Multi-chain chimeric polypeptides for treatment of neuroinflammatory disorders

Abstract

Provided herein are multi-chain chimeric polypeptides and their use in reducing neuroinflammation in a subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 330,660 filed on April 13, 2022 and U.S. Provisional Patent Application Serial No. 63 / 330,639 filed on April 13, 2022.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically as an XML file named 47039-0032WO1_SL_ST26.XML. This XML file was created on April 10, 2023, and is 83,459 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of biotechnology, and more particularly, to antigen binding molecules and the treatment of neuroinflammatory diseases. Background Art

[0006] Tissue factor (TF) is a 263-amino acid integral membrane glycoprotein with a molecular weight of approximately 46 kDa and a triggering protein of the extrinsic coagulation pathway. It is the primary initiator of coagulation in vivo. Tissue factor is normally isolated from circulating blood but initiates the coagulation cascade upon exposure to circulating coagulation serine proteases. Vascular injury exposes subendothelial cells expressing tissue factor, leading to 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 cleavage of FX to FXa and FIX to FIXa. The resulting proteolytic activity and active membrane surface area of ​​FXa subsequently prevent the efficient conversion of small amounts of prothrombin to thrombin. Thrombin generated by FXa initiates platelet activation and activates trace amounts of pro-cofactors (factor V (FV) and factor VIII (FVIII)) into active cofactors (factor Va (FVa) and factor VIIIa (FVIIIa)). FIXa complexes with FVIIIa on the platelet surface to form an intrinsic tenase complex, leading to the rapid generation of FXa. FXa and FVa complex together to form a prothrombinase complex on the surface of activated platelets, leading to the rapid cleavage of prothrombin into thrombin.

[0007] In addition to the tissue factor-FVIIa complex, a recent study demonstrated that the tissue factor-FVIIa-FXa complex can activate FVIII, thereby providing additional levels of FVIIIa during the initial phase. The extrinsic pathway is crucial in initiating coagulation by activating a limited amount of thrombin, while the intrinsic pathway maintains coagulation through a dramatic amplification of the initial signal.

[0008] Many tissue factors expressed on the cell surface are "encrypted" and must be "decrypted" to fully participate in coagulation. At present, the mechanism of "decryption" of tissue factors on the cell surface is still unclear, but the exposure of anionic phospholipids plays an important role in this process. Healthy cells actively sequester anionic phospholipids (such as phosphatidylserine (PS)) to the inner leaflet of the plasma membrane. Cell damage, activation or cytosolic Ca 2+ Upon increased levels, this bilayer asymmetry disappears, leading to increased exposure of PS on the outer leaflet and, consequently, increased specific activity of the tissue factor-FVIIa complex at the cell surface. While PS exposure is known to reduce the apparent Km for activation of FIX and FX by the tissue factor-FVIIa complex, other mechanisms may include conformational rearrangements of tissue factor or tissue factor-FVIIa and subsequent exposure of substrate binding sites. Summary of the Invention

[0009] Provided herein are methods for reducing the level of neuroinflammation in a tissue of a subject in need thereof, the method 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 an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first and second domains of the affinity domain pair; and the first target binding domain and the second target binding domain each specifically bind to a ligand for TGF-β receptor II (TGF-βRII).

[0010] Also provided herein are methods of treating a neuroinflammatory disorder in a subject, the method 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 an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first and second domains of the affinity domain pair; and the first target binding domain and the second target binding domain each specifically bind to a ligand for TGF-β receptor II (TGF-βRII).

[0011] Also provided herein are methods of reducing the rate of progression of a neuroinflammatory disorder in a subject, the method 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 an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first and second domains of the affinity domain pair; and the first target binding domain and the second target binding domain each specifically bind to a ligand for TGF-β receptor II (TGF-βRII).

[0012] In some embodiments, 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, 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, the soluble tissue factor domain and the first domain of the affinity domain pair are directly adjacent to each other in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide. In some embodiments, the second domain of the affinity domain pair and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide.

[0013] In some embodiments, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments, the first target binding domain and the second target binding domain specifically bind to different antigens. In some embodiments, one or both of the first target binding domain and the second target binding domain are antigen binding domains. In some embodiments, one or both of the first target binding domain and the second target binding domain are soluble TGF-β receptor II (TGF-βRII). In some embodiments, the first target binding domain and the second target binding domain are soluble TGF-βRII.

[0014] In some embodiments, the first chimeric polypeptide further comprises one or more additional target binding domains. In some embodiments, the second chimeric polypeptide further comprises one or more additional target binding domains.

[0015] In some embodiments, the soluble tissue factor domain is a soluble human tissue factor domain.In some embodiments, the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:1.

[0016] In some embodiments, the affinity domain pair is a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα), and soluble IL-15. In some embodiments, the affinity domain pair is selected from the group consisting of barnase and barnstar, PKA and AKAP, an adaptor / docking tag module based on a mutant RNase I fragment, and a SNARE module based on the interaction of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

[0017] In some embodiments, the first target binding domain comprises a first sequence that is at least 80% identical to SEQ ID NO: 2 and a second sequence that is at least 80% identical to SEQ ID NO: 2, wherein the first sequence and the second sequence are separated by a linker. In some embodiments, the first target binding domain comprises a first sequence that is at least 90% identical to SEQ ID NO: 2 and a second sequence that is at least 90% identical to SEQ ID NO: 2. In some embodiments, the first target binding domain comprises a first sequence of SEQ ID NO: 2 and a second sequence of SEQ ID NO: 2. In some embodiments, the linker comprises the sequence of SEQ ID NO: 3.

[0018] In some embodiments, the first target binding domain comprises a sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the first target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the first target binding domain comprises the sequence of SEQ ID NO: 4.

[0019] In some embodiments, the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 6. In some embodiments, the first chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 6. In some embodiments, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 6.

[0020] In some embodiments, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 7. In some embodiments, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 68. In some embodiments, the first chimeric polypeptide comprises the sequence of SEQ ID NO: 70.

[0021] In some embodiments, the second target binding domain comprises a first sequence that is at least 80% identical to SEQ ID NO: 2 and a second sequence that is at least 80% identical to SEQ ID NO: 2, wherein the first sequence and the second sequence are separated by a linker. In some embodiments, the second target binding domain comprises a first sequence that is at least 90% identical to SEQ ID NO: 2 and a second sequence that is at least 90% identical to SEQ ID NO: 2. In some embodiments, the second target binding domain comprises a first sequence that is at least 80% identical to SEQ ID NO: 2 and a second sequence that is at least 80% identical to SEQ ID NO: 2. In some embodiments, the linker comprises the sequence of SEQ ID NO: 3.

[0022] In some embodiments, the second target binding domain comprises a sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the second target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO: 4. In some embodiments, the second target binding domain comprises the sequence of SEQ ID NO: 4.

[0023] In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the second chimeric polypeptide comprises a sequence that is at least 90% identical to SEQ ID NO: 5. In some embodiments, the second chimeric polypeptide comprises the sequence of SEQ ID NO: 5. In some embodiments, the second chimeric polypeptide comprises the sequence of SEQ ID NO: 8.

[0024] In certain embodiments, tissue is one or more of brain, spinal cord, skeletal muscle or optic nerve.In certain embodiments, subject has been diagnosed or accredited as suffering from neuroinflammatory disease.In certain embodiments, subject has been accredited as having the risk of suffering from neuroinflammatory disease of increase.In certain embodiments, neuroinflammatory disease is selected from the group consisting of: Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), schizophrenia, autism, depression, major depressive disorder (MDD), dysthymia, bipolar disorder, mood disorders, anxiety, fibromyalgia, Huntington's disease, amyotrophic lateral sclerosis (ALS), acute disseminated encephalomyelitis (ADEM), acute optic neuritis (AON), transverse myelitis, neuromyelitis optica (NMO), Lewy body dementia (LBD) and sarcopenia.

[0025] As used herein, the term "neuroinflammation" refers to an inflammatory response within the central nervous system (CNS) (e.g., brain and / or spinal cord) and can be characterized by a large number of cellular and molecular changes within the brain or CNS (e.g., production of one or more cytokines, chemokines, reactive oxygen species, and second messengers). In some embodiments, neuroinflammation can be a chronic inflammation caused by toxic metabolites, autoimmunity, aging, microorganisms, viruses, traumatic brain injury, or spinal cord injury.

[0026] As used herein, the term "neuroinflammatory disorder" refers to a condition in which an immune response damages a component of the nervous system (e.g., brain, spinal cord, and / or optic nerve). In some embodiments, neuroinflammatory disorders may be associated with aging or traumatic brain injury. For example, neuroinflammatory disorders may include neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, and multiple sclerosis). In some instances, neuroinflammatory disorders may include, but are not limited to, fibromyalgia, Huntington's disease, Lewy body dementia (LBD), amyotrophic lateral sclerosis (ALS), acute disseminated encephalomyelitis (ADEM), acute optic neuritis (AON), transverse myelitis, or neuromyelitis optica (NMO). In some instances, neuroinflammatory disorders may include various mental illnesses (e.g., schizophrenia, autism, depression, and other mood disorders). In some embodiments, an increase in neuroinflammatory levels may accelerate the progression of brain aging and certain diseases (including sarcopenia).

[0027] As used herein, the term "chimeric" refers to a polypeptide that includes an amino acid sequence (e.g., domain) originally derived from two different sources (e.g., two different naturally occurring proteins, e.g., from the same or different species). For example, a chimeric polypeptide can include domains from at least two different naturally occurring human proteins. In some instances, a chimeric polypeptide can include a domain (e.g., scFv) that is a synthetic sequence and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide can include at least two different domains (e.g., two different scFvs) that are synthetic sequences.

[0028] 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, the antigen binding domain can bind to an antigen or epitope with a specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, the antigen binding domain can be an antibody or fragment thereof. In some embodiments, the antigen binding domain can include an alternative scaffold. Non-limiting examples of antigen binding domains are described herein. Additional examples of antigen binding domains are known in the art.

[0029] A "soluble tissue factor domain" refers to a polypeptide 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 a segment of a wild-type mammalian tissue factor protein (e.g., a wild-type human tissue factor protein) that lacks the transmembrane domain and the intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

[0030] The term "soluble interleukin receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that 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 include a sequence that has 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 the extracellular domain of a wild-type interleukin receptor and retains 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.

[0031] The term "soluble cytokine receptor" is used herein in the broadest sense to refer to a polypeptide that lacks a transmembrane domain (and optionally an intracellular domain) that is capable of binding to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble cytokine receptor can include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type cytokine receptor and retains 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.

[0032] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, complete antibodies (e.g., complete immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer. Other examples of antibodies are described herein. Additional examples of antibodies are known in the art.

[0033] "Affinity" refers to the total strength of non-covalent interactions between an antigen binding site and its binding partner (e.g., an antigen or epitope). Unless otherwise indicated, "affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between a member of an antigen binding domain and an antigen or epitope. The affinity of a molecule X for its partner Y can be expressed in terms of the dissociation equilibrium constant (K D ) is represented. The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. For example, surface plasmon resonance (SPR) techniques (e.g., ) or biolayer interferometry (e.g. Other methods for determining the affinity of an antigen binding domain and its corresponding antigen or epitope are known in the art.

[0034] As used herein, a "multi-chain polypeptide" refers to a polypeptide comprising two or more (e.g., three, four, five, six, seven, eight, nine, or ten) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), wherein the two or more protein chains are associated by non-covalent bonds to form a quaternary structure.

[0035] The term "affinity domain pair" refers to two different protein domains that bind specifically to each other with a K D Less than 1x10 -7 M (e.g., less than 1x 10 -8 M, less than 1x 10 -9 M, less than 1x 10 -10 M, or less than 1x 10 -11 M). In some instances, the affinity domain pair can be a pair of naturally occurring proteins. In some embodiments, the affinity domain pair can be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs are described herein.

[0036] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen-binding domain. An epitope can, for example, be composed of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that binding to the former may be lost in the presence of denaturing solvents, but binding to the latter may not be lost. An epitope can include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding. Methods for identifying epitopes bound by antigen-binding domains are known in the art.

[0037] The term "treating" means ameliorating at least one symptom of a disorder. Generally, treatment methods involve administering to a subject in need or identified as needing such treatment a therapeutically effective amount of a composition that alleviates at least one symptom of the disorder.

[0038] The phrase "progression rate of neuroinflammatory disorder" refers to the rate at which neuroinflammatory disorder begins to worsen or causes the gradual loss of neurons in the CNS region. For example, neuroinflammatory disorder can progress at a rapid rate (e.g., days or weeks) or at a slower rate (e.g., months or years). In some embodiments, the progression rate of neuroinflammatory disease can be measured by molecular imaging (e.g., positron emission tomography (PET)), monitoring microglial activation or astrocyte response (e.g., immunohistochemistry, immunocytochemistry) or magnetic resonance methods (e.g., magnetic resonance imaging (MRI) or magnetic resonance spectroscopy (MRS)) or by cognitive assessment.

[0039] 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 the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may 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 the event of a conflict, the present specification (including definitions) will prevail.

[0040] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Shown are exemplary diagrams of multi-chain chimeric polypeptides: (i) a first chimeric polypeptide comprising a first target binding domain (A), a soluble tissue factor domain, a first domain of an affinity domain pair (soluble interleukin IL-15), and an additional target binding domain (B); and (ii) a second chimeric polypeptide comprising a second domain of an affinity domain pair (IL-15 receptor α sushi domain), a second target binding domain (C), and an additional antigen binding domain (D). The top cartoon depicts the association of the first and second chimeric polypeptides via a pair of affinity domains. The bottom schematic shows the order of the domains in the first and second chimeric polypeptides.

[0042] Figure 2 Figures illustrating examples of multi-chain chimeric polypeptides are shown: (i) a first chimeric polypeptide comprising a first target binding domain (A), a soluble tissue factor domain (comprising five amino acid substitutions to remove binding of the soluble tissue factor domain to FVIIa), the first domain of an affinity domain pair (soluble interleukin IL-15 comprising a D8N or D8A amino acid substitution), and an additional target binding domain (B); and (ii) a second chimeric polypeptide comprising the second domain of an affinity domain pair (IL-15 receptor α sushi domain), a second target binding domain (C), and an additional antigen binding domain (D). The top cartoon depicts the association of the first and second chimeric polypeptides via a pair of affinity domains. The bottom schematic diagram shows the order of the domains in the first and second chimeric polypeptides. In other embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain can comprise or consist of a soluble wild-type human tissue factor domain (comprising or consisting of a contiguous sequence within wild-type human tissue factor).

