypeptides present in the multi-chain chimeric polypeptide are associated (e.g., non-covalently associated) to form any of the antigen-binding domains described herein (e.g., any of the antigen-binding fragments of the antibodies described herein), VHH-scAb, VHH-Fab, Dual 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, SEED body, LUZ-Y, Fcab, 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 The following conjugates can be formed: IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Miniantibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock-and-Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv1-PEG-scFv2. For a description of these conjugates, see Spiess et al., Mol. Immunol. 67:95-106, 2015, which is 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., antigen-binding fragments of 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., antigen-binding fragments of 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).

[0113] An "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[0114] 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.

[0115] An "F(ab')2" fragment contains two Fab fragments linked near the hinge region by a disulfide bond.

[0116] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to a multivalent and multispecific binding protein as described in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and U.S. Patent 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).

[0117] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0118] In some aspects, any of the antigen binding domains described herein are capable of binding to an antigen selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

[0119] Additional examples and aspects of antigen binding domains are known in the art.

[0120] soluble interleukin or cytokine protein In some embodiments of any of the multi-chain chimeric polypeptides described herein, the one or more additional target binding domains is a soluble interleukin or soluble cytokine protein. In some embodiments, the soluble interleukin or soluble cytokine protein is selected from the group consisting 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.

[0121] Human soluble IL-2 (SEQ ID NO:10) TIFF2025508939000008.tif24170

[0122] Human soluble IL-3 (SEQ ID NO:11) TIFF2025508939000009.tif21170

[0123] Human soluble IL-7 (SEQ ID NO: 12) TIFF2025508939000010.tif22170

[0124] Human soluble IL-8 (SEQ ID NO: 13) egavlprsak elrcqcikty skpfhpkfik elrviesgph canteiivkl sdgrelcldp kenwvqrvve kflkraens

[0125] Human soluble IL-10 (SEQ ID NO:14) TIFF2025508939000011.tif27170

[0126] Human soluble IL-15 (SEQ ID NO: 15) TIFF2025508939000012.tif22170

[0127] Human soluble IL-17 (SEQ ID NO: 16) TIFF2025508939000013.tif20170

[0128] Human soluble IL-18 (SEQ ID NO: 17) TIFF2025508939000014.tif19170

[0129] Human soluble PDGF-DD (SEQ ID NO:18) TIFF2025508939000015.tif46170

[0130] Human soluble SCF (SEQ ID NO:19) TIFF2025508939000016.tif33170

[0131] Human soluble FLT3L (SEQ ID NO:20) TIFF2025508939000017.tif32170

[0132] Non-limiting examples of soluble MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 are provided below.

[0133] Human soluble MICA (SEQ ID NO:21) TIFF2025508939000018.tif56170

[0134] Human soluble MICB (SEQ ID NO:22) TIFF2025508939000019.tif53170

[0135] Human soluble ULBP1 (SEQ ID NO:23) TIFF2025508939000020.tif26170

[0136] Human soluble ULBP2 (SEQ ID NO:24) TIFF2025508939000021.tif27170

[0137] Human soluble ULBP3 (SEQ ID NO:25) TIFF2025508939000022.tif26170

[0138] Human soluble ULBP4 (SEQ ID NO:26) TIFF2025508939000023.tif34170

[0139] Human soluble ULBP5 (SEQ ID NO:27) TIFF2025508939000024.tif28170

[0140] Human soluble ULBP6 (SEQ ID NO:28) TIFF2025508939000025.tif27170

[0141] Additional examples of soluble interleukin proteins and soluble cytokine proteins are known in the art.

[0142] Soluble receptors 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 receptor or a soluble cytokine receptor or a ligand receptor. In some embodiments, the first and / or second target binding domain can be a soluble TGF-β receptor II (TGF-βRII) (see, e.g., Yung et al., Am. J. Resp. Crit. Care Med. 194(9):1140-1151, 2016).

[0143] In some embodiments, the first target binding domain comprises a soluble TGF-β receptor (e.g., a soluble TGFRβRII (e.g., a soluble human TGFRβRII)). In some embodiments, the second target binding domain comprises a soluble TGF-β receptor (e.g., a soluble TGFRβRII (e.g., a soluble human TGFRβRII)). In some embodiments, the soluble human TGFRβRII comprises a first sequence of soluble human TGFRβRII and a second sequence of soluble human TGFRβRII. In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker comprises the sequence GGGGSGGGSGGGGGS (SEQ ID NO:7).

[0144] In some embodiments, the first sequence of the soluble human TGFRβRII is TIFF2025508939000026.tif17170.

[0145] In some embodiments, the second sequence of the soluble human TGFRβRII is TIFF2025508939000027.tif16170.

[0146] In some embodiments, the first sequence of the soluble human TGFRβRII is and encoded by a 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 TIFF2025508939000028.tif41170.

[0147] In some embodiments, the second sequence of the soluble human TGFRβRII is and encoded by a 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 TIFF2025508939000029.tif45170.

[0148] In some embodiments, the soluble human TGFRβRII is and encoded by a 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 TIFF2025508939000030.tif83170.

[0149] In some embodiments, the soluble human TGFRβII is TIFF2025508939000031.tif31170.

[0150] In some embodiments, the one or more additional target binding domains are selected from the group consisting of soluble TGF-β receptor II (TGF-βRII) (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., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389 / fimmu.2018.01150), soluble NKG2D (see, e.g., as described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018), soluble NKp30 ... al., Front.Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389 / fimmu.2018.01150), soluble NKp44 (see, e.g., those described in Costa et al., Front.Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389 / fimmu.2018.01150), soluble NKp46 (see, e.g., those described in Mandelboim et al., Nature 409:1055-1060, 2001; Costa et al., Front.Immunol., Vol. 9, Article 1150, May 29, 2018), 29, 2018; doi:10.3389 / fimmu.2018.01150), soluble DNAM-1 (see, e.g., those described in Costa et al., Front. Immunol., Vol. 9, Article 1150, May 29, 2018; doi:10.3389 / fimmu.2018.01150), scMHC1 (see, e.g., those described in Washburn et al., PLoS One 6(3):e18439, 2011), scMHCII (see, e.g., those described in Bishwajit et al., Cellular Immunol. 170(1):25-33, 1996), scTCR (see, e.g., those described in 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).

[0151] Additional examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0152] Additional target binding domains 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 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 aspects, 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 target binding domains described herein or known in the art) 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).

[0153] 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 directly 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 aspects, 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).

[0154] 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 the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) in the first chimeric polypeptide 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, 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 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, the 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 directly 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 aspects, 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 in the first chimeric polypeptide between at least one additional binding domain (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) 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 the 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 of any of the exemplary pairs of affinity domains described herein) directly abuts the soluble tissue factor domain and / or the first domain of the pair of affinity domains. In some aspects, the first chimeric polypeptide further comprises (i) a linker sequence (e.g., any of the exemplary soluble tissue factor domains described herein) disposed between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and 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 exemplary first domains of any of the exemplary pairs of affinity domains described herein); and / or (ii) a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) disposed between the first domain of the pair of affinity domains and any one of the one or more additional target binding domains positioned between the soluble tissue factor domain and the first domain of the pair of affinity domains.

[0155] 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 the 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 in the second chimeric polypeptide (e.g., any of the exemplary target binding domains described herein or known in the art) and the second domain of the pair of affinity domains (e.g., any of the 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 directly 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 aspects, the second chimeric polypeptide further comprises a linker sequence (e.g., an exemplary linker sequence 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).

[0156] 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, or ten 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, or ten 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, or ten 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.

[0157] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and the 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., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more) of the first target binding domain, the second target binding domain, and the 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 the one or more additional target binding domains are each antigen binding domains (e.g., scFvs or single-domain antibodies).

[0158] Paired affinity domains In some embodiments, the 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, whereby the first chimeric polypeptide and the second chimeric polypeptide associate through binding between the first and second domains of the pair of affinity domains. In some embodiments, the pair of affinity domains is a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. The sushi domain, also known as the short consensus repeat or type 1 glycoprotein motif, is a common motif in protein-protein interactions. Sushi domains have been identified on numerous protein-binding molecules, including complement components C1r, C1s, factor H, and C2m, as well as 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 IL-15 has a D8N or D8A amino acid substitution. In some embodiments where one member of the paired affinity domains is the alpha chain of the human IL-15 receptor (IL15Rα), the human IL15Rα is mature full-length IL15Rα. In some embodiments, the paired affinity domains are barnase and barstar. 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 mutated 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 interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492, 2003).

[0159] 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, 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 equilibrium dissociation 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 -12M, 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 (inclusive) D Any of a variety of different methods known in the art can be used to measure the K between the first domain of the pair of affinity domains and the second domain of the pair of affinity domains. D Values ​​can be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecular binding kinetic rate assays, etc.).

[0160] In some embodiments, a first chimeric polypeptide of a 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, can 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 65 amino acids in length, about 65 to 65 amino acids in length, about 66 to 67 amino acids in length, about 67 to 68 amino acids in length, about 68 to 69 amino acids in length, about 70 to 71 amino acids in length, about 72 to 73 amino acids in length, about 74 to 75 amino acids in length, about 76 to 77 amino acids in length, about 78 to 79 amino acids in length, about 79 to 80 amino acids in length, about 80 to 81 amino acids in length, about 81 to 82 amino acids in length, about 82 to 83 amino acids in length, about 83 to 84 amino acids in length, about 84 to 85 amino acids in length, about 85 to 86 amino acids in length, about 86 to 87 amino acids in length, about 87 to 88 amino acids in length, about 88 to 89 amino acids in length, about 90 to 91 amino acids in length, about 92 to 93 amino acids in length, about 93 to 94 amino acids in length, about 94 to 95 amino acids in length, about 95 to 96 amino acids in length, about 96 to 97 amino acids in length, about 9 amino acid length, about 60 to 100 amino acids length, about 65 to 100 amino acids length, about 70 to 100 amino acids length, about 75 to 100 amino acids length, about 80 to 100 amino acids length, about 85 to 100 amino acids length, about 90 to 100 amino acids length, about 95 to 100 amino acids length, about 10 to 95 amino acids length, about 10 to 90 amino acids length, about 10 to 85 amino acids length, about 10 to 80 amino acids length, about Length of 10 to 75 amino acids, length of about 10 to 70 amino acids, length of about 10 to 65 amino acids, length of about 10 to 60 amino acids, length of about 10 to 55 amino acids, length of about 10 to 50 amino acids, length of about 10 to 45 amino acids, length of about 10 to 40 amino acids, length of about 10 to 35 amino acids, length of about 10 to 30 amino acids, length of about 10 to 25 amino acids, length of about 10 to 20 amino acids, length of about 10 to 15 amino acids, length of about 2 The length may be 0 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.

[0161] In some embodiments, either the first domain and / or the second domain of a pair of affinity domains 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 domain and / or the second domain of the pair of affinity domains remains intact. For example, the sushi domain derived from the alpha chain of the human IL-15 receptor (IL15Rα) may contain one or more additional amino acids at its 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 its N-terminus and / or C-terminus while still retaining the ability to bind to sushi derived from the alpha chain of the human IL-15 receptor (IL15Rα).

[0162] Non-limiting examples of a sushi domain from the alpha chain of IL-15 receptor alpha (IL15Rα) can include 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 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO:29). In some embodiments, the sushi domain from the alpha chain of IL15Rα can include 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 ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO:29). It can be encoded by a nucleic acid comprising TIFF2025508939000032.tif27170.

[0163] In some embodiments, the soluble IL-15 is TIFF2025508939000033.tif21170. In some embodiments, the soluble IL-15 may 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 TIFF2025508939000033.tif21170. It can be encoded by a nucleic acid comprising the sequence of TIFF2025508939000034.tif46170.

[0164] signal sequence In some embodiments, a multi-chain chimeric polypeptide includes a first chimeric polypeptide that includes a signal sequence at its N-terminus. In some embodiments, a multi-chain chimeric polypeptide includes a second chimeric polypeptide that includes a signal sequence at its N-terminus. In some embodiments, both the first chimeric polypeptide of a multi-chain chimeric polypeptide and the second chimeric polypeptide of a multi-chain chimeric polypeptide include a signal sequence. As will be understood by those skilled in the art, a signal sequence is an amino acid present at the N-terminus of many endogenously produced proteins that directs the protein into the secretory pathway (the protein is directed to reside in a specific intracellular organelle, reside at the cell membrane, or be secreted from the cell). Signal sequences are heterologous and generally differ in their primary sequence. However, signal sequences are typically 16 to 30 amino acids in length and include a hydrophilic, usually positively charged, N-terminal region, a central hydrophobic domain, and a C-terminal region containing a cleavage site for a signal peptidase.