[0043] Figure 3 A schematic diagram of the TGFRt15-TGFR construct is shown.

[0044] Figure 4 Additional schematics of the TGFRt15-TGFR construct are shown.

[0045] Figure 5 Results showing inhibition of TGFβ1 by TGFRt15-TGFR and TGFR-Fc.

[0046] Figure 6 Shows the results of a 32D β-cell proliferation assay using TGFRt15-TGFR or recombinant IL-15

[0047] Figure 7A and 7B Shown are the results of detecting IL-15 and TGFβRII in TGFRt15-TGFR using ELISA with corresponding antibodies.

[0048] Figure 8 The line graph shows the chromatographic profile of cell culture supernatant containing TGFRt15-TGFR protein after binding and elution on anti-TF antibody resin.

[0049] Figure 9 The analytical SEC profile of TGFRt15-TGFR is shown.

[0050] Figure 10 Shown are TGFRt15-TGFR before and after deglycosylation analyzed by reducing SDS-PAGE.

[0051] Figure 11A and 11B Shown are spleen weights and percentages of immune cell types in TGFRt15-TGFR-treated and control-treated mice. Figure 11A Shown are spleen weights of mice treated with TGFRt15-TGFR compared to PBS controls. Figure 11B Shown are the percentages of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with TGFRt15-TGFR compared to PBS controls.

[0052] Figure 12A and 12B Shown are spleen weights and immune stimulation over 92 hours in mice treated with TGFRt15-TGFR. Figure 12A Shown are the spleen weights of mice treated with TGFRt15-TGFR at 16, 24, 48, 72, and 92 hours after treatment. Figure 12B Shown are the percentages of immune cells in mice treated with TGFRt15-TGFR at 16, 24, 48, 72, and 92 hours after treatment.

[0053] Figure 13Aand 13B Shown are the expressions of Ki67 and granzyme B over time in mice treated with TGFRt15-TGFR.

[0054] Figure 14 The enhanced cytotoxicity of TGFRt15-TGFR on splenocytes in C57BL / 6 mice was shown.

[0055] Figure 15 Shown are changes in tumor size in response to treatment with PBS, chemotherapy alone, TGFRt15-TGFR alone, or a combination of chemotherapy and TGFRt15-TGFR in a pancreatic cancer mouse model.

[0056] Figure 16 The cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFR is shown.

[0057] Figures 17A-17C In vivo stimulation of Tregs, NK cells, and CD8+ T cells in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFR is shown.

[0058] Figures 18A-18C Shown is immune stimulation in C57BL / 6 mice after treatment with TGFRt15-TGFR.

[0059] Figure 19A and Figure 19B In vivo induction of NK cell and CD8+ T cell proliferation in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFR was shown.

[0060] Figure 20A and 20B showed that NK cell cytotoxicity was enhanced after treatment of NK cells with TGFRt15-TGFR.

[0061] Figure 21A and 21B It was shown that ADCC activity of NK cells was enhanced after treatment of NK cells with TGFRt15-TGFR.

[0062] Figures 22A-22H The antitumor activity of TGFRt15-TGFR plus anti-TRP1 antibody (TA99) in combination with chemotherapy in a melanoma mouse model was shown.

[0063] Figures 23A-23C showed that TGFRt15-TGFR improved Western diet-induced hyperglycemia in ApoE- / - mice.

[0064] Figure 24Upregulation of CD44hi memory T cells after treatment with TGFRt15-TGFR is shown.

[0065] Figures 25A-25K It was shown that TGFRt15-TGFR (HCW9218) enhances immune-mediated bioactivity in diabetic db / db mice. Figures 25A-25E Shown are representative flow cytometry data demonstrating increases in immune cell surface markers on splenocytes from TGFRt15-TGFR(HCW9218)-treated mice at day 4 compared to controls. Figures 25F-25G Shown are representative flow cytometry data demonstrating an increase in the number of central and effector memory cells in the splenocyte population at day 4 when treated with TGFRt15-TGFR (HCW9218) compared to controls. Figure 25H Shown is the killing of Yac1 target cells by in vivo treated TGFRt15-TGFR (HCW9218) compared to control splenocytes. Figure 25I Shown is an increase in interferon (IFN)-γ release by CD3+ cells compared to controls following antigen-independent stimulation by splenocytes treated with TGFRt15-TGFR (HCW9218) in vivo and stimulated with CD3 / a-CD28 beads ex vivo. Figure 25J Shown are representative data for increases in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) data from splenocytes treated in vivo with PBS or TGFRt15-TGFR(HCW9218) or TGFRt15*-TGFR(HCW9228) and analyzed by Seahorse XFe Bioanalyzer (Agilent). Figure 25K ELISA data are shown showing a decrease in TGFβ1 and TGFβ2 but not TGFβ3 in plasma following TGFRt15-TGFR (HCW9218) or TGFRt15*-TGFR (HCW9228).

[0066] Figures 26A-26P We show that TGFRt15-TGFR (HCW9218) treatment reduces senescent pancreatic β islet cells and SASP factors to ameliorate type 2 diabetes in db / db mice. Figure 26A The protocol for TGFRt15-TGFR (HCW9218) treatment in the db / db mouse model is shown. Figure 26B Shown are the expressions of aging, SASP and beta cell index (related genes) in islet transcripts analyzed by quantitative PCR and normalized to control treatment. Figures 26C-26DImmunofluorescence staining of p21+ cells (yellow) and insulin+β islet cells (green) in pancreatic tissue sections is shown. Figure 26E The number of insulin-positive islet cells in tissue sections is shown. Figure 26F Shown are the numbers of p21+ senescent cells in beta islet cells in tissue sections. Figures 26G-26I Shown are aging, SASP, and β-cell index in the pancreas determined by quantitative PCR. Figure 26J Shown are fasting blood glucose levels after TGFRt15-TGFR(HCW9218) treatment. Figure 26K The HOMA-IR index after TGFRt15-TGFR(HCW9218) treatment is shown. Figure 26L Shown is a volcano plot of RNAseq analysis of liver from db / db mice. Figures 26M-26P Heatmaps of metabolic, aging, inflammatory, and vascular genes are shown.

[0067] Figures 27A-27R It was shown that TGFRt15-TGFR(HCW9218) stimulates immune cell activity and metabolic function in the liver of chronologically aged mice. Figure 27A Shown are the schedules for TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) treatment on days 4 and 10 in young and old mouse models. Figure 27B Composite unbiased t-SNE identifying B cells, T cells, and type 1 ILCs is shown. Figure 27C Shown are representative viSNE plots colored according to density of livers harvested on day 4. Gates are colored by population, and inset numbers represent the frequency of I-ILCs (ILC1 and NK cells). Figures 27D to 27E Shown are pooled data from N=3 independent experiments with N=6 mice / group demonstrating that Figure 27D ) and day 10 ( Figure 27E ) The frequency of total ILC-1 cells in the liver was increased. Figures 27F to 27I Shown on day 4 ( Figure 27F ) and day 10 ( Figure 27G ) in the liver and on day 4 ( Figure 27H ) and day 10 ( Figure 27I ) Total NK cell frequency in spleen. Figure 27J The percentage of positivity for the Ki-67 proliferation marker in the liver is shown. Figures 27K-27L The liver ( Figure 27K ) and spleen( Figure 27L ) in total CD8+ T cell frequencies. Figures 27M to 27NShown are the extracellular acidification rates (ECAR) of splenocytes from young and old mice stimulated in vivo with TGFRt15-TGFR (HCW9218) analyzed by Seahorse XFe Bioanalyzer ( Figure 27M ) and oxygen consumption rate (OCR) ( Figure 27N ) data are representative of the increase compared to the control. Figure 27O Shown are the ex vivo cytotoxic activities of splenocytes from young and old mice stimulated with TGFRt15-TGFR(HCW9218) in vivo against Yac1 target cells as measured by flow cytometry compared to controls (PBS or TGFRt15*-TGFR(HCW9228)). Figures 27P-27Q Shown are increases in interferon (IFN)-γ and (TNF)-α release by CD3+ cells following antigen-independent stimulation by splenocytes treated with TGFRt15-TGFR (HCW9218) in vivo and with α-CD3 / CD28 beads ex vivo compared to control old and young mice, as measured by the MAGPIXX multiplex system. Figure 27R Representative flow cytometry data showing the percentage increase in intracellular granzyme B levels are shown.

[0068] Figures 28A to 28F It was shown that TGFRt15-TGFR(HCW9218) reduced inflammation (SASP) and cellular senescence markers in the liver of chronologically aged mice after one or two subcutaneous doses of TGFRt15-TGFR(HCW9218) or PBS. Figure 28A Shown in a volcano plot are 60-day differences in total RNASeq of hepatocytes from aged mice treated with TGFRt15-TGFR(HCW9218) compared to control treatment. Figures 28B to 28D Shown are heat maps of senescence and inflammation, glucose production and fatty acid metabolism, and circadian rhythm-related genes differentially expressed in the liver after treatment with TGFRt15-TGFR (HCW9218) compared to control treatment (adjusted p-value < 0.05). Figures 28E to 28F Shown are the relative mRNA expressions of IL1α, PAI-1, Il6, and Tnfα in the kidney and Il1β, Il1α, PAI-1, Il6, and Tnfα in the liver analyzed by quantitative PCR on day 10 and / or day 60 after treatment with TGFRt15-TGFR (HCW9218) compared to the control.

[0069] 29A to 29I Two doses of TGFRt15-TGFR (HCW9218) were shown to stimulate metabolic function and reduce markers of inflammation (SASP) and cellular senescence for a prolonged period in chronologically aged mouse liver. Figure 29A Shown is a two-dose TGFRt15-TGFR(HCW9218) treatment regimen in female old C57BL / 6J mice aging over time (76 weeks) that were injected subcutaneously with 3 mg / kg of TGFRt15-TGFR(HCW9218) (n=5 to 8) or saline. Mice received a second dose of TGFRt15-TGFR(HCW9218) on day 60 and were sacrificed on day 120. Figures 29B to 29C Shown are representative data showing increases in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) data from splenocytes stimulated in vivo with two doses of TGFRt15-TGFR (HCW9218) compared to controls as measured by a Seahorse XFe bioanalyzer. Figure 29D Shown are relative mRNA expression of Il1α, Cdkn1a, Pai-1, Il1b, and Il6 in the liver determined by quantitative PCR on day 120 after treatment with one or two doses of TGFRt15-TGFR (HCW9218) compared to controls. Figures 29E to 29F Shown are ELISA data showing protein levels of IL-1α, IL-6, IL-8, PAI-1, and fibronectin in liver tissue determined by ELISA liver at day 120 after treatment with one or two doses of TGFRt15-TGFR (HCW9218) compared to controls. Figures 29G-29H Shown is immunofluorescence staining of liver tissue cells expressing p21+ (yellow) after treatment with two doses of TGFRt15-TGFR (HCW9218). Figure 29I Shown are heat maps of aging-, inflammation-, and circadian rhythm-related genes differentially expressed in the liver after treatment with TGFRt15-TGFR (HCW9218) compared to control treatment (adjusted p-value < 0.05).

[0070] Figures 30A-30E showed that TGFRt15-TGFR(HCW9218) reduced neuroinflammation and affected neuronal function in naturally aged mice. Figure 30A Shown are the relative mRNA expressions of the aging-related genes Cdkn1a, Il6, Il1α, cdkn2a, Il1β, Tnfα, and Il8 in the hippocampus of young and old mice 120 , determined using quantitative PCR. Figure 30B Figure 2 shows the relative mRNA expression of aging-related genes Cdkn1a, Il1α, Il6, Tnfα, Il1β, and Il18 in the hippocampus of aged mice treated with TGFRt15-TGFR (HCW9218) for 60 days compared to controls, as determined using quantitative PCR. 18S rRNA was used for normalization. Figure 30CShown is a volcano plot of total RNASeq in the hippocampus of 60-day-old mice following TGFRt15-TGFR(HCW9218) treatment compared to control. Figures 30D to 30E Shown are heatmaps of differentially expressed neuron-related and circadian rhythm-related and neuroinflammatory-related genes in the hippocampus following treatment with TGFRt15-TGFR (HCW9218) compared to control treatment (adjusted p-value < 0.05).

[0071] Figures 31A to 31I We show that the physical fitness of aged mice treated with both TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) was significantly maintained over time, and that TGFRt15-TGFR(HCW9218) was well tolerated by mice and non-human primates and had no long-term adverse effects on naturally aged mice. Figure 31A Shown are grip strength tests performed in aged mice treated with TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) compared to controls. Figure 31B Rotarod performance of mice treated with saline, TGFRt15-TGFR(HCW9218), and TGFRt15*-TGFR(HCW9228) is shown, and FIG31C shows the open field test for measuring total distance traveled performed in the same mice. Figures 31D to 31E Ki67 expression of CD4, CD8, Treg and CD16+ NK cells in blood from cynomolgus monkeys (10 / group) after two-dose treatment with TGFRt15-TGFR (HCW9218) or saline (study day 1 and day 15) is shown. Figure 31D ) and absolute numbers ( Figure 31E ) by flow cytometry analysis. Figure 31F The probabilities of survival where survival was monitored and analyzed using the log-rank test are shown. Figure 31G Body weights measured after 5 months are shown. Figures 31H-31I Representative flow cytometry data showing the percentages of CD8+ T cells and NK cells in the blood are shown.

[0072] Figures 32A-32D Shown are metabolic activities of splenocytes at day 2 and day 4 in naturally aged mice. Figures 32A to 32D It shows that on the second day ( FIG. 32A to FIG. 32B ) and Day 4 ( FIG. 32C to FIG. 32D ) Representative data on increases in metabolic parameters of splenocytes from db / db mice stimulated in vivo with one dose of TGFRt15-TGFR (HCW9218) compared to controls, analyzed by Seahorse XFe bioanalyzer.