[0165] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 32). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a signal sequence encoded by the nucleic acid sequence ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 33), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO: 34), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 35).

[0166] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO:36). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence having the amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO:37). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence having the amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO:38). In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence having the amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 39). One of skill in the art will recognize other suitable signal sequences for use in the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide described herein.

[0167] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a signal sequence that is about 10-100 amino acids in length. For example, the signal sequence can be about 10-100 amino acids in length, about 15-100 amino acids in length, about 20-100 amino acids in length, about 25-100 amino acids in length, about 30-100 amino acids in length, about 35-100 amino acids in length, about 40-100 amino acids in length, about 45-100 amino acids in length, about 50-100 amino acids in length, about 55-100 amino acids in length, about 60-100 amino acids in length, about 65-100 amino acids in length, about 70-100 amino acids in length, about 75-100 amino acids in length, about 80-100 amino acids in length, about 85-100 amino acids in length, about 90-100 amino acids in length, about 95-100 amino acids in length, about 10-95 amino acids in length, about 10-90 amino acids in length, about 10-85 amino acids in length, about 10-80 amino acids in length, about 10-75 amino acids in length, about 10 The amino acid length may be about 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 signal sequence is 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.

[0168] In some aspects, any of the signal sequences disclosed herein can include 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, as long as the function of the signal sequence remains intact. For example, a signal sequence having the amino acids MKCLLYLAFLFLGVNC (SEQ ID NO:36) can include one or more additional amino acids at the N-terminus or C-terminus while still retaining the ability to direct a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second polypeptide of the multi-chain chimeric polypeptide, or both, into the secretory pathway.

[0169] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, contain a signal sequence that directs the multi-chain chimeric polypeptide to the extracellular space. Such embodiments are useful for producing multi-chain chimeric polypeptides that are relatively easy to isolate and / or purify.

[0170] Peptide tags In some embodiments, a multi-chain chimeric polypeptide comprises a first chimeric polypeptide comprising a peptide tag (e.g., at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, a multi-chain chimeric polypeptide comprises a second chimeric polypeptide comprising a peptide tag (e.g., at the N-terminus or C-terminus of the second chimeric polypeptide). In some embodiments, both the first chimeric polypeptide of the multi-chain chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide comprise a peptide tag. In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both comprise two or more peptide tags.

[0171] Exemplary peptide tags that may be included in the first chimeric polypeptide of a multi-chain chimeric polypeptide, the second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, include, but are not limited to, AviTag (GLNDIFEAQKIEWHE; SEQ ID NO:40), calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO:41), polyglutamic acid tag (EEEEEE; SEQ ID NO:42), E-tag (GAPVPYPDPLEPR; SEQ ID NO:43), FLAG-tag (DYKDDDDK; SEQ ID NO:44), HA-tag, hemagglutinin-derived peptide (YPYDVPDYA; SEQ ID NO:45), his-tag (HHHHH (SEQ ID NO:46); HHHHHHH (SEQ ID NO:47); HHHHHHHH (SEQ ID NO:48); HHHHHHHHH (SEQ ID NO:49), and the like. NO:49); HHHHHHHHH (SEQ ID NO:50); or HHHHHHHHHH (SEQ ID NO:51)), myc-tag (EQKLISEEDL; SEQ ID NO:52), NE-tag (TKENPRSNQEESYDDNES; SEQ ID NO:53), S-tag (KETAAAKFERQHMDS; SEQ ID NO:54), SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO:55), Softag 1 (SLAELLNAGLGGS; SEQ ID NO:56), Softag 3 (TQDPSRVG; SEQ ID NO:57), Spot-tag (PDRVRAVSHWSS; SEQ ID NO:58), Strep-tag (WSHPQFEK; SEQ ID NO:59), TC-tag (CCPGCC; SEQ Tissue factor protein is a peptide tag, including, but not limited to, Ty-tag (EVHTNQDPLD; SEQ ID NO:61), V5-tag (GKPIPNPLLGLDST; SEQ ID NO:62), VSV-tag (YTDIEMNRLGK; SEQ ID NO:63), and Xpress-tag (DLYDDDDK; SEQ ID NO:64). In some embodiments, the tissue factor protein is a peptide tag.

[0172] A peptide tag, which may be contained in the first chimeric polypeptide of a multi-chain chimeric polypeptide, the second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, can be used in any of a variety of applications related to the multi-chain chimeric polypeptide. For example, the peptide tag can be used in purifying the multi-chain chimeric polypeptide. As a non-limiting example, the first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both, can contain a myc tag; a multi-chain chimeric polypeptide containing the myc-tagged first chimeric polypeptide, the myc-tagged second chimeric polypeptide, or both, can be purified using an antibody that recognizes the myc tag. One non-limiting example of an antibody that recognizes a myc tag is 9E10, available from the non-profit Developmental Studies Hybridoma Bank. As another non-limiting example, the first chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide of a multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both can comprise a histidine tag; a multi-chain chimeric polypeptide comprising a histidine-tagged first chimeric polypeptide, a histidine-tagged second chimeric polypeptide, or both, can be purified using a nickel or cobalt chelate. Those of skill in the art will recognize other suitable tags and agents that bind to those tags for use in purifying multi-chain chimeric polypeptides. In some embodiments, the peptide tag is removed from the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification. In some embodiments, the peptide tag is not removed from the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide after purification.

[0173] The peptide tag, which may be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, may be used, for example, in immunoprecipitation of the multi-chain chimeric polypeptide, imaging of the multi-chain chimeric polypeptide (e.g., via Western blotting, ELISA, flow cytometry, and / or immunohistochemistry), and / or solubilization of the multi-chain chimeric polypeptide.

[0174] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a peptide tag that is about 10 to 100 amino acids in length. For example, the peptide tag can 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 65-100 amino acids in length, about 70-100 amino acids in length, about 75-100 amino acids in length, about 80-100 amino acids in length, about 85-100 amino acids in length, about 90-100 amino acids in length, about 95-100 amino acids in length, about 10-95 amino acids in length, about 10-90 amino acids in length, about 10-85 amino acids in length, about 10-80 amino acids in length, about 10-75 amino acids in length, about 10 The amino acid length may be about 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 peptide tag is 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.

[0175] A peptide tag included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both can be of any suitable length. For example, a peptide tag can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids in length. In embodiments in which a multi-chain chimeric polypeptide includes two or more peptide tags, the two or more peptide tags can be the same length or different lengths. In some embodiments, any of the peptide tags disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminus and / or C-terminus, so long as the function of the peptide tag remains intact. For example, a myc tag having the amino acids EQKLISEEDL (SEQ ID NO:65) can include one or more additional amino acids (e.g., at the N-terminus and / or C-terminus of the peptide tag) while still retaining the ability to be bound by an antibody (e.g., 9E10).

[0176] Exemplary Multi-Chain Chimeric Polypeptides In some examples of multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of 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 in the first chimeric polypeptide.

[0177] 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 in the second chimeric polypeptide.

[0178] In some aspects of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary tissue factor domains described herein. In some aspects of these multi-chain chimeric polypeptides, the pair of affinity domains can be any of the exemplary pair of affinity domains described herein.

[0179] 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 a soluble TGF-β receptor (e.g., a soluble TGFRβRII, e.g., a soluble human TGFRβRII).

[0180] In some aspects of the multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first sequence of soluble human TGFRβRII and a second sequence of soluble human TGFRβRII. In some aspects of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a linker disposed between the first sequence of soluble human TGFRβRII and the second sequence of soluble human TGFRβRII. In some examples of these multi-chain chimeric polypeptides, the linker sequence comprises the sequence GGGGSGGGSGGGGGS (SEQ ID NO:7).

[0181] In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFRβRII receptor is TIFF2025508939000035.tif19170.

[0182] In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFRβRII receptor is TIFF2025508939000036.tif18170.

[0183] In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFRβRII receptor is and encoded by a 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 TIFF2025508939000037.tif42170.

[0184] In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFRβRII receptor is and encoded by a 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 TIFF2025508939000038.tif44170.

[0185] In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII receptor is and encoded by a 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 TIFF2025508939000039.tif84170.

[0186] In some embodiments of these multi-chain chimeric polypeptides, the human TGFRβRII receptor is TIFF2025508939000040.tif31170.

[0187] In some embodiments, the first chimeric polypeptide comprises: It may include a 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 TIFF2025508939000041.tif62170.

[0188] In some embodiments, the first chimeric polypeptide comprises: and encoded by a 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 TIFF2025508939000042.tif176170.

[0189] In some embodiments, the first chimeric polypeptide comprises: It may include a 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 TIFF2025508939000043.tif61170.

[0190] In some embodiments, the first chimeric polypeptide comprises: Encoded by a 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 TIFF2025508939000044.tif183170.

[0191] In some embodiments, the second chimeric polypeptide comprises: It may comprise a 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 TIFF2025508939000045.tif36170.

[0192] In some embodiments, the second chimeric polypeptide comprises: Encoded by a 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 TIFF2025508939000046.tif104170.

[0193] In some embodiments, the second chimeric polypeptide comprises: It may comprise a 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 TIFF2025508939000047.tif36170.

[0194] In some embodiments, the second chimeric polypeptide comprises: and encoded by a 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 TIFF2025508939000048.tif104170.

[0195] Nucleic Acids / Vectors Also provided herein are nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first nucleic acid can encode the first chimeric polypeptide and a second nucleic acid can encode the second chimeric polypeptide. In some embodiments, a single nucleic acid can encode both the first chimeric polypeptide and the second chimeric polypeptide.

[0196] Also provided herein are vectors comprising any of the nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some aspects, a first vector can comprise a nucleic acid encoding a first chimeric polypeptide, and a second vector can comprise a nucleic acid encoding a second chimeric polypeptide. In some aspects, a single vector can comprise a first nucleic acid encoding the first chimeric polypeptide and a second nucleic acid encoding the second chimeric polypeptide.

[0197] Any of the vectors described herein can be an expression vector. For example, the expression vector can include a promoter sequence operably linked to sequences encoding the first and second chimeric polypeptides.

[0198] Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vector), adeno-associated viral (AAV) vector, lentiviral vector, and retroviral vector), as well as any Gateway® vector. A vector can contain, for example, sufficient cis-acting elements for expression; other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. One of skill in the art would be able to select suitable vectors and mammalian cells for producing any of the multi-chain chimeric polypeptides described herein.

[0199] cell Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein encoding any of the multi-chain chimeric polypeptides described herein (e.g., encoding both the first and second chimeric polypeptides). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein encoding any of the first chimeric polypeptides described herein. Also provided are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the nucleic acids described herein encoding any of the second chimeric polypeptides described herein.

[0200] Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the vectors described herein encoding any of the multi-chain chimeric polypeptides described herein (e.g., encoding a first and second chimeric polypeptide). Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the vectors described herein encoding any of the first chimeric polypeptides described herein. Also provided herein are cells (e.g., any of the exemplary cells described herein or known in the art) comprising any of the vectors described herein encoding any of the second chimeric polypeptides described herein.

[0201] In some embodiments of any of the methods described herein, the cell may be a eukaryotic cell. As used herein, the term "eukaryotic cell" refers to a cell having a separate membrane-bound nucleus. Such cells may include, for example, mammalian (e.g., rodent, non-human primate, or human) cells, insect cells, fungal cells, or plant cells. In some embodiments, the eukaryotic cell is a yeast cell such as Saccharomyces cerevisiae. In some embodiments, the eukaryotic cell is a higher eukaryote, such as a mammalian cell, an avian cell, a plant cell, or an insect cell. Non-limiting examples of mammalian cells include Chinese hamster ovary cells and human embryonic kidney cells (e.g., HEK293 cells).

[0202] Methods for introducing nucleic acids and expression vectors into cells (e.g., eukaryotic cells) are known in the art. Non-limiting examples of methods that can be used to introduce nucleic acids into cells include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, imparefection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection.

[0203] Methods for producing multi-chain chimeric polypeptides Also provided herein are methods of producing any of the multi-chain chimeric polypeptides described herein, comprising culturing any of the cells described herein in a culture medium under conditions sufficient to result in the production of the multi-chain chimeric polypeptide, and recovering the multi-chain chimeric polypeptide from the cells and / or the culture medium.

[0204] Also provided herein is a method of producing any of the multi-chain chimeric polypeptides described herein, comprising culturing any of the cells described herein in a first culture medium under conditions sufficient to result in the production of a first chimeric polypeptide, recovering the first chimeric polypeptide from the cells and / or the first culture medium, culturing any of the cells described herein in a second culture medium under conditions sufficient to result in the production of a second chimeric polypeptide, recovering the second chimeric polypeptide from the cells and / or the second culture medium, and combining (e.g., mixing) the recovered first chimeric polypeptide and the recovered second chimeric polypeptide to form a multi-chain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein).

[0205] Recovery of the multi-chain chimeric polypeptide, the first chimeric polypeptide, or the second chimeric polypeptide from cells (e.g., eukaryotic cells) can be carried out using techniques well known in the art (e.g., ammonium sulfate precipitation, polyethylene glycol precipitation, ion exchange chromatography (anionic or cationic), chromatography based on hydrophobic interactions, metal affinity chromatography, ligand affinity chromatography, and size exclusion chromatography).

[0206] Methods for culturing cells are well known in the art. Cells can be maintained in vitro under conditions that favor proliferation, differentiation, and growth. Briefly, cells (e.g., any cell) can be expanded by contacting them with a cell culture medium containing growth factors and nutritional supplements necessary to support cell survival and growth.

[0207] Also provided herein is a multi-chain chimeric polypeptide (e.g., any of the multi-chain chimeric polypeptides described herein), a first chimeric polypeptide (e.g., any of the first chimeric polypeptides), or a second chimeric polypeptide (e.g., any of the second chimeric polypeptides described herein) produced by any of the methods described herein.

[0208] treatment method Provided herein are methods for treating unresectable, advanced / metastatic pancreatic cancer in a subject (e.g., any of the exemplary subjects described herein or known in the art), comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target-binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target-binding domain specifically binds to a ligand of TGF-βRII. Any of the exemplary multi-chain chimeric polypeptides described herein can be used in these methods.

[0209] In some embodiments, the methods described herein result in a reduction in tumor size and / or volume in a subject or population of subjects (e.g., at least a 1% reduction, at least a 5% reduction, at least a 10% reduction, at least a 15% reduction, at least a 20% reduction, at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, at least a 10 ... 85% reduction, at least 90% reduction, at least 95% reduction, or at least 99% reduction, or about 1% reduction to about 99% reduction, about 1% reduction to about 95% reduction, about 1% reduction to about 90% reduction, about 1% reduction to about 85% reduction, about 1% reduction to about 80% reduction, about 1% reduction to about 75% reduction, about 1% reduction to about 70% reduction, about 1% reduction to about 65% reduction, about 1% reduction to about 60% reduction, about 1% reduction to about 55% reduction, about 1% reduction to about 50% reduction, about 1% reduction to about 45% reduction, about 1% reduction to about 40% reduction, about 1% reduction to about 35% reduction, about 1% reduction to about 30% reduction, about 1% reduction to about 25% reduction % decrease, approx. 1% decrease ~ approx. 20% decrease, approx. 1% decrease ~ approx. 15% decrease, approx. 1% decrease ~ approx. 10% decrease, approx. 1% decrease ~ approx. 5% decrease, approx. 5% decrease ~ approx. 99% decrease, approx. Small to approximately 75% decrease, approximately 5% decrease to approximately 70% decrease, approximately 5% decrease to approximately 65% ​​decrease, approximately 5% decrease to approximately 60% decrease, approximately 5% decrease to approximately 55% decrease, approximately 5% decrease to approximately 50% decrease, approximately 5% decrease to approximately 45% decrease, approximately 5% decrease to approximately 40% decrease, approximately 5% decrease to approximately 35% decrease, approximately 5% decrease to approximately 30% decrease , about 5% decrease to about 25% decrease, about 5% decrease to about 20% decrease, about 5% decrease to about 15% decrease, about 5% decrease to about 10% decrease, about 10% decrease to about 99% decrease, about 10% decrease to about 95% decrease, about 10% decrease to about 90% decrease, about 10% decrease to about 85% decrease, about 10% decrease to about 80% decrease, 10% decrease to approx. 75% decrease, approx. 10% decrease to approx. 70% decrease, approx. 10% decrease to approx. 65% decrease, approx. 10% decrease to approx. 60% decrease, approx. 10% decrease to approx. 55% decrease, approx. 10% decrease to approx.About 10% reduction to about 30% reduction, about 10% reduction to about 25% reduction, about 10% reduction to about 20% reduction, about 10% reduction to about 15% reduction, about 15% reduction to about 99% reduction, about 15% reduction to about 95% reduction, about 15% reduction to about 90% reduction, about 15% reduction to about 85% reduction, about 15% reduction to about 80% reduction, about 15% reduction to about 75% reduction, about 15% reduction to about 70% reduction, about 15% reduction to about 65% reduction, about 15% reduction to about 60% reduction, about 15% reduction to about 55% reduction, about 15% reduction to about 50% reduction, about 15% reduction to about 45% reduction, about 15% reduction to about 40% reduction, about 15% reduction to about 35 % reduction, about 15% reduction to about 30% reduction, about 15% reduction to about 25% reduction, about 15% reduction to about 20% reduction, about 20% reduction to about 99% reduction, about 20% reduction to about 95% reduction, about 20% reduction to about 90% reduction, about 20% reduction to about 85% reduction, about 20% reduction to about 80% reduction, about 20% reduction to about 75% reduction, about 20% reduction to about 70% reduction, about 20% reduction to about 65% reduction, about 20% reduction to about 60% reduction, about 20% reduction to about 55% reduction, about 20% reduction to about 50% reduction, about 20% reduction to about 45% reduction, about 20% reduction to about 40% reduction, about 20% reduction to about 35% reduction, about 20% reduction to About 30% reduction, about 20% reduction to about 25% reduction, about 25% reduction to about 99% reduction, about 25% reduction to about 95% reduction, about 25% reduction to about 90% reduction, about 25% reduction to about 85% reduction, about 25% reduction to about 80% reduction, about 25% reduction to about 75% reduction, about 25% reduction to about 70% reduction, about 25% reduction to about 65% reduction, about 25% reduction to about 60% reduction, about 25% reduction to about 55% reduction, about 25% reduction to about 50% reduction, about 25% reduction to about 45% reduction, about 25% reduction to about 40% reduction, about 25% reduction to about 35% reduction, about 25% reduction to about 30% reduction, about 30% reduction to about 99% reduction, about 30 % reduction to about 95% reduction, about 30% reduction to about 90% reduction, about 30% reduction to about 85% reduction, about 30% reduction to about 80% reduction, about 30% reduction to about 75% reduction, about 30% reduction to about 70% reduction, about 30% reduction to about 65% reduction, about 30% reduction to about 60% reduction, about 30% reduction to about 55% reduction, about 30% reduction to about 50% reduction, about 30% reduction to about 45% reduction, about 30% reduction to about 40% reduction, about 30% reduction to about 35% reduction, about 35% reduction to about 99% reduction, about 35% reduction to about 95% reduction, about 35% reduction to about 90% reduction, about 35% reduction to about 85% reduction, about 35% reduction to about 80% reduction,About 35% reduction to about 75% reduction, about 35% reduction to about 70% reduction, about 35% reduction to about 65% reduction, about 35% reduction to about 60% reduction, about 35% reduction to about 55% reduction, about 35% reduction to about 50% reduction, about 35% reduction to about 45% reduction, about 35% reduction to about 40% reduction, about 40% reduction to about 99% reduction, about 40% reduction to about 95% reduction, about 40% reduction to about 90% reduction, about 40% reduction to about 85% reduction, about 40% reduction to about 80% reduction, about 40% reduction to about 75% reduction, about 40% reduction to about 70% reduction, about 40% reduction to about 65% reduction, about 40% reduction to about 60% reduction, about 40% reduction to about 55 % reduction, about 40% reduction to about 50% reduction, about 40% reduction to about 45% reduction, about 45% reduction to about 99% reduction, about 45% reduction to about 95% reduction, about 45% reduction to about 90% reduction, about 45% reduction to about 85% reduction, about 45% reduction to about 80% reduction, about 45% reduction to about 75% reduction, about 45% reduction to about 70% reduction, about 45% reduction to about 65% reduction, about 45% reduction to about 60% reduction, about 45% reduction to about 55% reduction, about 45% reduction to about 50% reduction, about 50% reduction to about 99% reduction, about 50% reduction to about 95% reduction, about 50% reduction to about 90% reduction, about 50% reduction to about 85% reduction, about 50% reduction to About 80% reduction, about 50% reduction to about 75% reduction, about 50% reduction to about 70% reduction, about 50% reduction to about 65% reduction, about 50% reduction to about 60% reduction, about 50% reduction to about 55% reduction, about 55% reduction to about 99% reduction, about 55% reduction to about 95% reduction, about 55% reduction to about 90% reduction, about 55% reduction to about 85% reduction, about 55% reduction to about 80% reduction, about 55% reduction to about 75% reduction, about 55% reduction to about 70% reduction, about 55% reduction to about 65% reduction, about 55% reduction to about 60% reduction, about 60% reduction to about 99% reduction, about 60% reduction to about 95% reduction, about 60% reduction to about 90% reduction, about 60 % reduction to about 85% reduction, about 60% reduction to about 80% reduction, about 60% reduction to about 75% reduction, about 60% reduction to about 70% reduction, about 60% reduction to about 65% reduction, about 65% reduction to about 99% reduction, about 65% reduction to about 95% reduction, about 65% reduction to about 90% reduction, about 65% reduction to about 85% reduction, about 65% reduction to about 80% reduction, about 65% reduction to about 75% reduction, about 65% reduction to about 70% reduction, about 70% reduction to about 99% reduction, about 70% reduction to about 95% reduction, about 70% reduction to about 90% reduction, about 70% reduction to about 85% reduction, about 70% reduction to about 80% reduction, about 70% reduction to about 75% reduction,about a 75% reduction to about a 99% reduction, about a 75% reduction to about a 95% reduction, about a 75% reduction to about a 90% reduction, about a 75% reduction to about a 85% reduction, about a 75% reduction to about a 80% reduction, about a 80% reduction to about a 99% reduction, about a 80% reduction to about a 95% reduction, about a 80% reduction to about a 90% reduction, about a 80% reduction to about a 85% reduction, about a 85% reduction to about a 99% reduction, about a 85% reduction to about a 95% reduction, about a 85% reduction to about a 90% reduction, about a 90% reduction to about a 99% reduction, about a 90% reduction to about a 95% reduction, or about a 95% reduction to about a 99% reduction) (e.g., compared to tumor size and / or volume before administration, or compared to a similar subject not receiving treatment or receiving a different treatment). In some embodiments, the size and / or volume of a tumor in a subject can be assessed by X-ray, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).

[0210] In some aspects, the methods described herein may result in a decrease in the rate of tumor growth in a subject or population of subjects (e.g., at least a 1% decrease, at least a 5% decrease, at least a 10% decrease, at least a 15% decrease, at least a 20% decrease, at least a 25% decrease, at least a 30% decrease, at least a 35% decrease, at least a 40% decrease, at least a 45% decrease, at least a 50% decrease, at least a 55% decrease, at least a 60% decrease, at least a 65% decrease, at least a 70% decrease, at least a 75% decrease, at least a 80% decrease, at least a 85% decrease, at least a 90% decrease, at least a 95% decrease, or at least a 99% decrease, or from about a 1% decrease to about a 99% decrease (or any subrange of this range described herein)) (e.g., compared to the rate of tumor growth in the subject prior to administration, or compared to similar subjects not receiving treatment or receiving a different treatment). In some embodiments, the rate of tumor growth in a subject can be determined by imaging the subject over time using, for example, X-rays, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).

[0211] Also provided herein are methods for improving objective response rates in subjects with unresectable, advanced / metastatic pancreatic cancer (e.g., any of the exemplary subjects described herein), comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target-binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target-binding domain specifically binds to a ligand of TGF-βRII. Any of the exemplary multi-chain chimeric polypeptides described herein can be used in these methods.

[0212] As used herein, the term "objective response rate" refers to the international criteria proposed by the Response Evaluation Criteria in Solid Tumors Committee (RECIST) v1.1 (as described in Eisenhauer et al., Eur. J. Cancer 45:228-247, 2009).