[0073] Figures 33A-33I TGFRt15-TGFR(HCW9218) was shown to enhance immune-mediated biological activities in aged mice. Figures 33A to 33B Representative data measured by flow cytometry are shown, which show that the blood of young and old mice ( Figure 33A ) and spleen( Figure 33B ) compared with the control. Figures 33C to 33D Blood of young and old mice measured by flow cytometry on day 4 ( Figure 33C ) and spleen( Figure 33D ) in NK cells, which are immune cell proliferation markers. Figures 33E to 33G Shown are representative flow cytometry data demonstrating increases in immune cell surface markers on liver immune cells in young and old mice at day 4 following treatment with TGFRt15-TGFR (HCW9218) compared to controls. Figures 33H to 33I Shown are pooled data from N=2 independent experiments with N=6 mice / group demonstrating that compared to control treatment ( Figure 33H ) compared with the liver on days 4 and 10 and the spleen on days 4 and 10 after treatment with TGFRt15-TGFR(HCW9218) ( Figure 33I ) in terms of the frequency of total CD4+ T cells in the .

[0074] Figures 34A-34C We show that TGFRt15-TGFR (HCW9218) stimulates immune cell activity and metabolic function and reduces markers of inflammation (SASP) and cellular senescence in chronologically aged mice. Figure 34A Shown is a heat map of immune pathway-related genes differentially expressed in the liver after treatment with TGFRt15-TGFR (HCW9218) compared to control treatment (adjusted p-value < 0.05). Figure 34B Shown are heat maps of aging-, inflammation-, and circadian rhythm-related genes differentially expressed in the liver after treatment with TGFRt15*-TGFR(HCW9228) compared to control treatment (adjusted p-value < 0.05). Figure 34C Shown are heat maps of differentially expressed aging-, inflammation-, and circadian rhythm-related genes in the liver after treatment with TGFRt15-TGFR(HCW9218) compared to TGFRt15*-TGFR(HCW9228) after 120 days (adjusted p-value < 0.05).

[0075] Figure 35AShown are heat maps of differentially expressed aging-, inflammation-, and circadian rhythm-related genes in the hippocampus following treatment with TGFRt15-TGFR (HCW9218) compared to control treatment (adjusted p-value < 0.05). Figure 35B Shown are cluster analyses of differentially expressed hippocampal genes in the hippocampus following TGFRt15-TGFR (HCW9218) treatment at day 120.

[0076] Figures 36A-36D Immunohistochemical staining for blood-brain barrier studies is shown. Figures 36A-36D Mice received subcutaneous injection of PBS or TGFRt15-TGFR (HCW9218) (3 mg / kg) and were euthanized the next day. Figure 36A )、73 weeks old( Figure 36B 36C) or 105 weeks old (Figure 36C) of the mouse brain were processed for immunohistochemical staining with anti-human tissue factor (TF) specific antibody. No positive staining was detected in the mouse brain. Figure 36D The antibody is shown to show specific staining for human TF in human brain sections.

[0077] Figures 37A-37C Behavioral studies show that TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) maintain minimal acutely significant performance in naturally aged mice. Figure 37A Demonstrates grip strength, Figure 37B The rotarod test is shown, and Figure 37C Shown are open field tests performed in aged mice treated with TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) to measure the acute effects on peak force values ​​30 days after the first dose of each respective treatment.

[0078] Figure 38A The graph shows one or more latencies in the rotarod test performed in a chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR (HCW9218) as described in Example 16.

[0079] Figure 38B The graph shows the latency to fall in the rotarod test, speed in the rotarod test, total distance traveled in the open field, total speed in the open field, time to turn in the pole test, and total time in the pole test in the chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR (HCW9218) as described in Example 16.

[0080] Figure 39The graphs show the feeding frequency (left graph) and feeding duration (right graph) in the open field in a chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR (HCW9218) as described in Example 16.

[0081] Figure 40 is a panel of anti-α-synuclein stained formalin-fixed paraffin-embedded tissue sections of striatum from a chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR (HCW9218) as described in Example 16.

[0082] Figure 41 The figures show the fluorescence area percentage of formalin-fixed paraffin-embedded tissue sections immunohistochemically stained for α-synuclein from the chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR(HCW9218) as described in Example 16 (left panel) and the DAB (3,3'-diaminobenzidine) area percentage of formalin-fixed paraffin-embedded tissue sections immunohistochemically stained for α-synuclein from the chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR(HCW9218) as described in Example 16 (right panel).

[0083] Figure 42 is a panel of anti-ionized calcium binding adapter molecule 1 (IBA1) stained striatal formalin-fixed paraffin-embedded tissue sections from a chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR (HCW9218) as described in Example 16.

[0084] Figure 43 The figures show the percentage of fluorescent area of ​​formalin-fixed paraffin-embedded tissue sections immunohistochemically stained for IBA1 from chemically induced Parkinson's disease mouse models that were untreated or treated with TGFRt15-TGFR(HCW9218) as described in Example 16 (left panel) and the percentage of area of ​​formalin-fixed paraffin-embedded tissue sections immunohistochemically stained for α-synuclein from chemically induced Parkinson's disease mouse models that were untreated or treated with TGFRt15-TGFR(HCW9218) as described in Example 16 (right panel).

[0085] Figure 44are two Western blots and graphs showing the expression or normalized expression levels of α-synuclein (top Western blot and graph) and P16 (bottom Western blot and graph) in brain samples from a chemically induced Parkinson's disease mouse model that was untreated or treated with TGFRt15-TGFR (HCW9218) as described in Example 16. DETAILED DESCRIPTION

[0086] Provided herein are methods of reducing the level of neuroinflammation in a tissue of a subject in need thereof; methods of treating a neuroinflammatory disorder in a subject; and methods of reducing the rate of progression of a neuroinflammatory disorder in a subject, the methods 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 an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first and second domains of the affinity domain pair; and the first target binding domain and the second target binding domain each specifically bind to a ligand for TGF-β receptor II (TGF-βRII).

[0087] 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 can each independently be about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 800 amino acids, or about 50 amino acids to about 950 amino acids. 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 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 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 1 00 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids,about 100 amino acids to about 340 amino acids, about 100 amino acids to about 320 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 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 10 00 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to about 461 amino acids, about 150 amino acids to about 470 amino acids, about 150 amino acids to about 481 amino acids, about 150 amino acids to about 490 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 510 amino acids, about 150 amino acids to about 520 amino acids, about 150 amino acids to about 530 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 5 about 420 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 380 amino acids, about 150 amino acids to about 360 amino acids, about 150 amino acids to about 340 amino acids, about 150 amino acids to about 320 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 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 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids,about 200 amino acids to about 460 amino acids, about 200 amino acids to about 440 amino acids, about 200 amino acids to about 420 amino acids, about 200 amino acids to about 400 amino acids, about 200 amino acids to about 380 amino acids, about 200 amino acids to about 360 amino acids, about 200 amino acids to about 340 amino acids, about 200 amino acids to about 320 amino acids, about 200 amino acids to about 300 amino acids, about 200 amino acids to about 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 3000 amino acids, amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 2000 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids, about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 1 about 500 amino acids, about 220 amino acids to about 480 amino acids, about 220 amino acids to about 460 amino acids, about 220 amino acids to about 440 amino acids, about 220 amino acids to about 420 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 380 amino acids, about 220 amino acids to about 360 amino acids, about 220 amino acids to about 340 amino acids, about 220 amino acids to about 320 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 100 amino acids, about 240 amino acids to about 3000 amino acids, about 240 amino acids to about 2500 amino acids, about 240 amino acids to about 2000 amino acids, about 240 amino acids to about 1500 amino acids, about 240 amino acids to about 1000 amino acids, about 240 amino acids to about 950 amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids,about 240 amino acids to about 500 amino acids, about 240 amino acids to about 480 amino acids, about 240 amino acids to about 460 amino acids, about 240 amino acids to about 440 amino acids, about 240 amino acids to about 420 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 380 amino acids, about 240 amino acids to about 360 amino acids, about 240 amino acids to about 340 amino acids, about 240 amino acids to about 320 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 3000 amino acids, about 24 ... amino acids, about 260 amino acids to about 2500 amino acids, about 260 amino acids to about 2000 amino acids, about 260 amino acids to about 1500 amino acids, about 260 amino acids to about 1000 amino acids, about 260 amino acids to about 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 1 about 500 amino acids, about 260 amino acids to about 480 amino acids, about 260 amino acids to about 460 amino acids, about 260 amino acids to about 440 amino acids, about 260 amino acids to about 420 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 380 amino acids, about 260 amino acids to about 360 amino acids, about 260 amino acids to about 340 amino acids, about 260 amino acids to about 320 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 3000 amino acids, about 280 amino acids to about 2500 amino acids, about 280 amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids,about 280 amino acids to about 460 amino acids, about 280 amino acids to about 440 amino acids, about 280 amino acids to about 420 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 380 amino acids, about 280 amino acids to about 360 amino acids, about 280 amino acids to about 340 amino acids, about 280 amino acids to about 320 amino acids, about 280 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 10 00 amino acids, about 300 amino acids to about 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 amino acids, about 300 amino acids to about 500 amino acids, about 300 amino acids to about 480 amino acids, about 300 amino acids to about 460 amino acids, about 300 amino acids to about 440 amino acids, about 300 amino acids to about 5 about 420 amino acids, about 300 amino acids to about 400 amino acids, about 300 amino acids to about 380 amino acids, about 300 amino acids to about 360 amino acids, about 300 amino acids to about 340 amino acids, about 300 amino acids to about 320 amino acids, about 320 amino acids to about 3000 amino acids, about 320 amino acids to about 2500 amino acids, about 320 amino acids to about 2000 amino acids, about 320 amino acids to about 1500 amino acids, about 320 amino acids to about 1000 amino acids, about 320 amino acids to about 950 amino acids, about 320 amino acids to about 900 amino acids, about 320 amino acids to about 850 amino acids, about 3 about 320 amino acids to about 800 amino acids, about 320 amino acids to about 750 amino acids, about 320 amino acids to about 700 amino acids, about 320 amino acids to about 650 amino acids, about 320 amino acids to about 600 amino acids, about 320 amino acids to about 550 amino acids, about 320 amino acids to about 500 amino acids, about 320 amino acids to about 480 amino acids, about 320 amino acids to about 460 amino acids, about 320 amino acids to about 440 amino acids, about 320 amino acids to about 420 amino acids, about 320 amino acids to about 400 amino acids, about 320 amino acids to about 380 amino acids, about 320 amino acids to about 360 amino acids,about 320 amino acids to about 340 amino acids, about 340 amino acids to about 3000 amino acids, about 340 amino acids to about 2500 amino acids, about 340 amino acids to about 2000 amino acids, about 340 amino acids to about 1500 amino acids, about 340 amino acids to about 1000 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 900 amino acids, about 340 amino acids to about 850 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 750 amino acids, about 340 amino acids to about 700 amino acids, about 340 amino acids to about 650 amino acids, about 340 amino acids to about 800 amino acids, about 340 amino acids to about 950 amino acids, about 340 amino acids to about 10 ... amino acids to about 600 amino acids, about 340 amino acids to about 550 amino acids, about 340 amino acids to about 500 amino acids, about 340 amino acids to about 480 amino acids, about 340 amino acids to about 460 amino acids, about 340 amino acids to about 440 amino acids, about 340 amino acids to about 420 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 360 amino acids to about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 4000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about 360 amino acids to about 1500 amino acids, about 340 amino acids to about 400 amino acids, about 340 amino acids to about 380 amino acids, about 340 amino acids to about 360 amino acids, about 3000 amino acids, about 360 amino acids to about 2500 amino acids, about 360 amino acids to about 2000 amino acids, about 360 amino acids to about 1500 amino acids, about amino acids, about 360 amino acids to about 1000 amino acids, about 360 amino acids to about 950 amino acids, about 360 amino acids to about 900 amino acids, about 360 amino acids to about 850 amino acids, about 360 amino acids to about 800 amino acids, about 360 amino acids to about 750 amino acids, about 360 amino acids to about 700 amino acids, about 360 amino acids to about 650 amino acids, about 360 amino acids to about 600 amino acids, about 360 amino acids to about 550 amino acids, about 360 amino acids to about 500 amino acids, about 360 amino acids to about 480 amino acids, about 360 amino acids to about 460 amino acids, about 360 amino acids to about about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 1500 amino acids, about 380 amino acids to about 1000 amino acids, about 380 amino acids to about 950 amino acids, about 380 amino acids to about 900 amino acids, about 380 amino acids to about 850 amino acids, about 380 amino acids to about 800 amino acids, about 380 amino acids to about 750 amino acids,about 380 amino acids to about 700 amino acids, about 380 amino acids to about 650 amino acids, about 380 amino acids to about 600 amino acids, about 380 amino acids to about 550 amino acids, about 380 amino acids to about 500 amino acids, about 380 amino acids to about 480 amino acids, about 380 amino acids to about 460 amino acids, about 380 amino acids to about 440 amino acids, about 380 amino acids to about 420 amino acids, about 380 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 5 about 1500 amino acids, about 400 amino acids to about 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 amino acids, about 400 amino acids to about 500 amino acids, about 400 amino acids to about 480 amino acids, about 400 amino acids to about 460 amino acids, about 4 00 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about about 420 amino acids to about 650 amino acids, about 420 amino acids to about 600 amino acids, about 420 amino acids to about 550 amino acids, about 420 amino acids to about 500 amino acids, about 420 amino acids to about 480 amino acids, about 420 amino acids to about 460 amino acids, about 420 amino acids to about 440 amino acids, about 440 amino acids to about 3000 amino acids, about 440 amino acids to about 2500 amino acids, about 440 amino acids to about 2000 amino acids, about 440 amino acids to about 1500 amino acids, about 440 amino acids to about 1000 amino acids, about 440 amino acids to about 950 amino acids, about 440 amino acids to about 900 amino acids,about 440 amino acids to about 850 amino acids, about 440 amino acids to about 800 amino acids, about 440 amino acids to about 750 amino acids, about 440 amino acids to about 700 amino acids, about 440 amino acids to about 650 amino acids, about 440 amino acids to about 600 amino acids, about 440 amino acids to about 550 amino acids, about 440 amino acids to about 500 amino acids, about 440 amino acids to about 480 amino acids, about 440 amino acids to about 460 amino acids, about 460 amino acids to about 3000 amino acids, about 460 amino acids to about 2500 amino acids, about 460 amino acids to about 2000 amino acids, about 460 amino acids to about about 1500 amino acids, about 460 amino acids to about 1000 amino acids, about 460 amino acids to about 950 amino acids, about 460 amino acids to about 900 amino acids, about 460 amino acids to about 850 amino acids, about 460 amino acids to about 800 amino acids, about 460 amino acids to about 750 amino acids, about 460 amino acids to about 700 amino acids, about 460 amino acids to about 650 amino acids, about 460 amino acids to about 600 amino acids, about 460 amino acids to about 550 amino acids, about 460 amino acids to about 500 amino acids, about 460 amino acids to about 480 amino acids, about 480 amino acids to about 3000 amino acids, about 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to 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 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids,about 500 amino acids to about 550 amino acids, about 550 amino acids to about 3000 amino acids, about 550 amino acids to about 2500 amino acids, about 550 amino acids to about 2000 amino acids, about 550 amino acids to about 1500 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 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 amino acids to about 1500 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 3000 amino acids, about 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to about about 1500 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 3000 amino acids, about 750 amino acids to about 2500 amino acids, about 750 amino acids to about 2000 amino acids, about 750 amino acids to about 1500 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids,about 750 amino acids to about 850 amino acids, about 750 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 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 3000 amino acids, about 850 amino acids to about 2500 amino acids, about 850 amino acids to about 2000 amino acids, about 850 amino acids to about 1500 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 3000 amino acids, about 900 amino acids to about 2500 amino acids, about 900 amino acids to about 2000 amino acids, about 900 amino acids to about 1500 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, about 950 amino acids to about 3000 amino acids, about 950 amino acids to about 2500 amino acids, about 950 amino acids to about 2000 amino acids, about 950 amino acids to about 1500 amino acids, about 950 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, about 1000 amino acids to about 1500 amino acids, about 1500 amino acids to about 3000 amino acids, about 1500 amino acids to about 2500 amino acids, about 1500 amino acids to about 2000 amino acids, about 2000 amino acids to about 3000 amino acids, about 2000 amino acids to about 2500 amino acids, or about 2500 amino acids to about 3000 amino acids. Schematics of exemplary multi-chain chimeric polypeptides provided herein are depicted at, Figure 1 and 2 middle.