[0213] In some embodiments, the methods include increasing the objective response rate in a subject or population of subjects (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase). increase, at least 95% increase, at least 100% increase, at least 110% increase, at least 120% increase, at least 130% increase, at least 140% increase, at least 150% increase, at least 160% increase, at least 170% increase, at least 180% increase, at least 190% increase, at least 200% increase, at least 210% increase, at least 220% increase, at least 230% increase, at least 240% increase, at least 250% increase, at least 260% increase, at least 270% increase, at least 280% increase, at least 2 90% increase, or at least 300% increase, or about 1% increase to about 300% increase, about 1% increase to about 280% increase, about 1% increase to about 260% increase, about 1% increase to about 240% increase, about 1% increase to about 220% increase, about 1% increase to about 200% increase, about 1% increase to about 180% increase, about 1% increase to about 160% increase, about 1% increase to about 140% increase, about 1% increase to about 120% increase, about 1% increase to about 100% increase, about 1% increase to about 95% increase, about 1% increase to about 90% increase, about 1% increase to about 85% increase, about 1% increase to about 80% increase, about 1% increase to about 75% increase, about 1% increase Increased to approximately 70% increase, increased to approximately 65% ​​increase, increased to approximately 60% increase, increased to approximately 55% increase, increased to approximately 50% increase, increased to approximately 45% increase, increased to approximately 40% increase, increased to approximately 35% increase, increased to approximately 30% increase, increased to approximately 25% increase, increased to approximately 20% increase, increased to approximately 15% increase, increased to approximately 10% increase, increased to approximately 5% increase, increased to approximately 300% increase, increased to approximately 280% increase, increased to approximately 260% increase, increased to approximately 240% increase, increased to approximately 220% increase,Approximately 5% increase to approximately 200% increase, approximately 5% increase to approximately 180% increase, approximately 5% increase to approximately 160% increase, approximately 5% increase to approximately 140% increase, approximately 5% increase to approximately 120% increase, approximately 5% increase to approximately 100% increase, approximately 5% increase to approximately 95% increase, approximately 5% increase to approximately 90% increase, approximately 5% increase to approximately 85% increase, approximately 5% increase to approximately 80% increase, approximately 5% increase to approximately 75% increase, approximately 5% increase to approximately 70% increase, approximately 5% increase to approximately 65% ​​increase, approximately 5% increase to approximately 60% increase, approximately 5% increase to approximately 55% increase, approximately 5% increase to approximately 50% increase, approximately 5% increase to approximately 45% increase, approximately 5% increase to approximately 40% increase, approximately 5% increase to approximately 35 % increase, approximately 5% increase to approximately 30% increase, approximately 5% increase to approximately 25% increase, approximately 5% increase to approximately 20% increase, approximately 5% increase to approximately 15% increase, approximately 5% increase to approximately 10% increase, approximately 10% increase to approximately 300% increase, approximately 10% increase to approximately 280% increase, approximately 10% increase to approximately 260% increase, approximately 10% increase to approximately 240% increase, approximately 10% increase to approximately 220% increase, approximately 10% increase to approximately 200% increase, approximately 10% increase to approximately 180% increase, approximately 10% increase to approximately 160% increase, approximately 10% increase to approximately 140% increase, approximately 10% increase to approximately 120% increase, approximately 10% increase to approximately 100% increase, approximately 10% increase to approximately 95% increase, approximately 1 0% increase to approximately 90% increase, approximately 10% increase to approximately 85% increase, approximately 10% increase to approximately 80% increase, approximately 10% increase to approximately 75% increase, approximately 10% increase to approximately 70% increase, approximately 10% increase to approximately 65% ​​increase, approximately 10% increase to approximately 60% increase, approximately 10% increase to approximately 55% increase, approximately 10% increase to approximately 50% increase, approximately 10% increase to approximately 45% increase, approximately 10% increase to approximately 40% increase, approximately 10% increase to approximately 35% increase, approximately 10% increase to approximately 30% increase, approximately 10% increase to approximately 25% increase, approximately 10% increase to approximately 20% increase, approximately 10% increase to approximately 15% increase, approximately 15% increase to approximately 300% increase, approximately 15% increase to approximately 280% increase, approximately 15% increase to approximately 260% increase, approximately 15% increase to approximately 240% increase, approximately 15% increase to approximately 220% increase, approximately 15% increase to approximately 200% increase, approximately 15% increase to approximately 180% increase, approximately 15% increase to approximately 160% increase, approximately 15% increase to approximately 140% increase, approximately 15% increase to approximately 120% increase, approximately 15% increase to approximately 100% increase, approximately 15% increase to approximately 95% increase, approximately 15% increase to approximately 90% increase, approximately 15% increase to approximately 85% increase, approximately 15% increase to approximately 80% increase, approximately 15% increase to approximately 75% increase, approximately 15% increase to approximately 70% increase, approximately 15% increase to approximately 65% ​​increase, approximately 15% increase to approximately 60% increase,Approximately 15% increase to approximately 55% increase, approximately 15% increase to approximately 50% increase, approximately 15% increase to approximately 45% increase, approximately 15% increase to approximately 40% increase, approximately 15% increase to approximately 35% increase, approximately 15% increase to approximately 30% increase, approximately 15% increase to approximately 25% increase, approximately 15% increase to approximately 20% increase, approximately 20% increase to approximately 300% increase, approximately 20% increase to approximately 280% increase, approximately 20% increase to approximately 260% increase, approximately 20% increase to approximately 240% increase, approximately 20% increase to approximately 220% increase, approximately 20% increase to approximately 200% increase, approximately 20% increase to approximately 180% increase, approximately 20% increase to approximately 160% increase, approximately 20% increase to approximately 140% increase, approximately 2 0% increase to approximately 120% increase, approximately 20% increase to approximately 100% increase, approximately 20% increase to approximately 95% increase, approximately 20% increase to approximately 90% increase, approximately 20% increase to approximately 85% increase, approximately 20% increase to approximately 80% increase, approximately 20% increase to approximately 75% increase, approximately 20% increase to approximately 70% increase, approximately 20% increase to approximately 65% ​​increase, approximately 20% increase to approximately 60% increase, approximately 20% increase to approximately 55% increase, approximately 20% increase to approximately 50% increase, approximately 20% increase to approximately 45% increase, approximately 20% increase to approximately 40% increase, approximately 20% increase to approximately 35% increase, approximately 20% increase to approximately 30% increase, approximately 20% increase to approximately 25% increase, approximately 25% increase to approximately 300% increase, approximately 25% increase to approximately 280% increase, approximately 25% increase to approximately 260% increase, approximately 25% increase to approximately 240% increase, approximately 25% increase to approximately 220% increase, approximately 25% increase to approximately 200% increase, approximately 25% increase to approximately 180% increase, approximately 25% increase to approximately 160% increase, approximately 25% increase to approximately 140% increase, approximately 25% increase to approximately 120% increase, approximately 25% increase to approximately 100% increase, approximately 25% increase to approximately 95% increase, approximately 25% increase to approximately 90% increase, approximately 25% increase to approximately 85% increase, approximately 25% increase to approximately 80% increase, approximately 25% increase to approximately 75% increase, approximately 25% increase to approximately 70% increase, approximately 25% increase to approximately 65% ​​increase Increased, approximately 25% increase to approximately 60% increase, approximately 25% increase to approximately 55% increase, approximately 25% increase to approximately 50% increase, approximately 25% increase to approximately 45% increase, approximately 25% increase to approximately 40% increase, approximately 25% increase to approximately 35% increase, approximately 25% increase to approximately 30% increase, approximately 30% increase to approximately 300% increase, approximately 30% increase to approximately 280% increase, approximately 30% increase to approximately 260% increase, approximately 30% increase to approximately 240% increase, approximately 30% increase to approximately 220% increase, approximately 30% increase to approximately 200% increase, approximately 30% increase to approximately 180% increase, approximately 30% increase to approximately 160% increase, approximately 30% increase to approximately 140% increase, approximately 30% increase to approximately 120% increase,Approximately 30% increase to approximately 100% increase, approximately 30% increase to approximately 95% increase, approximately 30% increase to approximately 90% increase, approximately 30% increase to approximately 85% increase, approximately 30% increase to approximately 80% increase, approximately 30% increase to approximately 75% increase, approximately 30% increase to approximately 70% increase, approximately 30% increase to approximately 65% ​​increase, approximately 30% increase to approximately 60% increase, approximately 30% increase to approximately 55% increase, approximately 30% increase to approximately 50% increase, approximately 30% increase to approximately 45% increase, approximately 30% increase to approximately 40% increase, approximately 30% increase to approximately 35% increase, approximately 35% increase to approximately 300% increase, approximately 35% increase to approximately 280% increase, approximately 35% increase to approximately 260% increase, approximately 35% increase Approximately 240% increase, approximately 35% increase to approximately 220% increase, approximately 35% increase to approximately 200% increase, approximately 35% increase to approximately 180% increase, approximately 35% increase to approximately 160% increase, approximately 35% increase to approximately 140% increase, approximately 35% increase to approximately 120% increase, approximately 35% increase to approximately 100% increase, approximately 35% increase to approximately 95% increase, approximately 35% increase to approximately 90% increase, approximately 35% increase to approximately 85% increase, approximately 35% increase to approximately 80% increase, approximately 35% increase to approximately 75% increase, approximately 35% increase to approximately 70% increase, approximately 35% increase to approximately 65% ​​increase, approximately 35% increase to approximately 60% increase, approximately 35% increase to approximately 55% increase, approximately 35% increase to approximately 50% increase, approximately 35% increase to approximately 45% increase, approximately 35% increase to approximately 40% increase, approximately 40% increase to approximately 300% increase, approximately 40% increase to approximately 280% increase, approximately 40% increase to approximately 260% increase, approximately 40% increase to approximately 240% increase, approximately 40% increase to approximately 220% increase, approximately 40% increase to approximately 200% increase, approximately 40% increase to approximately 180% increase, approximately 40% increase to approximately 160% increase, approximately 40% increase to approximately 140% increase, approximately 40% increase to approximately 120% increase, approximately 40% increase to approximately 100% increase, approximately 40% increase to approximately 95% increase, approximately 40% increase to approximately 90% increase, approximately 40% increase to approximately 85% increase, approximately 40% increase to approximately 80% increase Increased, approximately 40% increase to approximately 75% increase, approximately 40% increase to approximately 70% increase, approximately 40% increase to approximately 65% ​​increase, approximately 40% increase to approximately 60% increase, approximately 40% increase to approximately 55% increase, approximately 40% increase to approximately 50% increase, approximately 40% increase to approximately 45% increase, approximately 45% increase to approximately 300% increase, approximately 45% increase to approximately 280% increase, approximately 45% increase to approximately 260% increase, approximately 45% increase to approximately 240% increase, approximately 45% increase to approximately 220% increase, approximately 45% increase to approximately 200% increase, approximately 45% increase to approximately 180% increase, approximately 45% increase to approximately 160% increase, approximately 45% increase to approximately 140% increase, approximately 45% increase to approximately 120% increase,Approximately 45% increase to approximately 100% increase, approximately 45% increase to approximately 95% increase, approximately 45% increase to approximately 90% increase, approximately 45% increase to approximately 85% increase, approximately 45% increase to approximately 80% increase, approximately 45% increase to approximately 75% increase, approximately 45% increase to approximately 70% increase, approximately 45% increase to approximately 65% ​​increase, approximately 45% increase to approximately 60% increase, approximately 45% increase to approximately 55% increase, approximately 45% increase to approximately 50% increase, approximately 50% increase to approximately 300% increase, approximately 50% increase to approximately 280% increase, approximately 50% increase to approximately 260% increase, approximately 50% increase to approximately 240% increase, approximately 50% increase to approximately 220% increase, approximately 50% increase to approximately 200% increase, approximately 50% increase Increased to approximately 180% increase, increased to approximately 160% increase, increased to approximately 140% increase, increased to approximately 120% increase, increased to approximately 100% increase, increased to approximately 95% increase, increased to approximately 90% increase, increased to approximately 85% increase, increased to approximately 80% increase, increased to approximately 75% increase, increased to approximately 70% increase, increased to approximately 65% ​​increase, increased to approximately 60% increase, increased to approximately 55% increase, increased to approximately 300% increase, increased to approximately 280% increase, increased to approximately 260% increase, increased to approximately 2 40% increase, approximately 55% increase to approximately 220% increase, approximately 55% increase to approximately 200% increase, approximately 55% increase to approximately 180% increase, approximately 55% increase to approximately 160% increase, approximately 55% increase to approximately 140% increase, approximately 55% increase to approximately 120% increase, approximately 55% increase to approximately 100% increase, approximately 55% increase to approximately 95% increase, approximately 55% increase to approximately 90% increase, approximately 55% increase to approximately 85% increase, approximately 55% increase to approximately 80% increase, approximately 55% increase to approximately 75% increase, approximately 55% increase to approximately 70% increase, approximately 55% increase to approximately 65% ​​increase, approximately 55% increase to approximately 60% increase, approximately 60% increase to approximately 300% increase, approximately 60% increase to approximately 280% increase, approximately 60% increase to approximately 260% increase, approximately 60% increase to approximately 240% increase, approximately 60% increase to approximately 220% increase, approximately 60% increase to approximately 200% increase, approximately 60% increase to approximately 180% increase, approximately 60% increase to approximately 160% increase, approximately 60% increase to approximately 140% increase, approximately 60% increase to approximately 120% increase, approximately 60% increase to approximately 100% increase, approximately 60% increase to approximately 95% increase, approximately 60% increase to approximately 90% increase, approximately 60% increase to approximately 85% increase, approximately 60% increase to approximately 80% increase, approximately 60% increase to approximately 75% increase, approximately 60% increase to approximately 70% increase, approximately 60% increase to approximately 65% ​​increase, approximately 65% ​​increase to approximately 300% increase,Approximately 65% ​​increase to approximately 280% increase, approximately 65% ​​increase to approximately 260% increase, approximately 65% ​​increase to approximately 240% increase, approximately 65% ​​increase to approximately 220% increase, approximately 65% ​​increase to approximately 200% increase, approximately 65% ​​increase to approximately 180% increase, approximately 65% ​​increase to approximately 160% increase, approximately 65% ​​increase to approximately 140% increase, approximately 65% ​​increase to approximately 120% increase, approximately, 65% increase to approximately 100% increase, approximately 65% ​​increase to approximately 95% increase, approximately 65% ​​increase to approximately 90% increase, approximately 65% ​​increase to approximately 85% increase, approximately 65% ​​increase to approximately 80% increase, approximately 65% ​​increase to approximately 75% increase, approximately 65% ​​increase to approximately 70% increase, approximately 70% increase to approximately 300% increase, approximately 70% increase to approximately 280% increase, approximately 70% increase to approximately 260% increase, approximately 70% increase to approximately 240% increase, approximately 70% increase to approximately 220% increase, approximately 70% increase to approximately 200% increase, approximately 70% increase to approximately 180% increase, approximately 70% increase to approximately 160% increase, approximately 70% increase to approximately 140% increase, approximately 70% increase to approximately 120% increase, approximately 70% increase to approximately 100% increase, approximately 70% increase to approximately 95% increase, approximately 70% increase to approximately 90% increase, approximately 70% increase to approximately 85% increase, approximately 70% increase to approximately 80% increase, approximately 70% increase to approximately 75% increase, approximately 75% increase to approximately 300% increase, approximately 75% increase to approximately 280% increase, approximately 75% increase to approximately 260% increase, approximately 75% increase to approximately 240% increase, approximately 75% increase to approximately 220% increase, approximately 75% increase to approximately 200% increase, approximately 75% increase to approximately 180% increase, approximately 75% increase to approximately 160% increase, approximately 75% increase to approximately 140% increase, approximately 75% increase to approximately 120% increase, approximately 75% increase to approximately 100% increase, approximately 75% increase to approximately 95% increase, approximately 75% increase to approximately 90% increase, approximately 75% increase to approximately 85% increase, approximately 75% increase to approximately 80% increase, approximately 80% increase to approximately 300% increase, approximately 80% increase to approximately 280% increase, approximately 80% increase to approximately 260% increase, approximately 80% increase to approximately 240% increase, approximately 80% increase to approximately 220% increase, approximately 80% increase to approximately 200% increase, approximately 80% increase to approximately 180% increase, approximately 80% increase to approximately 160% increase, approximately 80% increase to approximately 140% increase, approximately 80% increase to approximately 120% increase, approximately 80% increase to approximately 100% increase, approximately 80% increase to approximately 95% increase, approximately 80% increase to approximately 90% increase, approximately 8 0% increase to approximately 85% increase, approximately 85% increase to approximately 300% increase, approximately 85% increase to approximately 280% increase, approximately 85% increase to approximately 260% increase, approximately 85% increase to approximately 240% increase, approximately 85% increase to approximately 220% increase, approximately 85% increase to approximately 200% increase, approximately 85% increase to approximately 180% increase, approximately 85% increase to approximately 160% increase, approximately 85% increase to approximately 140% increase, approximately 85% increase to approximately 120% increase, approximately 85% increase to approximately 100% increase, approximately 85% increase to approximately 95% increase, approximately 85% increase to approximately 90% increase, approximately 90% increase to approximately 300% increase, approximately 90% increase to approximately 280% increase, approximately 90% increase to approximately 260% increase,Approximately 90% increase to approximately 240% increase, approximately 90% increase to approximately 220% increase, approximately 90% increase to approximately 200% increase, approximately 90% increase to approximately 180% increase, approximately 90% increase to approximately 160% increase, approximately 90% increase to approximately 140% increase, approximately 90% increase to approximately 120% increase, approximately 90% increase to approximately 100% increase, approximately 90% increase to approximately 95% increase, approximately 95% increase to approximately 300% increase, approximately 95% increase to approximately 280% increase, approximately 95% increase to approximately 260% increase, approximately 95% increase to approximately 240% increase, approximately 95% increase to approximately 220% increase, approximately 95% increase to approximately 200% increase, approximately 95% increase to approximately 180% increase, approximately 95% increase Approximately 160% increase, approximately 95% increase to approximately 140% increase, approximately 95% increase to approximately 120% increase, approximately 95% increase to approximately 100% increase, approximately 100% increase to approximately 300% increase, approximately 100% increase to approximately 280% increase, approximately 100% increase to approximately 260% increase, approximately 100% increase to approximately 240% increase, approximately 100% increase to approximately 220% increase, approximately 100% increase to approximately 200% increase, approximately 100% increase to approximately 180% increase, approximately 100% increase to approximately 160% increase, approximately 100% increase to approximately 140% increase, approximately 100% increase to approximately 120% increase, approximately 120% increase to approximately 300% increase, approximately 120% increase to approximately 280% increase, approximately 1 20% increase to approximately 260% increase, approximately 120% increase to approximately 240% increase, approximately 120% increase to approximately 220% increase, approximately 120% increase to approximately 200% increase, approximately 120% increase to approximately 180% increase, approximately 120% increase to approximately 160% increase, approximately 120% increase to approximately 140% increase, approximately 140% increase to approximately 300% increase, approximately 140% increase to approximately 280% increase, approximately 140% increase to approximately 260% increase, approximately 140% increase to approximately 240% increase, approximately 140% increase to approximately 220% increase, approximately 140% increase to approximately 200% increase, approximately 140% increase to approximately 180% increase, approximately 140% increase to approximately 160% increase, approximately 160% increase to approximately 300% increase, approximately 160% increase to approximately 280% increase, approximately 160% increase to approximately 260% increase, approximately 160% increase to approximately 240% increase, approximately 160% increase to approximately 220% increase, approximately 160% increase to approximately 200% increase, approximately 160% increase to approximately 180% increase, approximately 180% increase to approximately 300% increase, approximately 180% increase to approximately 280% increase, approximately 180% increase to approximately 260% increase, approximately 180% increase to approximately 240% increase, approximately 180% increase to approximately 220% increase, approximately 180% increase to approximately 200% increase, approximately 200% increase to approximately 300% increase, approximately 200% increase to approximately 280% increase, approximately 200% increase to approximately 260% increase,about a 200% increase to about a 240% increase, about a 200% increase to about a 220% increase, about a 220% increase to about a 300% increase, about a 220% increase to about a 280% increase, about a 220% increase to about a 260% increase, about a 220% increase to about a 240% increase, about a 240% increase to about a 300% increase, about a 240% increase to about a 280% increase, about a 240% increase to about a 260% increase, about a 260% increase to about a 300% increase, about a 260% increase to about a 280% increase, or about a 280% increase to about a 300% increase) (compared to a similar subject not receiving the treatment or receiving a different treatment).

[0214] Also provided herein is a method of increasing progression-free survival or progression-free survival rate in a subject or population of subjects (e.g., any of the exemplary subjects described herein) with unresectable advanced / metastatic pancreatic cancer, comprising the step of administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding of the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII) and the second target binding domain specifically binds to a ligand of TGF-βRII. Any of the exemplary multi-chain chimeric polypeptides described herein can be used in these methods.

[0215] As used herein, the term "progression-free survival" refers to the length of time during and / or after treatment that a subject survives without cancer progression. Progression-free survival can be based on anatomical measurements, for example, of tumor size or volume, as determined using, for example, X-ray, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET). The term "progression-free survival rate" refers to the percentage of subjects who survive without cancer progression at a specified time (e.g., 6 months, 1 year, etc.) from the start of treatment.

[0216] In some embodiments, the methods described herein provide for an increase in progression-free survival or progression-free survival rate (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 110% increase, at least a 120% increase, at least a 130% increase, at least a 140% increase, at least a 150% increase, at least a 160% increase, at least a 200% increase, at least a 250% increase, at least a 300% increase, at least a 350% increase, at least a 400% increase, at least a 450% increase, at least a 500% increase, at least a 550% increase, at least a 600% increase, at least a 650% increase, at least a 700% increase, at least a 750% increase, at least a 800% increase, at least a 850% increase, at least a 900% increase, at least a 950% increase, at least a 1000% increase, at least a 1100% increase, at least a 1200% increase, at least a 1300% increase, at least a 1400% increase, at least a 1500% increase, at least a 1600 or at least a 170% increase, at least a 180% increase, at least a 190% increase, at least a 200% increase, at least a 210% increase, at least a 220% increase, at least a 230% increase, at least a 240% increase, at least a 250% increase, at least a 260% increase, at least a 270% increase, at least a 280% increase, at least a 290% increase, or at least a 300% increase, or about a 1% increase to about a 300% increase (or any subrange of this range described herein)) (e.g., compared to the progression-free survival or progression-free survival rate in the subject prior to administration of the multi-chain chimeric polypeptide, or compared to the progression-free survival or progression-free survival rate in a similar subject who has received another treatment (e.g., any of the exemplary first-line and / or second-line treatments for pancreatic cancer described herein).

[0217] Also provided herein are methods for increasing progression-free intervals in a subject or population of subjects (e.g., any of the exemplary subjects described herein) with unresectable, advanced / metastatic pancreatic cancer, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target-binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target-binding domain specifically binds to a ligand of TGF-βRII. Any of the exemplary multi-chain chimeric polypeptides described herein can be used in these methods.

[0218] As used herein, the term "progression-free interval" can refer to the length of time from initiation of treatment until the cancer progresses and / or metastasizes to other parts of the subject's body. Cancer progression and / or metastasis can be determined, for example, by imaging the subject using, for example, X-rays, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).

[0219] In some embodiments, the methods described herein provide for an increase in progression-free interval (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 110% increase, at least a 120% increase, at least a 130% increase, at least a 140% increase, at least a 150% increase, at least a 160% increase, at least a 170% increase, at least a 180% increase, at least a 190% increase, at least a 200% increase, at least a 210% increase, at least a 220% increase, at least a 230% increase, at least a 240% increase, at least a 250% increase, at least a 260% increase, at least a 270% increase, at least a 280% increase, at least a 290% increase, at least a 300% increase, at least a 310% increase, at least a 320% increase, at least a 330% increase, at least a 340% increase, at least a 350% increase, at least a 360% increase, at least a 370% increase, at least a 380% increase, at least a 390% increase, at least a 400% increase, % increase, at least a 170% increase, at least a 180% increase, at least a 190% increase, at least a 200% increase, at least a 210% increase, at least a 220% increase, at least a 230% increase, at least a 240% increase, at least a 250% increase, at least a 260% increase, at least a 270% increase, at least a 280% increase, at least a 290% increase, or at least a 300% increase, or about a 1% increase to about a 300% increase (or any subrange of this range described herein)) (e.g., as compared to the progression-free period in the subject prior to administration of the multi-chain chimeric polypeptide, or as compared to the progression-free period in a similar subject who has received another treatment (e.g., any of the exemplary first-line and / or second-line treatments for pancreatic cancer described herein)).

[0220] Also provided herein are methods for increasing the duration of response in a subject or a population of subjects with unresectable, advanced / metastatic pancreatic cancer (any of the exemplary subjects described herein), comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII. Any of the exemplary multi-chain chimeric polypeptides described herein can be used in these methods.

[0221] As used herein, the term "duration of response" can refer to the length of time from response to treatment to the progression of cancer in a subject.For example, duration of response can be a measure of the length of time that a tumor continues to respond to treatment without cancer growing or metastasizing.In some examples, the growth or metastasis of pancreatic cancer in a subject can be determined by imaging the subject, for example, using X-ray, ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET).