[0088] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any first target binding domain described herein) and the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) are directly 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 exemplary linker sequence described herein or known in the art) between the first target binding domain (e.g., any exemplary first target binding domain described herein) and the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) in the first chimeric polypeptide.

[0089] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and the first domain of the affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) are directly 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 exemplary linker sequence described herein or known in the art) between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and the first domain of the affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) in the first chimeric polypeptide.

[0090] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the affinity domain pair (e.g., any exemplary second domain of any exemplary affinity domain pair described herein) and the second target binding domain (e.g., any exemplary second target binding domain 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 exemplary linker sequence described herein or known in the art) between the second domain of the affinity domain pair (e.g., any exemplary second domain of any exemplary affinity domain pair described herein) and the second target binding domain (e.g., any exemplary second target binding domain described herein) in the second chimeric polypeptide.

[0091] 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. Other aspects of these chimeric polypeptides, nucleic acids, vectors, cells and methods are known in the art.

[0092] Tissue Factor

[0093] Human tissue factor is a 263 amino acid transmembrane protein consisting of three domains: (1) a 219 amino acid N-terminal extracellular domain (residues 1-219); (2) a 22 amino acid transmembrane domain (residues 220-242); and (3) a 21 amino acid cytoplasmic C-terminal tail (residues 242-263) (UniProtKB identifier: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, and an S-palmitoylation site at Cys245. Deletions or mutations in the cytoplasmic domain have not been found to affect the coagulation activity of tissue factor. Tissue factor has an S-palmitoylation site at Cys245 in the intracellular domain of the protein. Cys245 is located at the amino-terminal end of the intracellular domain and close to the membrane surface. The tissue factor transmembrane domain consists of a single-spanning α-helix.

[0094] The extracellular domain of tissue factor, composed of two type III fibronectin domains, is connected to the transmembrane domain by a six-amino acid linker. This linker provides conformational flexibility, decoupling the extracellular domain of tissue factor from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module consists of two overlapping β-sheets: the top sheet domain contains three antiparallel β-strands, and the bottom sheet domain contains four β-strands. The β-strands are connected by β-loops between strands βA and βB, βC and βD, and βE and βF, all of which are conformationally conserved in both modules. Three short α-helical segments connect the β-strands. A unique feature of tissue factor is the 17-amino acid β-hairpin between strands β10 and β11, which is not a common element in the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is not present in the C-terminal domain and is unique to tissue factor. This fibronectin type III domain structure is characteristic of the immunoglobulin-like protein fold family and is conserved in a variety of extracellular proteins.

[0095] Once zymogen FVII binds to tissue to form the active tissue factor-FVIIa complex, it is rapidly converted to FVIIa by limited proteolysis. FVIIa circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII) and can also bind directly to tissue factor. The allosteric interaction between tissue factor and FVIIa on the tissue factor-FVIIa complex greatly increases the enzymatic activity of FVIIa: the rate of hydrolysis of small chromogenic peptide substrates is increased by approximately 20-fold to 100-fold, and the rate of activation of the natural macromolecular substrates FIX and FX is increased by nearly a million-fold. Consistent with allosteric activation of the FVIIa active site upon tissue factor binding, formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., after exposure of phosphatidyl-L-serine on the membrane surface) increases the rate of activation of FIX or FX, as measured by Ca. 2+ The tissue factor-FVIIa-phospholipid complex resulted in an overall approximately million-fold increase in FX activation, a key regulatory point in the coagulation cascade, relative to free FVIIa.

[0096] FVII is a single-chain polypeptide of approximately 50 kDa, consisting of 406 amino acid residues, with an N-terminal γ-carboxyglutamic acid (GLA)-rich domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII is expressed through the Ile- 154 -Arg 152 FVIIa is activated by specific proteolytic cleavage of the Cys bond. This cleavage results in the light chain and heavy chain being separated by Cys 135 and Cys 262 FVIIa is held together by a single disulfide bond. 2+ The GLA domain binds to phospholipid membranes in a C-dependent manner. Immediately adjacent to the C-terminus of the GLA domain is an aromatic stack and two EGF domains. The aromatic stack connects GLA to the binding of a single Ca 2+ ion domain of EGF1. 2+ Occupancy of the binding site increases FVIIa amidolytic activity and tissue factor association. The catalytic triad consists of His 193 、Asp 242 and Ser 344 Composed of a single Ca 2+ The binding of ions is crucial for its catalytic activity. FVII is proteolytically activated to FVIIa, which is released at Ile 153 The newly formed amino terminus folds back and inserts into the activation pocket, where it binds to Asp 343The carboxylate salts of FVIIa form a salt bridge, generating an oxyanion pore. This salt bridge formation is crucial for FVIIa activity. However, upon proteolytic activation, oxyanion pore formation does not occur in free FVIIa. Consequently, FVIIa circulates in a zymogen-like state, which is poorly recognized by plasma protease inhibitors, resulting in a circulation half-life of approximately 90 minutes.

[0097] The tissue factor-mediated positioning of the FVIIa active site above the membrane surface is important for FVIIa to orient toward its cognate substrate. Free FVIIa adopts a stable extended structure when bound to the membrane, with its active site located approximately After FVIIa binds to tissue factor, the FVa active site is relocated closer to the membrane. This regulation may facilitate the correct alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using FVIIa without the GLA domain, it has been shown that the active site remains located at a similar distance above the membrane, demonstrating that tissue factor is able to fully support FVIIa active site positioning even in the absence of FVIIa-membrane interactions. Other data suggest that tissue factor supports full FVIIa proteolytic activity as long as the tissue factor extracellular domain is somehow tethered to the membrane surface. However, raising the FVIIa active site above the membrane surface to greater than It significantly reduces the ability of the tissue factor-FVIIa complex to activate FX, but does not attenuate the tissue factor-FVIIa amidolytic activity.

[0098] 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 substitution identified a limited number of residue positions at which alanine substitution resulted in a 5- to 10-fold decrease in FVIIa binding affinity. The majority of these residue side chains 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 in a broad region at the boundary between the two modules. In the C-module, the residue Arg located on the protruding BC loop is located in the C-module. 135 and Phe 140 Provides independent contact with FVIIa. 133 Located at the bottom of the finger structure and fills the gap between the two modules. 20 、Thr 60 、Asp 58 and Ile22 The main cluster of important binding residues provides continuity. 60 Only partially exposed to solvent, it may play a local structural role rather than making significant contacts with the ligand. The binding site extends to the concave side of the intermodule angle and involves Glu 24 and Gln 110 , and possibly the more distant residue Val 207 The binding region extends from Asp58 to the region bound by Lys 48 、Lys 46 、Gln 37 、Asp 44 and Trp 45 Trp 45 and Asp 44 does not interact independently with FVIIa, indicating that Trp 45 The effect of mutation at this position may reflect the effect of the side chain on the adjacent Asp 44 and Gln 37 The interaction region further includes two surface-exposed aromatic residues, Phe 76 and Tyr 78 , which form part of the hydrophobic cluster in the N-module.

[0099] The known physiological substrates of tissue factor-FVIIa are FVII, FIX and FX and certain protease-activated receptors. Mutational analysis has identified a number of residues that, when mutated, support intact FVIIa amidolytic activity towards small peptide substrates but lack the ability to support activation of macromolecular substrates (i.e., FVII, FIX and FX) (Ruf et al., J Biol Chem [Biochemistry] 267(31):22206-22210, 1992; Ruf et al., J Biol Chem [Biochemistry] 267(9):6375-6381, 1992; Huang et al., J Biol Chem [Biochemistry] 271(36):21752-21757, 1996; Kirchhofer et al., Biochemistry [Biochemistry] 39(25):7380-7387, 2000). The tissue factor loop region at residues 159-165, as well as residues within or adjacent to this flexible loop, has been shown to be critical for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate binding exosite region of tissue factor that is quite distant from the FVIIa active site. Substitution of glycine residues with slightly larger alanine residues significantly impairs tissue factor-FVIIa proteolytic activity. This suggests that the flexibility provided by glycine is crucial for the residues 159-165 loop to recognize macromolecular substrates of tissue factor.

[0100] Lys residue 165 and Lys 166 It has also been shown to be important for substrate recognition and binding. Mutation of any of these residues to alanine results in a significant reduction in tissue factor cofactor function. Lys 165 and Lys 166 With their backs to each other, Lys 165 In most tissue factor-FVIIa structures, it points toward FVIIa, while Lys 166 Pointing to the substrate binding exosite region in the crystal structure. Lys 165 and Gla 35 The putative salt bridge formation between them supports the idea that tissue factor interacts with the GLA domain of FVIIa to regulate substrate recognition. These results suggest that the C-terminal portion of the tissue factor extracellular domain directly interacts with the GLA domains of FIX and FX (and possibly the adjacent EGF1 domain), and that the presence of the FVIIa GLA domain may directly or indirectly regulate these interactions.

[0101] Soluble tissue factor domain

[0102] In some embodiments of any polypeptide, composition or method described herein, the soluble tissue factor domain can be a wild-type tissue factor polypeptide lacking a signal sequence, a transmembrane domain and an intracellular domain. In some instances, the soluble tissue factor domain can be a tissue factor mutant, wherein the wild-type tissue factor polypeptide lacks a signal sequence, a transmembrane domain and an intracellular domain, and is further modified at the selected amino acid. In some instances, the soluble tissue factor domain can be a soluble human tissue factor domain. In some instances, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some instances, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domain, mouse soluble tissue factor domain, rat soluble tissue factor domain and mutant soluble tissue factor domain are as follows.

[0103] Exemplary soluble human tissue factor domain (SEQ ID NO: 1)

[0104] SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0105] Exemplary nucleic acid encoding soluble human tissue factor domain (SEQ ID NO:9)

[0106] AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

[0107] Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 10)

[0108] SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0109] Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 11)

[0110] SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0111] Exemplary soluble mouse tissue factor domain (SEQ ID NO: 12)

[0112] agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtn lgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge

[0113] Exemplary soluble rat tissue factor domain (SEQ ID NO: 13)

[0114] agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

[0115] 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, 10, 11, 12, or 13. In some embodiments, the soluble tissue factor domain can comprise the sequence of SEQ ID NO: 1, 10, 11, 12, or 13, wherein one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) are removed from its N-terminus and / or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids are removed from its C-terminus.

[0116] As understood in the art, those skilled in the art understand that mutations in amino acids that are conserved between different mammalian species are more likely to reduce the activity and / or structural stability of a protein, while mutations in amino acids that are not conserved between different mammalian species are less likely to reduce the activity and / or structural stability of a protein.

[0117] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain is unable to bind Factor VIIa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive Factor X into Factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multi-chain chimeric polypeptide does not stimulate coagulation in a mammal.

[0118] In some instances, the soluble tissue factor domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain.

[0119] 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 an amino acid position corresponding to amino acid position 20 of a mature, wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of a mature, wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of a mature, wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of a mature, wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of a mature, wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of a mature, wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of a mature, wild-type human tissue factor protein. In some embodiments, the mutant soluble tissue factor has an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

[0120] 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:9.