[0222] In some embodiments, the methods described herein provide for an increase in duration of response in a subject or population of subjects (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 110% increase, at least a 120% increase, at least a 130% increase, at least a 140% increase, at least a 150% increase, at least a 160% increase, at least a 170% increase, at least a 180% increase, at least a 190% increase, at least a 200% increase, at least a 210% increase, at least a 220% increase, at least a 230% increase, at least a 240% increase, at least a 250% increase, at least a 260% increase, at least a 270% increase, at least a 280% increase, at least a 290% increase, at least a 300% increase, at least a 310% increase, at least a 320% increase, at least a 330% increase, at least a 340% increase, at least a 350% increase, at least a 360% increase, at least a 370% increase, at least a 380% increase, at least a 390% increase, at least a The multi-chain chimeric polypeptide may result in a 180% increase, at least a 190% increase, at least a 200% increase, at least a 210% increase, at least a 220% increase, at least a 230% increase, at least a 240% increase, at least a 250% increase, at least a 260% increase, at least a 270% increase, at least a 280% increase, at least a 290% increase, or at least a 300% increase, or an increase of about 1% to about 300% (or any subrange of this range described herein)) (e.g., compared to the duration of response in the subject prior to administration of the multi-chain chimeric polypeptide (e.g., in response prior to administration of a first-line and / or second-line therapy for pancreatic cancer), or compared to the duration of response in a similar subject who has received another treatment (e.g., any of the exemplary first-line and / or second-line treatments for pancreatic cancer described herein)).

[0223] Also provided herein are methods for increasing overall survival in a population of subjects with unresectable, advanced / metastatic pancreatic cancer (e.g., any of the exemplary subjects described herein), comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the multi-chain chimeric polypeptide comprises: (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 through the binding between the first domain and the second domain of the pair of affinity domains, and wherein the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII. Any of the exemplary multi-chain chimeric polypeptides described herein can be used in these methods.

[0224] As used herein, the term "overall survival" can refer to the length of time from either the date of diagnosis or the date of treatment initiation that a group of subjects is still alive. Overall survival can determine how long a group of subjects receiving a cancer treatment regimen live compared to another group of similar subjects in a control group (e.g., receiving another treatment, e.g., a first-line and / or second-line treatment for pancreatic cancer, e.g., an exemplary first-line and / or second-line treatment for pancreatic cancer described herein).

[0225] In some embodiments, the methods described herein result in an increase in overall survival in a subject (e.g., at least a 1% increase, at least a 5% increase, at least a 10% increase, at least a 15% increase, at least a 20% increase, at least a 25% increase, at least a 30% increase, at least a 35% increase, at least a 40% increase, at least a 45% increase, at least a 50% increase, at least a 55% increase, at least a 60% increase, at least a 65% increase, at least a 70% increase, at least a 75% increase, at least a 80% increase, at least a 85% increase, at least a 90% increase, at least a 95% increase, at least a 100% increase, at least a 110% increase, at least a 120% increase, at least a 130% increase, at least a 140% increase, at least a 150% increase, at least a 160% increase, at least a 170% increase, at least a 180% increase, at least a 200% increase, at least a 210% increase, at least a 220% increase, at least a 230% increase, at least a 240% increase, at least a 250% increase, at least a 260% increase, at least a 270% increase, at least a 280% increase, at least a 290% increase, at least a 300% increase, at least a 310% increase, at least a 320% increase, at least a 330% increase, at least a 340% increase, at least a 350% increase, at least a 360% increase, at least a 370% increase, at least a 380% increase, at least a 390% increase, at least a 400% increase, at least a 410% increase or at least a 100% increase, at least a 190% increase, at least a 200% increase, at least a 210% increase, at least a 220% increase, at least a 230% increase, at least a 240% increase, at least a 250% increase, at least a 260% increase, at least a 270% increase, at least a 280% increase, at least a 290% increase, or at least a 300% increase, or about a 1% increase to about a 300% increase (or any subrange of this range described herein)) (e.g., as compared to the progression-free period in the subject prior to administration of the multi-chain chimeric polypeptide, or as compared to another treatment (e.g., as compared to another population of similar subjects in a control group (e.g., receiving another treatment, e.g., a first-line and / or second-line treatment for pancreatic cancer, e.g., an exemplary first-line and / or second-line treatment for pancreatic cancer described herein)).

[0226] As used herein, the term "subject" can refer to an organism, typically a mammal (e.g., a human). In some embodiments, a subject is a patient.

[0227] In some embodiments, the subject is 18 years of age or older (e.g., 19 years of age or older, 20 years of age or older, 25 years of age or older, 30 years of age or older, 35 years of age or older, 40 years of age or older, 45 years of age or older, 50 years of age or older, 55 years of age or older, 60 years of age or older, 65 years of age or older, 70 years of age or older, 75 years of age or older, 80 years of age or older, 85 years of age or older, 90 years of age or older, 95 years of age or older, or 100 years of age).

[0228] In some embodiments, the subject has previously been treated with a standard first-line systemic therapy for pancreatic cancer, and the subject's pancreatic cancer has progressed on and / or been intolerant to the previous therapy. In some embodiments, the subject has previously been treated with a standard first-line systemic therapy for pancreatic cancer, and the subject was intolerant to the first-line systemic therapy.

[0229] In some aspects, the standard first-line systemic therapy includes one or more of FOLFIRINOX, modified FOLFINIROX, gemcitabine, albumin-bound paclitaxel, cisplatin, erlotinib, capecitabine, docetaxel, fluoropyrimidines, and oxaliplatin.

[0230] In some aspects, the first-line systemic therapy comprises one of (i) FOLFIRINOX, (ii) modified FOLFIRINOX, (iii) gemcitabine and albumin-bound paclitaxel, (iv) gemcitabine and erlotinib, (v) gemcitabine, (vi) gemcitabine and capecitabine, (vii) gemcitabine, docetaxel, and capecitabine, and (viii) a fluoropyrimidine and oxaliplatin.

[0231] In some embodiments, the subject has previously been identified as having a BRCA1, BRCA2, or PALB2 mutation, and therefore the first-line systemic therapy comprises one of: (i) FOLFIRINOX, (ii) modified FOLFIRINOX, and (iii) gemcitabine and cisplatin.

[0232] In some embodiments, the subject has previously been treated with a second-line or post-line systemic therapy for pancreatic cancer, and the subject's pancreatic cancer has progressed on the previous treatment and / or been intolerant to the previous treatment. In some embodiments, the second-line or post-line systemic therapy includes one or more of another first-line systemic therapy (e.g., any of the exemplary first-line systemic therapies described herein), 5-fluorouracil, leucovorin, liposomal irinotecan, irinotecan, FOLFIRINOX, modified FOLFIRINOX, oxaliplatin, FOLFOX, capecitabine, gemcitabine, albumin-bound paclitaxel, cisplatin, erlotinib, pembrolizumab, larotrectinib, and entrectinib. In some embodiments, the second-line or post-line systemic therapy is another first-line systemic therapy (e.g., any of the exemplary first-line systemic therapies described herein).

[0233] In some aspects, the second-line or back-line systemic therapy comprises one of: (i) 5-fluorouracil, leucovorin, and liposomal irinotecan, (ii) 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI), (iii) FOLFIRINOX or modified FOLFIRINOX, (iv) oxaliplatin, 5-fluorouracil, and leucovorin (OFF), (v) FOLFOX, (vi) capecitabine and oxaliplatin, (vii) capecitabine, and (viii) 5-fluorouracil continuous infusion.

[0234] In some embodiments, the subject has previously been treated with a fluoropyrimidine-based therapy, such that the second-line or post-line systemic therapy comprises one of: (i) gemcitabine, (ii) gemcitabine and albumin-bound paclitaxel, and (iii) gemcitabine with erlotinib.

[0235] In some embodiments, the subject has been previously treated with a fluoropyrimidine-based therapy and previously identified as having a BRCA1, BRCA2, or PALB2 mutation, so the second-line or post-line systemic therapy comprises gemcitabine and cisplatin. In some embodiments, the subject has been previously treated with a fluoropyrimidine-based therapy and has not received prior treatment with irinotecan, so the second-line or post-line systemic therapy comprises 5-fluorouracil, leucovorin, and liposomal irinotecan.

[0236] In some embodiments, the subject has previously been identified as having an MSI-H or dMMR tumor, and therefore the second-line or post-line systemic therapy comprises pembrolizumab. In some embodiments, the subject has previously been identified as having an NTRK gene fusion, and therefore the second-line or post-line systemic therapy comprises larotrectinib or entrectinib.

[0237] In some embodiments, the subject has distant metastatic disease. In some embodiments, the subject has adequate cardiac, pulmonary, hepatic, and renal function. In some embodiments, the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, or 2.

[0238] In some embodiments, the subject has a life expectancy of at least 12 weeks (at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 22 weeks, or at least 24 weeks) prior to the administering step. In some embodiments, the subject has measurable disease as assessed by imaging studies prior to the administering step. In some embodiments, the subject has received prior radiation therapy at least 4 weeks prior to the administering step. In some embodiments, any acute effects of any prior therapy in the subject have been reduced to baseline or to a grade of 1 or less on the NCI CTCAE v5.0 prior to the administering step.

[0239] In some embodiments, the subject has an absolute neutrophil count of 1,500 or more per microliter; a platelet count of 100,000 or more per microliter; a hemoglobin level of 9 g or more per deciliter; a glomerular filtration rate (GFR) of more than 40 mL / min or a serum creatinine level of 1.5 x the upper limit of normal (ULN); a total bilirubin level of 2.0 x the ULN or less, or 3.0 x the ULN or less if the subject has Gilbert's syndrome; and aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels of 2.5 x the ULN or less, or 5.0 x the ULN or less if liver metastases are present. In some embodiments, the subject has a pulmonary function test (PFT) level of greater than 50% forced expiratory volume (FEV1) if they have symptomatic or previously known functional impairment.

[0240] In some embodiments, the subject is a female and the female has a negative pregnancy test within 14 days prior to the administering step, hi some embodiments, the female is using birth control or is surgically sterilized for at least 14 days prior to and during the administering step.

[0241] In some embodiments, the subject is male and the subject uses a barrier method of contraception during and for at least 28 days after the administering step.

[0242] In some embodiments, the subject does not have a history of clinically significant vascular disease. In some embodiments, the subject does not have a corrected QT interval (QTc) of 470 milliseconds or greater by Fridericia correction.

[0243] In some embodiments, the subject does not have untreated CNS metastases. In some embodiments, the subject has not received prior treatment for CNS metastases, and the subject is neurologically stable for at least two weeks prior to the administering step. In some embodiments, the subject does not receive a corticosteroid during the administering step. In some embodiments, the subject receives a stable or tapering dose of a corticosteroid of 10 mg or less daily during the administering step.

[0244] In some embodiments, the subject has not undergone surgery, radiation therapy, chemotherapy, other immunotherapy, or investigational therapy within 14 days prior to the administering step. In some embodiments, the subject has no other prior malignancies, except for adequately treated basal or squamous cell skin cancer, cervical intraepithelial neoplasia, adequately treated stage I or II cancer in which the subject is currently in complete remission, or any other cancer in which the subject is disease-free for three years following surgical treatment.

[0245] In some embodiments, the subject has no known hypersensitivity or history of allergic reaction caused by compounds of similar chemical or biological composition to the multi-chain chimeric polypeptide, hi some embodiments, the subject has not received prior treatment with a TGF-beta antagonist or IL-15 or an analog thereof.

[0246] In some embodiments, the subject is not receiving concurrent herbal or unconventional therapy. In some embodiments, the subject does not have an autoimmune disease requiring active treatment. In some embodiments, the subject does not have a condition requiring systemic corticosteroid therapy or immunosuppressive therapy within 14 days of the administering step. In some embodiments, the subject does not have an active autoimmune disease and is receiving an inhaled or topical steroid dose of 10 mg or less prednisone equivalent daily, or an adrenal replacement steroid dose.

[0247] In some embodiments, the subject does not have an active systemic infection requiring parenteral antibiotic therapy. In some embodiments, the subject has not previously received an organ allograft or allogeneic transplant. In some embodiments, the subject has not been identified or diagnosed as being HIV positive or having AIDS.

[0248] In some embodiments, the subject is female, and the female is not pregnant or breastfeeding. In some embodiments, the subject does not have any ongoing toxicity from prior therapy. In some embodiments, ongoing toxicity is greater than Grade 1 using NCI CTCAE v5.0 or above baseline. In some embodiments, ongoing toxicity excludes peripheral neuropathy, alopecia, and fatigue.

[0249] In some embodiments, the subject does not have a psychotic illness.

[0250] In some embodiments, the multi-chain chimeric polypeptide is administered subcutaneously to the subject. In some embodiments, the subject receives a single dose of the multi-chain chimeric polypeptide. In some embodiments, the single dose is about 0.1 mg of the multi-chain chimeric polypeptide per kg of the subject's body weight (mg / kg), about 0.25 mg / kg, about 0.5 mg / kg, about 0.8 mg / kg, or about 1.2 mg / kg.