[0121] In some embodiments, the total length of the soluble tissue factor domain can be from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 160 amino acids, or from about 20 amino acids to about 175 amino acids. amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 2 0 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids,about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 40 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 215 amino acids, about 40 amino acids to about 210 amino acids, about 40 amino acids to about 205 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 195 amino acids, about 40 amino acids 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 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids, about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 50 amino acids to about 60 about 220 amino acids, about 50 amino acids to about 215 amino acids, about 50 amino acids to about 210 amino acids, about 50 amino acids to about 205 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids,about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 75 amino acids, about 50 amino acids to about 1 amino acids, about 50 amino acids to about 70 amino acids, about 50 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 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 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 220 amino acids, about 70 amino acids to about 215 amino acids, about 70 amino acids to about 210 amino acids, about 70 amino acids to about 205 amino acids, about 70 amino acids to about 200 amino acids, about 70 amino acids to about 195 amino acids, about 70 amino acids to about 190 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 180 amino acids, about 70 amino acids to about 175 amino acids, about 70 amino acids to about 170 amino acids,about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 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 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids,about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids, about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 100 amino acids to about 220 amino acids, about 100 amino acids to about 215 amino acids, about 100 amino acids to about 210 amino acids, about 100 amino acids to about 2 to about 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 1 from about 10 amino acids to about 180 amino acids, from about 110 amino acids to about 175 amino acids, from about 110 amino acids to about 170 amino acids, from about 110 amino acids to about 165 amino acids, from about 110 amino acids to about 160 amino acids, from about 110 amino acids to about 155 amino acids, from about 110 amino acids to about 150 amino acids, from about 110 amino acids to about 145 amino acids, from about 110 amino acids to about 140 amino acids, from about 110 amino acids to about 135 amino acids, from about 110 amino acids to about 130 amino acids, from about 110 amino acids to about 125 amino acids, from about 110 amino acids to about 120 amino acids, from about 110 amino acids to about 115 amino acids,about 115 amino acids to about 220 amino acids, about 115 amino acids to about 215 amino acids, about 115 amino acids to about 210 amino acids, about 115 amino acids to about 205 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids 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 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 175 amino acids, about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 215 amino acids, about 125 amino acids to about 210 amino acids, about 125 amino acids to about 205 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids,about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 215 amino acids, about 130 amino acids to about 210 amino acids, about 130 amino acids to about 205 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 215 amino acids, about 135 amino acids to about 210 amino acids, about 135 amino acids to about 205 amino acids, about 135 amino acids to about 250 amino acids. about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 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 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about 155 amino acids, about 140 amino acids to about 150 amino acids,about 140 amino acids to about 145 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 215 amino acids, about 145 amino acids to about 210 amino acids, about 145 amino acids to about 205 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids, about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 215 amino acids, about 150 amino acids to about 210 amino acids, about 150 amino acids to about 205 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 215 amino acids, about 155 amino acids to about 210 amino acids, about 155 amino acids to about 205 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids, about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 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 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids,about 165 amino acids to about 220 amino acids, about 165 amino acids to about 215 amino acids, about 165 amino acids to about 210 amino acids, about 165 amino acids to about 205 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids, about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 215 amino acids, about 170 amino acids to about 210 amino acids, about 170 amino acids to about 205 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 215 amino acids, about 175 amino acids to about 210 amino acids, about 175 amino acids to about 205 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 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 180 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 amino acids to about 190 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 215 amino acids, about 190 amino acids to about 210 amino acids, about 190 amino acids to about 205 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 215 amino acids, about 195 amino acids to about 210 amino acids, about 195 amino acids to about 205 amino acids,about 195 amino acids to about 200 amino acids, about 200 amino acids to about 220 amino acids, about 200 amino acids to about 215 amino acids, about 200 amino acids to about 210 amino acids, about 200 amino acids to about 205 amino acids, about 205 amino acids to about 220 amino acids, about 205 amino acids to about 215 amino acids, about 205 amino acids to about 210 amino acids, about 210 amino acids to about 220 amino acids, about 210 amino acids to about 215 amino acids, or about 215 amino acids to about 220 amino acids.

[0122] Adapter sequence

[0123] In some embodiments, the linker sequence can 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 instances, the linker sequence is a synthetic linker sequence.

[0124] In some embodiments of any multi-chain chimeric polypeptides described herein, the first chimeric polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences, such as any of the exemplary linker sequences described herein or known in the art). In some embodiments of any multi-chain chimeric polypeptides described herein, the second chimeric polypeptide can include one, two, three, four, five, six, seven, eight, nine, or ten linker sequences (e.g., the same or different linker sequences, such as any of the exemplary linker sequences described herein or known in the art).

[0125] In some embodiments, the total length of the linker sequence can be from 1 amino acid to about 100 amino acids, from 1 amino acid to about 90 amino acids, from 1 amino acid to about 80 amino acids, from 1 amino acid to about 70 amino acids, from 1 amino acid to about 60 amino acids, from 1 amino acid to about 50 amino acids, from 1 amino acid to about 45 amino acids, from 1 amino acid to about 40 amino acids, from 1 amino acid to about 35 amino acids, from 1 amino acid to about 30 amino acids, from 1 amino acid to about 25 amino acids, from 1 amino acid to about 24 amino acids, from 1 amino acid to about 22 amino acids, from 1 amino acid to about 20 amino acids, from 1 amino acid to about 18 amino acids, from 1 amino acid to about 16 amino acids, from 1 amino acid to about 17 amino acids. to about 14 amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 8 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 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 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 24 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino acids to 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 4 amino acids to about 16 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 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 4 amino acids to about 18 ...from about 6 amino acids to about 80 amino acids, from about 6 amino acids to about 70 amino acids, from about 6 amino acids to about 60 amino acids, from about 6 amino acids to about 50 amino acids, from about 6 amino acids to about 45 amino acids, from about 6 amino acids to about 40 amino acids, from about 6 amino acids to about 35 amino acids, from about 6 amino acids to about 30 amino acids, from about 6 amino acids to about 25 amino acids, from about 6 amino acids to about 24 amino acids, from about 6 amino acids to about 22 amino acids, from about 6 amino acids to about 20 amino acids, from about 6 amino acids to about 18 amino acids, from about 6 amino acids to about 16 amino acids, from about 6 amino acids to about 14 amino acids, from about 6 amino acids to about 12 amino acids, from about 6 amino acids to about 16 amino acids 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 45 amino acids, about 8 amino acids to about 40 amino acids, about 8 amino acids to about 35 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 24 amino acids, about 8 amino acids to about 22 amino acids, about 8 amino acids to about 20 amino acids, about 8 amino acids to about 18 amino acids , about 8 amino acids to about 16 amino acids, about 8 amino acids to about 14 amino acids, about 8 amino acids to about 12 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 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids to about 25 amino acids, about 10 amino acids to about 24 amino acids, about 10 amino acids to about 22 amino acids, about 10 amino acids to about 20 amino acids, about 10 amino acids to about 18 amino acids, about 10 amino acids to about 16 amino acids, about 10 amino acids to about 14 amino acids, about 10 amino acids to about 12 amino acids, about 12 amino acids to about 100 amino acids, about 12 amino acids to about 90 amino acids, about 12 amino acids to about 80 amino acids, about 12 amino acids to about 70 amino acids, about 12 amino acids to about 60 amino acids, about 12 amino acids to about 50 amino acids, about 12 amino acids to about 45 amino acids, about 12 amino acids to about 40 amino acids, about 12 amino acids to about 35 amino acids,from about 12 amino acids to about 30 amino acids, from about 12 amino acids to about 25 amino acids, from about 12 amino acids to about 24 amino acids, from about 12 amino acids to about 22 amino acids, from about 12 amino acids to about 20 amino acids, from about 12 amino acids to about 18 amino acids, from about 12 amino acids to about 16 amino acids, from about 12 amino acids to about 14 amino acids, from about 14 amino acids to about 100 amino acids, from about 14 amino acids to about 90 amino acids, from about 14 amino acids to about 80 amino acids, from about 14 amino acids to about 70 amino acids, from about 14 amino acids to about 60 amino acids, from about 14 amino acids to about 50 amino acids, from about 14 amino acids to about 45 amino acids, from about 14 amino acids to about about 40 amino acids, about 14 amino acids to about 35 amino acids, about 14 amino acids to about 30 amino acids, about 14 amino acids to about 25 amino acids, about 14 amino acids to about 24 amino acids, about 14 amino acids to about 22 amino acids, about 14 amino acids to about 20 amino acids, about 14 amino acids to about 18 amino acids, about 14 amino acids to about 16 amino acids, about 16 amino acids to about 100 amino acids, about 16 amino acids to about 90 amino acids, about 16 amino acids to about 80 amino acids, about 16 amino acids to about 70 amino acids, about 16 amino acids to about 60 amino acids, about 16 amino acids to about 50 amino acids, about 16 amino acids to about 45 amino acids, from about 16 amino acids to about 40 amino acids, from about 16 amino acids to about 35 amino acids, from about 16 amino acids to about 30 amino acids, from about 16 amino acids to about 25 amino acids, from about 16 amino acids to about 24 amino acids, from about 16 amino acids to about 22 amino acids, from about 16 amino acids to about 20 amino acids, from about 16 amino acids to about 18 amino acids, from about 18 amino acids to about 100 amino acids, from about 18 amino acids to about 90 amino acids, from about 18 amino acids to about 80 amino acids, from about 18 amino acids to about 70 amino acids, from about 18 amino acids to about 60 amino acids, from about 18 amino acids to about 50 amino acids, from about 18 amino acids to about 45 amino acids, from about 18 amino acids to about 5 about 40 amino acids, about 18 amino acids to about 35 amino acids, about 18 amino acids to about 30 amino acids, about 18 amino acids to about 25 amino acids, about 18 amino acids to about 24 amino acids, about 18 amino acids to about 22 amino acids, about 18 amino acids to about 20 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 45 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 35 amino acids,about 20 amino acids to about 30 amino acids, about 20 amino acids to about 25 amino acids, about 20 amino acids to about 24 amino acids, about 20 amino acids to about 22 amino acids, about 22 amino acids to about 100 amino acids, about 22 amino acids to about 90 amino acids, about 22 amino acids to about 80 amino acids, about 22 amino acids to about 70 amino acids, about 22 amino acids to about 60 amino acids, about 22 amino acids to about 50 amino acids, about 22 amino acids to about 45 amino acids, about 22 amino acids to about 40 amino acids, about 22 amino acids to about 35 amino acids, about 22 amino acids to about 30 amino acids, about 22 amino acids to about 25 amino acids, about 22 amino acids to about 24 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids,about 60 amino acids to about 90 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 70 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 90 amino acids, or about 90 amino acids to about 100 amino acids.

[0126] 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), such as 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, such as 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, such as 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, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more GGSG sequences.

[0127] In some embodiments, the linker sequence may comprise or consist of: GGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the linker sequence may be encoded by a nucleic acid comprising or consisting of: GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 14). In some embodiments, the linker sequence may comprise or consist of: GGGSGGGS (SEQ ID NO: 15).

[0128] target binding domain

[0129] 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 one or more target binding domains can be an antigen binding domain that specifically binds to a ligand of TGF-βRII (e.g., any exemplary antigen binding domain described herein or known in the art) or a soluble interleukin or cytokine receptor that specifically binds to a ligand of TGF-βRII (e.g., any exemplary soluble interleukin receptor or soluble cytokine receptor described herein).

[0130] 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 can each independently have the following total number of amino acids: about 5 amino acids to about 1000 amino acids, about 5 amino acids to about 950 amino acids, about 5 amino acids to about 900 amino acids, about 5 amino acids to about 850 amino acids, about 5 amino acids to about 800 amino acids, about 5 amino acids to about 750 amino acids, about 5 amino acids to about 700 amino acids, about 5 amino acids to about 650 amino acids, about 5 amino acids to about 600 amino acids, about 5 amino acids to about 550 amino acids, about 5 amino acids to about 500 amino acids, or about 5 amino acids to about 500 amino acids. amino acids, about 5 amino acids to about 450 amino acids, about 5 amino acids to about 400 amino acids, about 5 amino acids to about 350 amino acids, 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 195 amino acids, about 5 amino acids to about 190 amino acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about 170 amino acids, about 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115 amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about 105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 95 amino acids, about 5 amino acids to about 9 0 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, about 5 amino acids to about 10 amino acids,about 10 amino acids to about 1000 amino acids, about 10 amino acids to about 950 amino acids, about 10 amino acids to about 900 amino acids, about 10 amino acids to about 850 amino acids, about 10 amino acids to about 800 amino acids, about 10 amino acids to about 750 amino acids, about 10 amino acids to about 700 amino acids, about 10 amino acids to about 650 amino acids, about 10 amino acids to about 600 amino acids, about 10 amino acids to about 550 amino acids, about 10 amino acids to about 500 amino acids, about 10 amino acids to about 450 amino acids, about 10 amino acids to about 400 amino acids, about 10 amino acids to about 350 amino acids, about 10 amino acids to about 5 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 195 amino acids, about 10 amino acids to about 190 amino acids, about 10 amino acids to about 185 amino acids, about 10 amino acids to about 180 amino acids, about 10 amino acids to about 175 amino acids, about 10 amino acids to about 170 amino acids, about 10 amino acids to about 165 amino acids, about 10 amino acids to about 160 amino acids, about 10 amino acids to about 155 amino acids, about 10 amino acids to about 150 amino acids, about 10 amino acids to about 145 amino acids, about 10 amino acids to about 140 amino acids, about 10 amino acids to about 135 amino acids, about 10 amino acids to about 130 amino acids, about 10 amino acids to about 125 amino acids, about 10 amino acids to about 120 amino acids, about 10 amino acids to about 115 amino acids, about 10 amino acids to about 110 amino acids, about 10 amino acids to about 105 amino acids, about 10 amino acids to about 100 amino acids, about 10 amino acids to about 95 amino acids, about 10 amino acids to about 90 amino acids, about 10 amino acids to about 85 amino acids, about 10 amino acids to about 80 amino acids, about 10 amino acids to about 75 amino acids, about 10 amino acids to about 70 amino acids, about 10 amino acids to about 65 amino acids, about 10 amino acids to about 60 amino acids, about 10 amino acids to about 55 amino acids, about 10 amino acids to about 50 amino acids, about 10 amino acids to about 45 amino acids, about 10 amino acids to about 40 amino acids, about 10 amino acids to about 35 amino acids, about 10 amino acids to about 30 amino acids, about 10 amino acids 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 1000 amino acids, about 15 amino acids to about 950 amino acids,about 15 amino acids to about 900 amino acids, about 15 amino acids to about 850 amino acids, about 15 amino acids to about 800 amino acids, about 15 amino acids to about 750 amino acids, about 15 amino acids to about 700 amino acids, about 15 amino acids to about 650 amino acids, about 15 amino acids to about 600 amino acids, about 15 amino acids to about 550 amino acids, about 15 amino acids to about 500 amino acids, about 15 amino acids to about 450 amino acids, about 15 amino acids to about 400 amino acids, about 15 amino acids to about 350 amino acids, about 15 amino acids to about 300 amino acids, about 15 amino acids to about 280 amino acids, about 15 amino acids to about 260 amino acids, about 15 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids to about 125 amino acids, about 15 amino acids to about 120 amino acids, about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 95 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids , about 15 amino acids to about 65 amino acids, about 15 amino acids to about 60 amino acids, about 15 amino acids to about 55 amino acids, about 15 amino acids to about 50 amino acids, about 15 amino acids to about 45 amino acids, about 15 amino acids to about 40 amino acids, about 15 amino acids to about 35 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 amino acids to about 1000 amino acids, about 20 amino acids to about 950 amino acids, about 20 amino acids to about 900 amino acids, about 20 amino acids to about 850 amino acids, about 20 amino acids to about 800 amino acids,about 20 amino acids to about 750 amino acids, about 20 amino acids to about 700 amino acids, about 20 amino acids to about 650 amino acids, about 20 amino acids to about 600 amino acids, about 20 amino acids to about 550 amino acids, about 20 amino acids to about 500 amino acids, about 20 amino acids to about 450 amino acids, about 20 amino acids to about 400 amino acids, about 20 amino acids to about 350 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, about 20 amino acids to about 220 amino acids, about 20 amino acids to about 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 2 from about 25 amino acids to about 800 amino acids, from about 25 amino acids to about 750 amino acids, from about 25 amino acids to about 700 amino acids, from about 25 amino acids to about 650 amino acids, from about 25 amino acids to about 600 amino acids,about 25 amino acids to about 550 amino acids, about 25 amino acids to about 500 amino acids, about 25 amino acids to about 450 amino acids, about 25 amino acids to about 400 amino acids, about 25 amino acids to about 350 amino acids, about 25 amino acids to about 300 amino acids, about 25 amino acids to about 280 amino acids, about 25 amino acids to about 260 amino acids, about 25 amino acids to about 240 amino acids, about 25 amino acids to about 220 amino acids, about 25 amino acids to about 200 amino acids, about 25 amino acids to about 195 amino acids, about 25 amino acids to about 190 amino acids, about 25 amino acids to about 185 amino acids, about 25 amino acids to about 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 25 amino acids to about 110 amino acids, about 2 5 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids about 30 amino acids to about 1000 amino acids, about 30 amino acids to about 950 amino acids, about 30 amino acids to about 900 amino acids, about 30 amino acids to about 850 amino acids, about 30 amino acids to about 800 amino acids, about 30 amino acids to about 750 amino acids, about 30 amino acids to about 700 amino acids, about 30 amino acids to about 650 amino acids, about 30 amino acids to about 600 amino acids, about 30 amino acids to about 550 amino acids, about 30 amino acids to about 500 amino acids, about 30 amino acids to about 450 amino acids, about 30 amino acids to about 400 amino acids, about 30 amino acids to about 350 amino acids,about 30 amino acids to about 300 amino acids, about 30 amino acids to about 280 amino acids, about 30 amino acids to about 260 amino acids, about 30 amino acids to about 240 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids, about 30 amino acids to about 105 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 95 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 85 amino acids, about 30 amino acids to about 1 80 amino acids, about 30 amino acids to about 75 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 65 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 55 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 1000 amino acids, about 35 amino acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35 amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids, about 35 amino acids to about 500 amino acids, about 35 amino acids to about 600 amino acids, about 35 amino acids to about 55 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 1000 amino acids, about 35 amino acids to about 950 amino acids, about 35 amino acids to about 900 amino acids, about 35 amino acids to about 850 amino acids, about 35 amino acids to about 800 amino acids, about 35 amino acids to about about 35 amino acids to about 750 amino acids, about 35 amino acids to about 700 amino acids, about 35 amino acids to about 650 amino acids, about 35 amino acids to about 600 amino acids, about 35 amino acids to about 550 amino acids, about 35 amino acids to about 500 amino acids, about 35 amino acids to about 450 amino acids, about 35 amino acids to about 400 amino acids, about 35 amino acids to about 350 amino acids, about 35 amino acids to about 300 amino acids, about 35 amino acids to about 280 amino acids, about 35 amino acids to about 260 amino acids, about 35 amino acids to about 240 amino acids, about 35 amino acids to about 220 amino acids, about 35 amino acids to about 200 amino acids,about 35 amino acids to about 195 amino acids, about 35 amino acids to about 190 amino acids, about 35 amino acids to about 185 amino acids, about 35 amino acids to about 180 amino acids, about 35 amino acids to about 175 amino acids, about 35 amino acids to about 170 amino acids, about 35 amino acids to about 165 amino acids, about 35 amino acids to about 160 amino acids, about 35 amino acids to about 155 amino acids, about 35 amino acids to about 150 amino acids, about 35 amino acids to about 145 amino acids, about 35 amino acids to about 140 amino acids, about 35 amino acids to about 135 amino acids, about 35 amino acids to about 130 amino acids, about 35 amino acids to about 1 25 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 1000 amino acids, about 40 amino acids to about 950 amino acids, about 40 amino acids to about 900 amino acids, about 40 amino acids to about 850 amino acids, about 40 amino acids to about 800 amino acids, about 40 amino acids to about 750 amino acids, about 40 amino acids to about 700 amino acids, about 40 amino acids to about 650 amino acids, about 40 amino acids to about 600 amino acids, about 40 amino acids to about 550 amino acids, about 40 amino acids to about 500 amino acids, about 40 amino acids to about 450 amino acids, about 40 amino acids about 40 amino acids to about 195 amino acids, about 40 amino acids 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 175 amino acids, about 40 amino acids to about 170 amino acids, about 40 amino acids to about 165 amino acids,about 40 amino acids to about 160 amino acids, about 40 amino acids to about 155 amino acids, about 40 amino acids to about 150 amino acids, about 40 amino acids to about 145 amino acids, about 40 amino acids to about 140 amino acids, about 40 amino acids to about 135 amino acids, about 40 amino acids to about 130 amino acids, about 40 amino acids to about 125 amino acids, about 40 amino acids to about 120 amino acids, about 40 amino acids to about 115 amino acids, about 40 amino acids to about 110 amino acids, about 40 amino acids to about 105 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 95 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids, about 45 amino acids to about 700 amino acids, about 45 amino acids to about 650 amino acids, about 45 amino acids to about 600 amino acids, about 45 amino acids to about 550 amino acids, about 45 amino acids to about 500 amino acids, about 45 amino acids to about 450 amino acids, about 45 amino acids to about 400 amino acids, about 45 amino acids to about 350 amino acids, about 45 amino acids to about 300 amino acids, about 45 amino acids to about 280 amino acids, about 45 amino acids to about 260 amino acids, about 45 amino acids to about 240 amino acids, about 45 amino acids to about 220 amino acids, about 45 amino acids to about 200 amino acids, about 45 amino acids to about about 45 amino acids to about 195 amino acids, about 45 amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids,about 45 amino acids to about 120 amino acids, about 45 amino acids to about 115 amino acids, about 45 amino acids to about 110 amino acids, about 45 amino acids to about 105 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 95 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 85 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 75 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 65 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 55 amino acids, about 45 amino acids to about 50 amino acids, about 5 0 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 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 30 0 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 145 amino acids, about 50 amino acids to about 140 amino acids, about 50 amino acids to about 135 amino acids, about 50 amino acids to about 130 amino acids, about 50 amino acids to about 125 amino acids, about 50 amino acids to about 120 amino acids, about 50 amino acids to about 115 amino acids, about 50 amino acids to about 110 amino acids, about 50 amino acids to about 105 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 95 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 85 amino acids, about 50 amino acids to about 80 amino acids,about 50 amino acids to about 75 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 65 amino acids, about 50 amino acids to about 60 amino acids, about 50 amino acids to about 55 amino acids, about 55 amino acids to about 1000 amino acids, about 55 amino acids to about 950 amino acids, about 55 amino acids to about 900 amino acids, about 55 amino acids to about 850 amino acids, about 55 amino acids to about 800 amino acids, about 55 amino acids to about 750 amino acids, about 55 amino acids to about 700 amino acids, about 55 amino acids to about 650 amino acids, about 55 amino acids to about 600 amino acids, about 55 amino acids to about 550 amino acids, amino acids, about 55 amino acids to about 500 amino acids, about 55 amino acids to about 450 amino acids, about 55 amino acids to about 400 amino acids, about 55 amino acids to about 350 amino acids, about 55 amino acids to about 300 amino acids, about 55 amino acids to about 280 amino acids, about 55 amino acids to about 260 amino acids, about 55 amino acids to about 240 amino acids, about 55 amino acids to about 220 amino acids, about 55 amino acids to about 200 amino acids, about 55 amino acids to about 195 amino acids, about 55 amino acids to about 190 amino acids, about 55 amino acids to about 185 amino acids, about 55 amino acids to about 180 amino acids, about 55 amino acids about 55 amino acids to about 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids, about 55 amino acids to about 100 amino acids, about 55 amino acids to about 95 amino acids, about 55 amino acids to about 90 amino acids, about 55 amino acids to about 85 amino acids, about 55 amino acids to about 80 amino acids, about 55 amino acids to about 75 amino acids, about 55 amino acids to about 70 amino acids, about 55 amino acids to about 65 amino acids, about 55 amino acids to about 60 amino acids, about 60 amino acids to about 1000 amino acids, about 60 amino acids to about 950 amino acids, about 60 amino acids to about 900 amino acids, about 60 amino acids to about 850 amino acids, about 60 amino acids to about 800 amino acids, about 60 amino acids to about 750 amino acids,about 60 amino acids to about 700 amino acids, about 60 amino acids to about 650 amino acids, about 60 amino acids to about 600 amino acids, about 60 amino acids to about 550 amino acids, about 60 amino acids to about 500 amino acids, about 60 amino acids to about 450 amino acids, about 60 amino acids to about 400 amino acids, about 60 amino acids to about 350 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 19 5 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 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids to about 1 100 amino acids, about 60 amino acids to about 120 amino acids, about 60 amino acids to about 115 amino acids, about 60 amino acids to about 110 amino acids, about 60 amino acids to about 105 amino acids, about 60 amino acids to about 100 amino acids, about 60 amino acids to about 95 amino acids, about 60 amino acids to about 90 amino acids, about 60 amino acids to about 85 amino acids, about 60 amino acids to about 80 amino acids, about 60 amino acids to about 75 amino acids, about 60 amino acids to about 70 amino acids, about 60 amino acids to about 65 amino acids, about 65 amino acids to about 1000 amino acids, about 65 amino acids to about 950 amino acids, about 65 amino acids to about 900 amino acids, about 65 amino acids to about about 65 amino acids to about 850 amino acids, about 65 amino acids to about 800 amino acids, about 65 amino acids to about 750 amino acids, about 65 amino acids to about 700 amino acids, about 65 amino acids to about 650 amino acids, about 65 amino acids to about 600 amino acids, about 65 amino acids to about 550 amino acids, about 65 amino acids to about 500 amino acids, about 65 amino acids to about 450 amino acids, about 65 amino acids to about 400 amino acids, about 65 amino acids to about 350 amino acids, about 65 amino acids to about 300 amino acids, about 65 amino acids to about 280 amino acids, about 65 amino acids to about 260 amino acids, about 65 amino acids to about 240 amino acids,about 65 amino acids to about 220 amino acids, about 65 amino acids to about 200 amino acids, about 65 amino acids to about 195 amino acids, about 65 amino acids to about 190 amino acids, about 65 amino acids to about 185 amino acids, about 65 amino acids to about 180 amino acids, about 65 amino acids to about 175 amino acids, about 65 amino acids to about 170 amino acids, about 65 amino acids to about 165 amino