[0251] In some embodiments, the subject receives two or more doses of the multi-chain chimeric polypeptide over the course of treatment, hi some embodiments, at least one of the two or more doses is 0.1 mg of the multi-chain chimeric polypeptide per kg of the subject's body weight (mg / kg), about 0.25 mg / kg, about 0.5 mg / kg, about 0.8 mg / kg, or about 1.2 mg / kg.

[0252] In some embodiments, the treatment period is about 4 weeks. [Example]

[0253] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0254] Example 1: Generation and characterization of TGFRt15-TGFRs fusion protein Fusion protein complexes composed of TGFβ receptor II / IL-15RαSu and TGFβ receptor II / TF / IL-15 fusion proteins were generated (Figures 1 and 2). Human TGFβ receptor II (Ile24-Asp159), tissue factor 219, and IL-15 sequences were obtained from the UniProt website, and DNA for these sequences was synthesized by Genewiz. Specifically, two TGFβ receptor II sequences were joined using a G4S(3) linker to generate a single-chain form of TGFβ receptor II, which was then directly ligated to the N-terminal coding region of tissue factor 219 followed by the N-terminal coding region of IL-15 to generate the construct.

[0255] The nucleic acid and protein sequences of a construct containing two TGFβ receptor IIs linked to the N-terminus of tissue factor 219 followed by the N-terminus of IL-15 are shown below.

[0256] The nucleic acid sequences (including signal peptide sequences) of the two TGFβ receptor II / TF / IL-15 constructs are as follows: (signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (Two human TGFβ receptor II fragments) TIFF2025508939000049.tif115170(Human tissue factor 219) TIFF2025508939000050.tif89170 (human IL-15) TIFF2025508939000051.tif48170

[0257] The amino acid sequence of the TGFβ receptor II / TF / IL-15 fusion protein (including the leader sequence) is as follows: (signal peptide) MKWVTFISLLFLFSSAYS (human TGFβ receptor II) TIFF2025508939000052.tif40170(Human tissue factor 219) TIFF2025508939000053.tif32170 (human IL-15) TIFF2025508939000054.tif20170

[0258] A construct was also made by attaching two TGFβ receptor IIs directly to the IL-15RαSu chain, which was synthesized by Genewiz. The nucleic acid and protein sequences of the construct containing TGFβ receptor IIs linked to the N-terminus of IL-15RαSu are shown below.

[0259] The nucleic acid sequence of the TGFβ receptor II / IL-15RαSu construct (including the signal peptide sequence) is as follows: (signal peptide) ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTCTCCAGCGCCTACTCC (Two human TGFβ receptor II fragments) TIFF2025508939000055.tif115170 (human IL-15Rα sushi domain) TIFF2025508939000056.tif25170

[0260] The amino acid sequences of the two TGFβ receptor II / IL-15RαSu constructs (including the signal peptide sequence) are as follows: (signal peptide) MKWVTFISLLFLFSSAYS (Two human TGFβ receptor II extracellular domains) TIFF2025508939000057.tif38170 (human IL-15Rα sushi domain) ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR

[0261] In some cases, the leader peptide is cleaved from the intact polypeptide to generate a mature form that may be soluble or secreted.

[0262] The TGFβR / IL-15Rα and TGFβR / TF / IL-15 constructs were cloned into modified retroviral expression vectors as previously described (Hughes MS, Yu YY, Dudley ME, Zheng Z, Robbins PF, Li Y, et al. Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther 2005;16:457-72), and the expression vectors were transfected into CHO-K1 cells. Coexpression of the two constructs in CHO-K1 cells allowed the formation and secretion of soluble TGFβR / TF / IL-15:TGFβR / IL-15Rα protein complexes (termed TGFRt15-TGFRs), which could be purified by anti-TF IgG1 affinity chromatography and other methods.

[0263] Effect of TGFRt15-TGFRs on TGFβ1 activity in HEK-Blue TGFβ cells To assess the activity of TGFβRII in TGFRt15-TGFRs, the effect of TGFRt15-16s21 on the activity of TGFβ1 in HEK-Blue TGFβ cells was analyzed. HEK-Blue TGFβ cells (Invivogen) were washed twice with prewarmed PBS and cultured at 5 × 10 in test medium (DMEM, 10% heat-inactivated FCS, 1 × glutamine, 1 × anti-anti, and 2 × glutamine). 5 In a flat-bottom 96-well plate, 50 μL of cells were added to each well (2.5 × 10 4Cells / well), followed by 50 μL of 0.1 nM TGFβ1 (R&D Systems) was added. TGFRt15-16s21 or TGFR-Fc (R&D Systems), prepared in a 1:3 serial dilution, were then added to the plate to reach a total volume of 200 μL. After 24 h of incubation at 37 °C, 40 μL of induced HEK-Blue TGFβ cell supernatant was added to 160 μL of pre-warmed QUANTI-Blue (Invivogen) in a flat-bottom 96-well plate and incubated at 37 °C for 1 to 3 h. OD values ​​were then determined at 620–655 nM using a plate reader (Multiscan Sky) (Figure 3). The IC of each protein sample was 50 The IC values ​​of TGFRt15-TGFRs and TGFR-Fc were calculated using GraphPad Prism 7.04. 50 were 216.9 pM and 460.6 pM, respectively. These results indicated that the TGFβRII domain in TGFRt15-TGFRs could block the activity of TGFβ1 in HEK-Blue TGFβ cells.

[0264] IL-15 in TGFRt15-TGFRs promotes proliferation of 32D β cells containing IL-2Rβ and the common γ chain To evaluate the activity of IL-15 in TGFRt15-TGFRs, we compared the IL-15 activity of TGFRt15-TGFRs with that of recombinant IL-15 using 32Dβ cells expressing IL2Rβ and the common γ chain, and assessed their effect on promoting cell proliferation. IL-15-dependent 32Dβ cells were washed five times with IMDM-10% FBS and plated in wells at 2 × 10 4Cells were seeded at 10 ... 50 TGFRt15-TGFRs and IL-15 promoted the proliferation of 32D β cells, with the concentrations of TGFRt15-TGFRs and IL-15 being 1901 pM and 10.63 pM, respectively.

[0265] Detection of IL-15 and TGFβRII domains in TGFRt15-TGFRs with corresponding antibodies using ELISA A 96-well plate was coated with 100 μL (8 μg / mL) of anti-TF IgG1 in R5 (coating buffer) and incubated for 2 hours at room temperature (RT). The plate was washed three times and blocked with 100 μL of 1% BSA in PBS. TGFRt15-TGFRs were added in a 1:3 serial dilution and incubated for 60 minutes at RT. After three washes, 50 ng / mL of biotinylated anti-IL-15 antibody (BAM247, R&D Systems) or 200 ng / mL of biotinylated anti-TGFbRII antibody (BAF241, R&D Systems) was added to the wells and incubated for 60 minutes at RT. Plates were then washed three times, and 100 μL of 0.25 μg / mL HRP-SA (Jackson ImmunoResearch) was added per well and incubated at RT for 30 minutes. Plates were then washed four times and incubated with 100 μL of ABTS for 2 minutes at RT. Absorbance was read at 405 nm. As shown in Figures 5A and 5B, the IL-15 and TGFβRII domains in TGFRt15-TGFRs were detected by the respective antibodies.

[0266] Purification and elution chromatography of TGFRt15-TGFRs from an anti-TF antibody affinity column TGFRt15-TGFRs harvested from cell cultures were loaded onto an anti-TF antibody affinity column equilibrated with 5 column volumes of PBS. After loading the sample, the column was washed with 5 column volumes of PBS and subsequently eluted with 6 column volumes of 0.1 M acetic acid (pH 2.9). The A280 elution peak was collected and then neutralized with 1 M Tris base to pH 7.5-8.0. The neutralized sample was then buffer-exchanged into PBS using an Amicon centrifugal filter with a 30 kDa molecular weight cutoff. As shown in Figure 6, the anti-TF antibody affinity column bound TGFRt15-TGFRs containing TF as a fusion partner. The buffer-exchanged protein sample was stored at 2-8 °C for further biochemical analysis and biological activity testing. After each elution, the anti-TF antibody affinity column was stripped using 6 column volumes of 0.1 M glycine (pH 2.5). It was then neutralized with 5 column volumes of PBS and 7 column volumes of 20% ethanol for storage. The anti-TF antibody affinity column was connected to a GE Healthcare AKTA Avant system. The flow rate for all steps was 4 mL / min except for the elution step, which was 2 mL / min.

[0267] Analytical size exclusion chromatography (SEC) analysis of TGFRt15-TGFRs A Superdex 200 Increase 10 / 300 GL gel filtration column (GE Healthcare) was connected to an AKTA Avant system (GE Healthc...

Claims

1. 1. A method of treating unresectable advanced / metastatic pancreatic cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (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, the first chimeric polypeptide and the second chimeric polypeptide are associated through binding between the first domain and the second domain of the pair of affinity domains; and the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII; The method.

2. 1. A method for improving objective response rate in a subject with unresectable advanced / metastatic pancreatic cancer, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (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, the first chimeric polypeptide and the second chimeric polypeptide are associated through binding between the first domain and the second domain of the pair of affinity domains; and the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII; The method.

3. 1. A method of increasing progression-free survival or progression-free survival rate in a subject or population of subjects with unresectable advanced / metastatic pancreatic cancer, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (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, the first chimeric polypeptide and the second chimeric polypeptide are associated through binding between the first domain and the second domain of the pair of affinity domains; and the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII; The method.

4. 1. A method of increasing progression-free interval in a subject or population of subjects with unresectable advanced / metastatic pancreatic cancer, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (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, the first chimeric polypeptide and the second chimeric polypeptide are associated through binding between the first domain and the second domain of the pair of affinity domains; and the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII; The method.

5. 1. A method of increasing duration of response in a subject or population of subjects with unresectable advanced / metastatic pancreatic cancer, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (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, the first chimeric polypeptide and the second chimeric polypeptide are associated through binding between the first domain and the second domain of the pair of affinity domains; and the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII; The method.

6. 1. A method of increasing overall survival in a population of subjects with unresectable advanced / metastatic pancreatic cancer, the method comprising administering to each subject of the population a therapeutically effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (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, the first chimeric polypeptide and the second chimeric polypeptide are associated through binding between the first domain and the second domain of the pair of affinity domains; and the first target binding domain specifically binds to a ligand of TGF-β receptor II (TGF-βRII), and the second target binding domain specifically binds to a ligand of TGF-βRII; The method.

7. The method of any one of claims 1 to 6, wherein the subject is aged 18 years or older.

8. 8. The method of any one of claims 1 to 7, wherein the subject has previously been treated with a standard first-line systemic therapy for pancreatic cancer, and the subject's pancreatic cancer has progressed on and / or been intolerant to the previous treatment.

9. The method of any one of claims 1 to 7, wherein the subject has previously been treated with a standard first-line systemic therapy for pancreatic cancer, and the subject was intolerant to the first-line systemic therapy.

10. 10. The method of claim 8 or 9, wherein the standard first line systemic therapy comprises one or more of FOLFIRINOX, modified FOLFINIROX, gemcitabine, albumin-bound paclitaxel, cisplatin, erlotinib, capecitabine, docetaxel, fluoropyrimidines, and oxaliplatin.

11. said first-line systemic therapy comprising: (i) FOLFIRINOX, (ii) FOLFIRINOX with modifications; (iii) gemcitabine and albumin-bound paclitaxel; (iv) gemcitabine and erlotinib, (v) gemcitabine, (vi) gemcitabine and capecitabine, (vii) gemcitabine, docetaxel, and capecitabine, and (viii) fluoropyrimidine and oxaliplatin The method of claim 10, comprising one of:

12. the subject has previously been identified as having a BRCA1, BRCA2, or PALB2 mutation, and the first-line systemic therapy comprises: (i) FOLFIRINOX, (ii) FOLFIRINOX with modifications; and (iii) gemcitabine and cisplatin The method of claim 10, comprising one of:

13. 13. The method of any one of claims 1-12, wherein the subject has previously been treated with second-line or back-line systemic therapy for pancreatic cancer, and the subject's pancreatic cancer has progressed on and / or been intolerant to the previous treatment.

14. 14. The method of claim 13, wherein the second line or rear line systemic therapy comprises one or more of another first line systemic therapy, 5-fluorouracil, leucovorin, liposomal irinotecan, irinotecan, FOLFIRINOX, modified FOLFIRINOX, oxaliplatin, FOLFOX, capecitabine, gemcitabine, albumin-bound paclitaxel, cisplatin, erlotinib, pembrolizumab, larotrectinib, and entrectinib.