acids, about 65 amino acids to about 160 amino acids, about 65 amino acids to about 155 amino acids, about 65 amino acids to about 150 amino acids, about 65 amino acids to about 145 amino acids, about 65 amino acids to about 140 amino acids, about 65 amino acids to about 13 5 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 95 0 amino acids, about 70 amino acids to about 900 amino acids, about 70 amino acids to about 850 amino acids, about 70 amino acids to about 800 amino acids, about 70 amino acids to about 750 amino acids, about 70 amino acids to about 700 amino acids, about 70 amino acids to about 650 amino acids, about 70 amino acids to about 600 amino acids, about 70 amino acids to about 550 amino acids, about 70 amino acids to about 500 amino acids, about 70 amino acids to about 450 amino acids, about 70 amino acids to about 400 amino acids, about 70 amino acids to about 350 amino acids, about 70 amino acids to about 300 amino acids, about 70 amino acids to about 280 amino acids, about 70 amino acids to about about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 165 amino acids, about 70 amino acids to about 160 amino acids, about 70 amino acids to about 155 amino acids, about 70 amino acids to about 150 amino acids, about 70 amino acids to about 145 amino acids,about 70 amino acids to about 140 amino acids, about 70 amino acids to about 135 amino acids, about 70 amino acids to about 130 amino acids, about 70 amino acids to about 125 amino acids, about 70 amino acids to about 120 amino acids, about 70 amino acids to about 115 amino acids, about 70 amino acids to about 110 amino acids, about 70 amino acids to about 105 amino acids, about 70 amino acids to about 100 amino acids, about 70 amino acids to about 95 amino acids, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 70 amino acids to about 75 amino acids, about 75 amino acids to about 1000 amino acids, amino acids, about 75 amino acids to about 950 amino acids, about 75 amino acids to about 900 amino acids, about 75 amino acids to about 850 amino acids, about 75 amino acids to about 800 amino acids, about 75 amino acids to about 750 amino acids, about 75 amino acids to about 700 amino acids, about 75 amino acids to about 650 amino acids, about 75 amino acids to about 600 amino acids, about 75 amino acids to about 550 amino acids, about 75 amino acids to about 500 amino acids, about 75 amino acids to about 450 amino acids, about 75 amino acids to about 400 amino acids, about 75 amino acids to about 350 amino acids, about 75 amino acids to about 300 amino acids, about 75 amino acids to about 280 amino acids, about 75 amino acids to about 260 amino acids, about 75 amino acids to about 240 amino acids, about 75 amino acids to about 220 amino acids, about 75 amino acids to about 200 amino acids, about 75 amino acids to about 195 amino acids, about 75 amino acids to about 190 amino acids, about 75 amino acids to about 185 amino acids, about 75 amino acids to about 180 amino acids, about 75 amino acids to about 175 amino acids, about 75 amino acids to about 170 amino acids, about 75 amino acids to about 165 amino acids, about 75 amino acids to about 160 amino acids, about 75 amino acids to about 155 amino acids, about 75 amino acids to about 150 amino acids, about 75 amino acids to about 145 amino acids, about 75 amino acids to about 140 amino acids, about 75 amino acids to about 135 amino acids, about 75 amino acids to about 130 amino acids, about 75 amino acids to about 125 amino acids, about 75 amino acids to about 120 amino acids, about 75 amino acids to about 115 amino acids, about 75 amino acids to about 110 amino acids, about 75 amino acids to about 105 amino acids, about 75 amino acids to about 100 amino acids, about 75 amino acids to about 95 amino acids, about 75 amino acids to about 90 amino acids, about 75 amino acids to about 85 amino acids, about 75 amino acids to about 80 amino acids, about 80 amino acids to about 1000 amino acids,about 80 amino acids to about 950 amino acids, about 80 amino acids to about 900 amino acids, about 80 amino acids to about 850 amino acids, about 80 amino acids to about 800 amino acids, about 80 amino acids to about 750 amino acids, about 80 amino acids to about 700 amino acids, about 80 amino acids to about 650 amino acids, about 80 amino acids to about 600 amino acids, about 80 amino acids to about 550 amino acids, about 80 amino acids to about 500 amino acids, about 80 amino acids to about 450 amino acids, about 80 amino acids to about 400 amino acids, about 80 amino acids to about 350 amino acids, about 80 amino acids to about 300 amino acids, about 80 amino acids to about 280 amino acids, about 80 amino acids to about 260 amino acids, about 80 amino acids to about 240 amino acids, about 80 amino acids to about 220 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 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 85 amino acids to about 1000 amino acids, about 85 amino acids to about 950 amino acids, about 85 amino acids to about 900 amino acids, about 85 amino acids to about 850 amino acids, about 85 amino acids to about 800 amino acids, about 85 amino acids to about 750 amino acids, about 85 amino acids to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 260 amino acids,about 85 amino acids to about 240 amino acids, about 85 amino acids to about 220 amino acids, about 85 amino acids to about 200 amino acids, about 85 amino acids to about 195 amino acids, about 85 amino acids to about 190 amino acids, about 85 amino acids to about 185 amino acids, about 85 amino acids to about 180 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 170 amino acids, about 85 amino acids to about 165 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 155 amino acids, about 85 amino acids to about 150 amino acids, about 85 amino acids to about 145 amino acids, about 85 amino acids to about 156 amino acids, about 85 amino acids to about 160 amino acids, about 85 amino acids to about 175 amino acids, about 85 amino acids to about 17 ... about 140 amino acids, about 85 amino acids to about 135 amino acids, about 85 amino acids to about 130 amino acids, about 85 amino acids to about 125 amino acids, about 85 amino acids to about 120 amino acids, about 85 amino acids to about 115 amino acids, about 85 amino acids to about 110 amino acids, about 85 amino acids to about 105 amino acids, about 85 amino acids to about 100 amino acids, about 85 amino acids to about 95 amino acids, about 85 amino acids to about 90 amino acids, about 90 amino acids to about 1000 amino acids, about 90 amino acids to about 950 amino acids, about 90 amino acids to about 900 amino acids, about 90 amino acids to about 850 amino acids, about 90 amino acids to about 800 amino acids, about 90 amino acids to about 750 amino acids, about 90 amino acids to about 700 amino acids, about 90 amino acids to about 650 amino acids, about 90 amino acids to about 600 amino acids, about 90 amino acids to about 550 amino acids, about 90 amino acids to about 500 amino acids, about 90 amino acids to about 450 amino acids, about 90 amino acids to about 400 amino acids, about 90 amino acids to about 350 amino acids, about 90 amino acids to about 300 amino acids, about 90 amino acids to about 280 amino acids, about 90 amino acids to about 260 amino acids, about 90 amino acids to about 240 amino acids, about 90 amino acids to about 1 about 220 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids, about 90 amino acids to about 150 amino acids, about 90 amino acids to about 145 amino acids, about 90 amino acids to about 140 amino acids, about 90 amino acids to about 135 amino acids,about 90 amino acids to about 130 amino acids, about 90 amino acids to about 125 amino acids, about 90 amino acids to about 120 amino acids, about 90 amino acids to about 115 amino acids, about 90 amino acids to about 110 amino acids, about 90 amino acids to about 105 amino acids, about 90 amino acids to about 100 amino acids, about 90 amino acids to about 95 amino acids, about 95 amino acids to about 1000 amino acids, about 95 amino acids to about 950 amino acids, about 95 amino acids to about 900 amino acids, about 95 amino acids to about 850 amino acids, about 95 amino acids to about 800 amino acids, about 95 amino acids to about 750 amino acids, about 95 amino acids to about 70 0 amino acids, about 95 amino acids to about 650 amino acids, about 95 amino acids to about 600 amino acids, about 95 amino acids to about 550 amino acids, about 95 amino acids to about 500 amino acids, about 95 amino acids to about 450 amino acids, about 95 amino acids to about 400 amino acids, about 95 amino acids to about 350 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 280 amino acids, about 95 amino acids to about 260 amino acids, about 95 amino acids to about 240 amino acids, about 95 amino acids to about 220 amino acids, about 95 amino acids to about 200 amino acids, about 95 amino acids to about 195 amino acids, about 95 amino acids to about 250 amino acids, about 95 amino acids to about 260 amino acids, about 95 amino acids to about 270 amino acids, about 95 amino acids to about 280 amino acids, about 95 amino acids to about 290 amino acids, about 95 amino acids to about 291 amino acids, about 95 amino acids to about 292 amino acids, about 95 amino acids to about 293 amino acids, about 95 amino acids to about 294 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 300 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 190 amino acids, about 95 amino acids to about 185 amino acids, about 95 amino acids to about 180 amino acids, about 95 amino acids to about 175 amino acids, about 95 amino acids to about 170 amino acids, about 95 amino acids to about 165 amino acids, about 95 amino acids to about 160 amino acids, about 95 amino acids to about 155 amino acids, about 95 amino acids to about 150 amino acids, about 95 amino acids to about 145 amino acids, about 95 amino acids to about 140 amino acids, about 95 amino acids to about 135 amino acids, about 95 amino acids to about 130 amino acids, about 95 amino acids to about 125 amino acids, about 95 amino acids to about 120 amino acids , about 95 amino acids to about 115 amino acids, about 95 amino acids to about 110 amino acids, about 95 amino acids to about 105 amino acids, about 95 amino acids to about 100 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 100 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 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 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 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 1 about 110 amino acids, about 100 amino acids to about 105 amino acids, about 105 amino acids to about 1000 amino acids, about 105 amino acids to about 950 amino acids, about 105 amino acids to about 900 amino acids, about 105 amino acids to about 850 amino acids, about 105 amino acids to about 800 amino acids, about 105 amino acids to about 750 amino acids, about 105 amino acids to about 700 amino acids, about 105 amino acids to about 650 amino acids, about 105 amino acids to about 600 amino acids, about 105 amino acids to about 550 amino acids, about 105 amino acids to about 500 amino acids, about 105 amino acids to about 450 amino acids, about 105 amino acids to about 400 amino acids, about 105 amino acids to about 350 amino acids, about 105 amino acids to about 300 amino acids, about 105 amino acids to about 280 amino acids, about 105 amino acids to about 260 amino acids, about 105 amino acids to about 240 amino acids, about 105 amino acids to about 220 amino acids, about 105 amino acids to about 200 amino acids, about 105 amino acids to about 195 amino acids, about 105 amino acids to about 190 amino acids, about 105 amino acids to about 185 amino acids, about 105 amino acids to about 180 amino acids, about 105 amino acids to about 175 amino acids, about 105 amino acids to about 170 amino acids,about 105 amino acids to about 165 amino acids, about 105 amino acids to about 160 amino acids, about 105 amino acids to about 155 amino acids, about 105 amino acids to about 150 amino acids, about 105 amino acids to about 145 amino acids, about 105 amino acids to about 140 amino acids, about 105 amino acids to about 135 amino acids, about 105 amino acids to about 130 amino acids, about 105 amino acids to about 125 amino acids, about 105 amino acids to about 120 amino acids, about 105 amino acids to about 115 amino acids, about 105 amino acids to about 110 amino acids, about 110 amino acids to about 1000 amino acids, about 110 amino acids to about 950 amino acids, amino acids, about 110 amino acids to about 900 amino acids, about 110 amino acids to about 850 amino acids, about 110 amino acids to about 800 amino acids, about 110 amino acids to about 750 amino acids, about 110 amino acids to about 700 amino acids, about 110 amino acids to about 650 amino acids, about 110 amino acids to about 600 amino acids, about 110 amino acids to about 550 amino acids, about 110 amino acids to about 500 amino acids, about 110 amino acids to about 450 amino acids, about 110 amino acids to about 400 amino acids, about 110 amino acids to about 350 amino acids, about 110 amino acids to about 300 amino acids, about 110 amino acids to about about 280 amino acids, about 110 amino acids to about 260 amino acids, about 110 amino acids to about 240 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 110 amino acids to about 180 amino acids, about 110 amino acids to about 175 amino acids, about 110 amino acids to about 170 amino acids, about 110 amino acids to about 165 amino acids, about 110 amino acids to about 160 amino acids, about 110 amino acids to about 155 amino acids, about 110 amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115 amino acids to about 800 amino acids, about 115 amino acids to about 750 amino acids,about 115 amino acids to about 700 amino acids, about 115 amino acids to about 650 amino acids, about 115 amino acids to about 600 amino acids, about 115 amino acids to about 550 amino acids, about 115 amino acids to about 500 amino acids, about 115 amino acids to about 450 amino acids, about 115 amino acids to about 400 amino acids, about 115 amino acids to about 350 amino acids, about 115 amino acids to about 300 amino acids, about 115 amino acids to about 280 amino acids, about 115 amino acids to about 260 amino acids, about 115 amino acids to about 240 amino acids, about 115 amino acids to about 220 amino acids, about 115 amino acids to about 200 amino acids, about 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to about 1 about 130 amino acids, about 115 amino acids to about 125 amino acids, about 115 amino acids to about 120 amino acids, about 120 amino acids to about 1000 amino acids, about 120 amino acids to about 950 amino acids, about 120 amino acids to about 900 amino acids, about 120 amino acids to about 850 amino acids, about 120 amino acids to about 800 amino acids, about 120 amino acids to about 750 amino acids, about 120 amino acids to about 700 amino acids, about 120 amino acids to about 650 amino acids, about 120 amino acids to about 600 amino acids, about 120 amino acids to about 550 amino acids, about 120 amino acids to about 500 amino acids, about 120 amino acids to about 450 amino acids, about 120 amino acids to about 400 amino acids, about 120 amino acids to about 350 amino acids, about 120 amino acids to about 300 amino acids, about 120 amino acids 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 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 175 amino acids,about 120 amino acids to about 170 amino acids, about 120 amino acids to about 165 amino acids, about 120 amino acids to about 160 amino acids, about 120 amino acids to about 155 amino acids, about 120 amino acids to about 150 amino acids, about 120 amino acids to about 145 amino acids, about 120 amino acids to about 140 amino acids, about 120 amino acids to about 135 amino acids, about 120 amino acids to about 130 amino acids, about 120 amino acids to about 125 amino acids, about 125 amino acids to about 1000 amino acids, about 125 amino acids to about 950 amino acids, about 125 amino acids to about 900 amino acids, about 125 amino acids to about 850 amino acids, about 125 amino acids to about 800 amino acids, about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700 amino acids, about 125 amino acids to about 650 amino acids, about 125 amino acids to about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125 amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350 amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to about 280 amino acids, about 125 amino acids to about 260 amino acids, about 125 amino acids to about about 240 amino acids, about 125 amino acids to about 220 amino acids, about 125 amino acids to about 200 amino acids, about 125 amino acids to about 195 amino acids, about 125 amino acids to about 190 amino acids, about 125 amino acids to about 185 amino acids, about 125 amino acids to about 180 amino acids, about 125 amino acids to about 175 amino acids, about 125 amino acids to about 170 amino acids, about 125 amino acids to about 165 amino acids, about 125 amino acids to about 160 amino acids, about 125 amino acids to about 155 amino acids, about 125 amino acids to about 150 amino acids, about 125 amino acids to about 145 amino acids, about 125 amino acids to about 140 amino acids, about 125 amino acids to about 135 amino acids, about 125 amino acids to about 130 amino acids, about 130 amino acids to about 1000 amino acids, about 130 amino acids to about 950 amino acids, about 130 amino acids to about 900 amino acids, about 130 amino acids to about 850 amino acids, about 130 amino acids to about 800 amino acids, about 130 amino acids to about 750 amino acids, about 130 amino acids to about 700 amino acids, about 130 amino acids to about 650 amino acids, about 130 amino acids to about 600 amino acids, about 130 amino acids to about 550 amino acids, about 130 amino acids to about 500 amino acids,about 130 amino acids to about 450 amino acids, about 130 amino acids to about 400 amino acids, about 130 amino acids to about 350 amino acids, about 130 amino acids to about 300 amino acids, about 130 amino acids to about 280 amino acids, about 130 amino acids to about 260 amino acids, about 130 amino acids to