15. 15. The method of claim 14, wherein the second line or rear line systemic therapy is another first line systemic therapy.

16. The second line or rear line systemic therapy (i) 5-fluorouracil, leucovorin, and liposomal irinotecan; (ii) 5-fluorouracil, leucovorin, and irinotecan (FOLFIRI); (iii) FOLFIRINOX or modified FOLFIRINOX; (iv) oxaliplatin, 5-fluorouracil, and leucovorin (OFF); (v) FOLFOX, (vi) capecitabine and oxaliplatin, (vii) capecitabine, and (viii) 5-fluorouracil continuous infusion The method of claim 14 , comprising one of:

17. The subject has been previously treated with a fluoropyrimidine-based therapy, and the second-line or post-line systemic therapy comprises: (i) gemcitabine, (ii) gemcitabine and albumin-bound paclitaxel, and (iii) gemcitabine with erlotinib The method of claim 14 , comprising one of:

18. 15. The method of claim 14, wherein the subject has been previously treated with a fluoropyrimidine-based therapy and previously identified as having a BRCA1, BRCA2, or PALB2 mutation, and the second-line or rear-line systemic therapy comprises gemcitabine and cisplatin.

19. 15. The method of claim 14, wherein the subject has been previously treated with a fluoropyrimidine-based therapy and has not received prior treatment with irinotecan, and the second-line or back-line systemic therapy comprises 5-fluorouracil, leucovorin, and liposomal irinotecan.

20. 15. The method of claim 14, wherein the subject was previously identified as having an MSI-H or dMMR tumor and the second line or rear line systemic therapy comprises pembrolizumab.

21. 15. The method of claim 14, wherein the subject was previously identified as having a NTRK gene fusion and the second line or rear line systemic therapy comprises larotrectinib or entrectinib.

22. The method of any one of claims 1 to 21, wherein the subject has distant metastatic disease.

23. The method of any one of claims 1 to 22, wherein the subject has adequate cardiac, pulmonary, hepatic and renal function.

24. 24. The method of any one of claims 1 to 23, wherein the subject has an Eastern Cooperative Oncology Group (ECOG) performance status of 0, 1, or 2.

25. The method of any one of claims 1 to 24, wherein the subject has a life expectancy of at least 12 weeks prior to the administering step.

26. The method of any one of claims 1 to 25, wherein the subject has been determined to have measurable disease as assessed by imaging studies prior to said administering step.

27. The method of any one of claims 1 to 26, wherein the subject has undergone prior radiation therapy at least 4 weeks prior to the administering step.

28. 28. The method of any one of claims 1-27, wherein any acute effects of any prior therapy in the subject have been reduced to baseline or to an NCI CTCAE v5.0 Grade of 1 or less prior to the administering step.

29. The object is an absolute neutrophil count of 1,500 or more per microliter; a platelet count of 100,000 or more per microliter; Hemoglobin level of 9g or more per deciliter A glomerular filtration rate (GFR) of more than 40 mL / min or a serum creatinine level of less than 1.5 x the upper limit of normal (ULN); A total bilirubin level of less than or equal to 2.0 x ULN, or, if the subject has Gilbert's syndrome, less than or equal to 3.0 x ULN; and Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels of 2.5x ULN or less, or 5.0x ULN or less if liver metastases are present The method according to any one of claims 1 to 28, comprising:

30. 30. The method of any one of claims 1 to 29, wherein the subject, if symptomatic or with previously known impairment, has a pulmonary function test (PFT) value above 50% forced expiratory volume (FEV1).

31. The method of any one of claims 1 to 30, wherein the subject is a female and the female has a negative pregnancy test within 14 days prior to the administering step.

32. 32. The method of claim 31, wherein said female is on contraception or has been surgically sterilized for at least 14 days prior to and during said administering step.

33. 31. The method of any one of claims 1 to 30, wherein the subject is male and the subject is using a barrier method of contraception during and for at least 28 days after the administering step.

34. The method of any one of claims 1 to 33, wherein the subject has no history of clinically significant vascular disease.

35. 35. The method of any one of claims 1 to 34, wherein the subject does not have a corrected QT interval (QTc) of 470 milliseconds or greater by Fridericia correction.

36. The method of any one of claims 1 to 35, wherein the subject does not have untreated CNS metastases.

37. 36. The method of any one of claims 1 to 35, wherein the subject has undergone prior treatment for CNS metastases and the subject is neurologically stable for at least two weeks prior to the administering step.

38. 38. The method of any one of claims 1 to 37, wherein the subject does not receive a corticosteroid during the administering step.

39. 38. The method of any one of claims 1-37, wherein the subject receives a stable or tapering dose of a corticosteroid of 10 mg or less daily during the administering step.

40. 40. The method of any one of claims 1-39, wherein the subject has not undergone surgery, radiation therapy, chemotherapy, other immunotherapy, or an investigational therapy within 14 days prior to said administering step.

41. The object is Adequately treated basal cell or squamous cell skin cancer, Cervical carcinoma in situ, a well-treated stage I or II cancer in which the subject is currently in complete remission; or Any other cancer, wherein the subject is disease-free for three years after surgical treatment.

41. The method of any one of claims 1 to 40, wherein the patient is free of any other preceding malignancies, except for:

42. The method of any one of claims 1 to 41, wherein the subject has no known hypersensitivity or history of allergic reactions caused by compounds of similar chemical or biological composition to the multi-chain chimeric polypeptide.

43. The method of any one of claims 1 to 42, wherein the subject has not had prior treatment with a TGF-beta antagonist or IL-15 or an analogue thereof.

44. 44. The method of any one of claims 1 to 43, wherein the subject is not receiving any concomitant herbal or unconventional therapy.

45. The method of any one of claims 1 to 44, wherein the subject does not have an autoimmune disease requiring active treatment.

46. 46. ​​The method of any one of claims 1 to 45, wherein the subject does not have a condition requiring systemic treatment with corticosteroids or immunosuppressive treatment within 14 days of the administering step.

47. 47. The method of any one of claims 1 to 46, wherein the subject does not have an active autoimmune disease and is receiving an inhaled or topical steroid dosage of up to 10 mg of prednisone equivalent daily, or an adrenal replacement steroid dosage.

48. The method of any one of claims 1 to 47, wherein the subject does not have an active systemic infection requiring parenteral antibiotic therapy.

49. The method of any one of claims 1 to 48, wherein the subject has not previously received an organ allograft or allogeneic transplant.

50. 50. The method of any one of claims 1 to 49, wherein the subject has not been identified or diagnosed as being HIV positive or having AIDS.

51. The method of any one of claims 1 to 50, wherein the subject is a female, and the female is not pregnant or breastfeeding.

52. The method of any one of claims 1 to 51, wherein the subject is free of any ongoing toxicity from prior therapy.

53. 53. The method of claim 52, wherein the ongoing toxicity is greater than Grade 1 using NCI CTCAE v5.0 or greater than baseline.

54. 53. The method of claim 52, wherein the continuing toxicity excludes peripheral neuropathy, alopecia, and fatigue.

55. The method of any one of claims 1 to 54, wherein the subject does not have a psychotic illness.

56. The method of any one of claims 1 to 55, wherein said first target binding domain and said soluble tissue factor domain are directly adjacent to each other in said first chimeric polypeptide.

57. The method of any one of claims 1 to 55, wherein 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.

58. The method of any one of claims 1 to 57, wherein 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.

59. The method of any one of claims 1 to 57, 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.

60. The method of any one of claims 1 to 59, wherein the second domain of the pair of affinity domains and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

61. The method of any one of claims 1 to 59, wherein 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.

62. 62. The method of any one of claims 1 to 61, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

63. 62. The method of any one of claims 1 to 61, wherein one or both of the first target binding domain and the second target binding domain are soluble interleukin or cytokine receptors.

64. The method of any one of claims 1 to 63, wherein the first chimeric polypeptide further comprises one or more additional target binding domains.

65. The method of any one of claims 1 to 64, wherein the second chimeric polypeptide further comprises one or more additional target binding domains.

66. The method of any one of claims 1 to 65, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

67. 67. The method of claim 66, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:

1.

68. The method of any one of claims 1 to 67, wherein the pair of affinity domains is a sushi domain derived from the alpha chain of the human IL-15 receptor (IL-15Rα) and soluble IL-15.

69. 69. The method of any one of claims 1 to 68, wherein the first target binding domain comprises a soluble TGF-βRII.

70. 70. The method of claim 69, wherein the first target binding domain comprises a first sequence that is at least 80% identical to SEQ ID NO:66 and a second sequence that is at least 80% identical to SEQ ID NO:66, wherein the first and second sequences are separated by a linker.

71. 71. The method of claim 70, wherein the first target binding domain comprises a first sequence that is at least 90% identical to SEQ ID NO:66 and a second sequence that is at least 90% identical to SEQ ID NO:

66.

72. 72. The method of claim 71 , wherein the first target binding domain comprises a first sequence of SEQ ID NO:66 and a second sequence of SEQ ID NO:

66.

73. 71. The method of claim 70, wherein the linker comprises the sequence of SEQ ID NO:

7.

74. 70. The method of claim 69, wherein the first target binding domain comprises a sequence at least 80% identical to SEQ ID NO:

69.

75. 75. The method of claim 74, wherein the first target binding domain comprises a sequence at least 90% identical to SEQ ID NO:

69.

76. 76. The method of claim 75, wherein the first target binding domain comprises the sequence of SEQ ID NO:

69.

77. The method of any one of claims 1 to 76, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO:

70.

78. 78. The method of claim 77, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

70.

79. 79. The method of claim 78, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

70.

80. 80. The method of claim 79, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

72.

81. 56. The method of any one of claims 1 to 55, wherein the second target binding domain comprises a soluble TGF-βRII.

82. 82. The method of claim 81 , wherein the second target binding domain comprises a first sequence that is at least 80% identical to SEQ ID NO: 66 and a second sequence that is at least 80% identical to SEQ ID NO: 66, wherein the first and second sequences are separated by a linker.

83. 83. The method of claim 82, wherein the second target binding domain comprises a first sequence that is at least 90% identical to SEQ ID NO:66 and a second sequence that is at least 90% identical to SEQ ID NO:

66.

84. 84. The method of claim 83, wherein the second target binding domain comprises a first sequence of SEQ ID NO:66 and a second sequence of SEQ ID NO:

66.

85. 83. The method of claim 82, wherein the linker comprises the sequence of SEQ ID NO:

7.

86. 82. The method of claim 81 , wherein the second target binding domain comprises a sequence at least 80% identical to SEQ ID NO:

69.

87. 87. The method of claim 86, wherein the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO:

69.

88. 88. The method of claim 87, wherein the second target binding domain comprises the sequence of SEQ ID NO:

69.

89. The method of any one of claims 1 to 88, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

74.

90. 90. The method of claim 89, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO:

70.

91. 90. The method of claim 89, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

74.

92. 90. The method of claim 89, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

70.

93. 92. The method of claim 91 , wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO:

74.

94. 93. The method of claim 92, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

70.

95. The method of any one of claims 1 to 94, wherein the multi-chain chimeric polypeptide is administered subcutaneously to the subject.

96. The method of any one of claims 1 to 95, wherein the subject receives a single administration of the multi-chain chimeric polypeptide.

97. 97. The method of claim 96, wherein the single dose is 0.1 mg of the multi-chain chimeric polypeptide per kg of the subject's body weight (mg / kg).

98. 97. The method of claim 96, wherein the single dose is 0.25 mg / kg.

99. 97. The method of claim 96, wherein the single dose is 0.5 mg / kg.

100. 97. The method of claim 96, wherein the single dose is 0.8 mg / kg.

101. 97. The method of claim 96, wherein the single dose is 1.2 mg / kg.

102. The method of any one of claims 1 to 95, wherein the subject is administered the multi-chain chimeric polypeptide two or more times over a treatment period.

103. 103. The method of claim 102, wherein at least one of the two or more administrations is 0.1 mg of the multi-chain chimeric polypeptide per kg body weight (mg / kg) of the subject.

104. 103. The method of claim 102, wherein at least one of the two or more administrations is 0.25 mg / kg.

105. 103. The method of claim 102, wherein at least one of the two or more administrations is 0.5 mg / kg.

106. 103. The method of claim 102, wherein at least one of the two or more administrations is 0.8 mg / kg.

107. 103. The method of claim 102, wherein at least one of the two or more administrations is 1.2 mg / kg.

108. 108. The method of any one of claims 102 to 107, wherein the treatment period is about 4 weeks.

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