about 240 amino acids, about 130 amino acids to about 220 amino acids, about 130 amino acids to about 200 amino acids, about 130 amino acids to about 195 amino acids, about 130 amino acids to about 190 amino acids, about 130 amino acids to about 185 amino acids, about 130 amino acids to about 180 amino acids, about 130 amino acids to about 175 amino acids, about 130 amino acids to about 170 amino acids, about 130 amino acids to about 165 amino acids, about 130 amino acids to about 160 amino acids, about 130 amino acids to about 155 amino acids, about 130 amino acids to about 150 amino acids, about 130 amino acids to about 145 amino acids, about 130 amino acids to about 140 amino acids, about 130 amino acids to about 135 amino acids, about 135 amino acids to about 1000 amino acids, about 135 amino acids to about 950 amino acids, about 135 amino acids to about 900 amino acids, about 135 amino acids to about 850 amino acids, about 135 amino acids to about 800 amino acids, about 135 amino acids to about about 750 amino acids, about 135 amino acids to about 700 amino acids, about 135 amino acids to about 650 amino acids, about 135 amino acids to about 600 amino acids, about 135 amino acids to about 550 amino acids, about 135 amino acids to about 500 amino acids, about 135 amino acids to about 450 amino acids, about 135 amino acids to about 400 amino acids, about 135 amino acids to about 350 amino acids, about 135 amino acids to about 300 amino acids, about 135 amino acids to about 280 amino acids, about 135 amino acids to about 260 amino acids, about 135 amino acids to about 240 amino acids, about 135 amino acids to about 220 amino acids, about 135 amino acids to about 200 amino acids, about 135 amino acids to about 195 amino acids, about 135 amino acids to about 190 amino acids, about 135 amino acids to about 185 amino acids, about 135 amino acids to about 180 amino acids, about 135 amino acids to about 175 amino acids, about 135 amino acids to about 170 amino acids, about 135 amino acids to about 165 amino acids, about 135 amino acids to about 160 amino acids, about 135 amino acids to about 155 amino acids, about 135 amino acids to about 150 amino acids, about 135 amino acids to about 145 amino acids, about 135 amino acids to about 140 amino acids, about 140 amino acids to about 1000 amino acids,about 140 amino acids to about 950 amino acids, about 140 amino acids to about 900 amino acids, about 140 amino acids to about 850 amino acids, about 140 amino acids to about 800 amino acids, about 140 amino acids to about 750 amino acids, about 140 amino acids to about 700 amino acids, about 140 amino acids to about 650 amino acids, about 140 amino acids to about 600 amino acids, about 140 amino acids to about 550 amino acids, about 140 amino acids to about 500 amino acids, about 140 amino acids to about 450 amino acids, about 140 amino acids to about 400 amino acids, about 140 amino acids to about 350 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 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 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to about about 155 amino acids, about 140 amino acids to about 150 amino acids, about 140 amino acids to about 145 amino acids, about 145 amino acids to about 1000 amino acids, about 145 amino acids to about 950 amino acids, about 145 amino acids to about 900 amino acids, about 145 amino acids to about 850 amino acids, about 145 amino acids to about 800 amino acids, about 145 amino acids to about 750 amino acids, about 145 amino acids to about 700 amino acids, about 145 amino acids to about 650 amino acids, about 145 amino acids to about 600 amino acids, about 145 amino acids to about 550 amino acids, about 145 amino acids to about 500 amino acids, about 145 amino acids to about 450 amino acids, about 145 amino acids to about 400 amino acids, about 145 amino acids to about 350 amino acids, about 145 amino acids to about 300 amino acids, about 145 amino acids to about 280 amino acids, about 145 amino acids to about 260 amino acids, about 145 amino acids to about 240 amino acids, about 145 amino acids to about 220 amino acids, about 145 amino acids to about 200 amino acids, about 145 amino acids to about 195 amino acids, about 145 amino acids to about 190 amino acids, about 145 amino acids to about 185 amino acids, about 145 amino acids to about 180 amino acids, about 145 amino acids to about 175 amino acids,about 145 amino acids to about 170 amino acids, about 145 amino acids to about 165 amino acids, about 145 amino acids to about 160 amino acids, about 145 amino acids to about 155 amino acids, about 145 amino acids to about 150 amino acids, about 150 amino acids to about 1000 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 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 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to about about 185 amino acids, about 150 amino acids to about 180 amino acids, about 150 amino acids to about 175 amino acids, about 150 amino acids to about 170 amino acids, about 150 amino acids to about 165 amino acids, about 150 amino acids to about 160 amino acids, about 150 amino acids to about 155 amino acids, about 155 amino acids to about 1000 amino acids, about 155 amino acids to about 950 amino acids, about 155 amino acids to about 900 amino acids, about 155 amino acids to about 850 amino acids, about 155 amino acids to about 800 amino acids, about 155 amino acids to about 750 amino acids, about 155 amino acids to about 700 amino acids, about 155 amino acids to about 650 amino acids, about 155 amino acids to about 600 amino acids, about 155 amino acids to about 550 amino acids, about 155 amino acids to about 500 amino acids, about 155 amino acids to about 450 amino acids, about 155 amino acids to about 400 amino acids, about 155 amino acids to about 350 amino acids, about 155 amino acids to about 300 amino acids, about 155 amino acids to about 280 amino acids, about 155 amino acids to about 260 amino acids, about 155 amino acids to about 240 amino acids, about 155 amino acids to about 220 amino acids, about 155 amino acids to about 200 amino acids, about 155 amino acids to about 195 amino acids,about 155 amino acids to about 190 amino acids, about 155 amino acids to about 185 amino acids, about 155 amino acids to about 180 amino acids, about 155 amino acids to about 175 amino acids, about 155 amino acids to about 170 amino acids, about 155 amino acids to about 165 amino acids, about 155 amino acids to about 160 amino acids, about 160 amino acids to about 1000 amino acids, about 160 amino acids to about 950 amino acids, about 160 amino acids to about 900 amino acids, about 160 amino acids to about 850 amino acids, about 160 amino acids to about 800 amino acids, about 160 amino acids to about 750 amino acids, about 160 amino acids to about 700 amino acids, about 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids, about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350 amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids 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 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 160 amino acids to about 175 amino acids, about 160 amino acids to about 170 amino acids, about 160 amino acids to about 165 amino acids, about 165 amino acids to about 1000 amino acids, about 165 amino acids to about 950 amino acids, about 165 amino acids to about 900 amino acids, about 165 amino acids to about 850 amino acids, about 165 amino acids to about 800 amino acids, about 165 amino acids to about 750 amino acids, about 165 amino acids to about 700 amino acids, about 165 amino acids to about 650 amino acids, about 165 amino acids to about 600 amino acids, about 165 amino acids to about 550 amino acids, about 165 amino acids to about 500 amino acids, about 165 amino acids to about 450 amino acids, about 165 amino acids to about 400 amino acids, about 165 amino acids to about 350 amino acids, about 165 amino acids to about 300 amino acids, about 165 amino acids to about 280 amino acids, about 165 amino acids to about 260 amino acids, about 165 amino acids to about 240 amino acids, about 165 amino acids to about 220 amino acids, about 165 amino acids to about 200 amino acids, about 165 amino acids to about 195 amino acids,about 165 amino acids to about 190 amino acids, about 165 amino acids to about 185 amino acids, about 165 amino acids to about 180 amino acids, about 165 amino acids to about 175 amino acids, about 165 amino acids to about 170 amino acids, about 170 amino acids to about 1000 amino acids, about 170 amino acids to about 950 amino acids, about 170 amino acids to about 900 amino acids, about 170 amino acids to about 850 amino acids, about 170 amino acids to about 800 amino acids, about 170 amino acids to about 750 amino acids, about 170 amino acids to about 700 amino acids, about 170 amino acids to about 650 amino acids, about 170 amino acids to about 600 amino acids, about 170 amino acids to about 550 amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170 amino acids to about 350 amino acids, about 170 amino acids to about 300 amino acids, about 170 amino acids to about 280 amino acids, about 170 amino acids to about 260 amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to about about 185 amino acids, about 170 amino acids to about 180 amino acids, about 170 amino acids to about 175 amino acids, about 175 amino acids to about 1000 amino acids, about 175 amino acids to about 950 amino acids, about 175 amino acids to about 900 amino acids, about 175 amino acids to about 850 amino acids, about 175 amino acids to about 800 amino acids, about 175 amino acids to about 750 amino acids, about 175 amino acids to about 700 amino acids, about 175 amino acids to about 650 amino acids, about 175 amino acids to about 600 amino acids, about 175 amino acids to about 550 amino acids, about 175 amino acids to about 500 amino acids, about 175 amino acids to about 450 amino acids, about 175 amino acids to about 400 amino acids, about 175 amino acids to about 350 amino acids, about 175 amino acids to about 300 amino acids, about 175 amino acids to about 280 amino acids, about 175 amino acids to about 260 amino acids, about 175 amino acids to about 240 amino acids, about 175 amino acids to about 220 amino acids, about 175 amino acids to about 200 amino acids, about 175 amino acids to about 195 amino acids, about 175 amino acids to about 190 amino acids, about 175 amino acids to about 185 amino acids, about 175 amino acids to about 180 amino acids, about 180 amino acids to about 1000 amino acids,about 180 amino acids to about 950 amino acids, about 180 amino acids to about 900 amino acids, about 180 amino acids to about 850 amino acids, about 180 amino acids to about 800 amino acids, about 180 amino acids to about 750 amino acids, about 180 amino acids to about 700 amino acids, about 180 amino acids to about 650 amino acids, about 180 amino acids to about 600 amino acids, about 180 amino acids to about 550 amino acids, about 180 amino acids to about 500 amino acids, about 180 amino acids to about 450 amino acids, about 180 amino acids to about 400 amino acids, about 180 amino acids to about 350 amino acids, about 180 amino acids to about 300 amino acids, amino acids, about 180 amino acids to about 280 amino acids, about 180 amino acids to about 260 amino acids, about 180 amino acids to about 240 amino acids, about 180 amino acids to about 220 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 1000 amino acids, about 185 amino acids to about 950 amino acids, about 185 amino acids to about 900 amino acids, about 185 amino acids to about 850 amino acids, about 185 amino acids to about 800 amino acids, about 185 amino acids to about about 750 amino acids, about 185 amino acids to about 700 amino acids, about 185 amino acids to about 650 amino acids, about 185 amino acids to about 600 amino acids, about 185 amino acids to about 550 amino acids, about 185 amino acids to about 500 amino acids, about 185 amino acids to about 450 amino acids, about 185 amino acids to about 400 amino acids, about 185 amino acids to about 350 amino acids, about 185 amino acids to about 300 amino acids, about 185 amino acids to about 280 amino acids, about 185 amino acids to about 260 amino acids, about 185 amino acids to about 240 amino acids, about 185 amino acids to about 220 amino acids, about 185 amino acids to about 200 amino acids, about 185 amino acids to about 195 amino acids, about 185 amino acids to about 190 amino acids, about 190 amino acids to about 1000 amino acids, about 190 amino acids to about 950 amino acids, about 190 amino acids to about 900 amino acids, about 190 amino acids to about 850 amino acids, about 190 amino acids to about 800 amino acids, about 190 amino acids to about 750 amino acids, about 190 amino acids to about 700 amino acids, about 190 amino acids to about 650 amino acids, about 190 amino acids to about 600 amino acids, about 190 amino acids to about 550 amino acids, about 190 amino acids to about 500 amino acids,about 190 amino acids to about 450 amino acids, about 190 amino acids to about 400 amino acids, about 190 amino acids to about 350 amino acids, about 190 amino acids to about 300 amino acids, about 190 amino acids to about 280 amino acids, about 190 amino acids to about 260 amino acids, about 190 amino acids to about 240 amino acids, about 190 amino acids to about 220 amino acids, about 190 amino acids to about 200 amino acids, about 190 amino acids to about 195 amino acids, about 195 amino acids to about 1000 amino acids, about 195 amino acids to about 950 amino acids, about 195 amino acids to about 900 amino acids, about 195 amino acids to about 850 amino acids, amino acids, about 195 amino acids to about 800 amino acids, about 195 amino acids to about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to about about 240 amino acids, about 195 amino acids to about 220 amino acids, about 195 amino acids to about 200 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 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 280 amino acids, about 200 amino acids to about 260 amino acids, about 200 amino acids to about 240 amino acids, about 200 amino acids to about 220 amino acids, about 220 amino acids to about 1000 amino acids, about 220 amino acids to about 950 amino acids, about 220 amino acids to about 900 amino acids, about 220 amino acids to about 850 amino acids, about 220 amino acids to about 800 amino acids, about 220 amino acids to about 750 amino acids,about 220 amino acids to about 700 amino acids, about 220 amino acids to about 650 amino acids, about 220 amino acids to about 600 amino acids, about 220 amino acids to about 550 amino acids, about 220 amino acids to about 500 amino acids, about 220 amino acids to about 450 amino acids, about 220 amino acids to about 400 amino acids, about 220 amino acids to about 350 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 950 amino acids, about 220 amino acids to about 1500 amino acids, about 220 amino acids to about 2500 amino acids, about 220 amino acids to about 30 ... amino acids, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to about 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 950 amino acids, about 260 amino acids to about 900 amino acids, about 260 amino acids to about 850 amino acids, about 260 amino acids to about 800 amino acids, about 260 amino acids to about 750 amino acids, about 260 amino acids to about 700 amino acids, about 260 amino acids to about 650 amino acids, about 260 amino acids to about 600 amino acids, about 260 amino acids to about 550 amino acids, about 260 amino acids to about 500 amino acids, about 260 amino acids to about 450 amino acids, about 260 amino acids to about 400 amino acids, about 260 amino acids to about 350 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 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids,about 280 amino acids to about 500 amino acids, about 280 amino acids to about 450 amino acids, about 280 amino acids to about 400 amino acids, about 280 amino acids to about 350 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 950 amino acids, about 300 amino acids to about 900 amino acids, about 300 amino acids to about 850 amino acids, about 300 amino acids to about 800 amino acids, about 300 amino acids to about 750 amino acids, about 300 amino acids to about 700 amino acids, about 300 amino acids to about 650 amino acids, about 300 amino acids to about 600 amino acids, about 300 amino acids to about 550 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 1000 amino acids, about 350 amino acids to about 950 amino acids, about 350 amino acids to about 900 amino acids, about 350 amino acids to about 850 amino acids, about 350 amino acids to about 800 amino acids, about 350 amino acids to about 750 amino acids, about 350 amino acids to about 700 amino acids, about 350 amino acids to about 650 amino acids, about 350 amino acids to about 1 about 600 amino acids, about 350 amino acids to about 550 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 1000 amino acids, about 400 amino acids to about 950 amino acids, about 400 amino acids to about 900 amino acids, about 400 amino acids to about 850 amino acids, about 400 amino acids to about 800 amino acids, about 400 amino acids to about 750 amino acids, about 400 amino acids to about 700 amino acids, about 400 amino acids to about 650 amino acids, about 400 amino acids to about 600 amino acids, about 400 amino acids to about 550 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 1000 amino acids, about 450 amino acids to about 950 amino acids, about 450 amino acids to about 900 amino acids, about 450 amino acids to about 850 amino acids, about 450 amino acids to about 800 amino acids, about 450 amino acids to about 750 amino acids, about 450 amino acids to about 700 amino acids, about 450 amino acids to about 650 amino acids, about 450 amino acids to about 600 amino acids, about 450 amino acids to about 550 amino acids, about 450 amino acids to about 500 amino acids,about 500 amino acids to about 1000 amino acids, about 500 amino acids to about 950 amino acids, about 500 amino acids to about 900 amino acids, about 500 amino acids to about 850 amino acids, about 500 amino acids to about 800 amino acids, about 500 amino acids to about 750 amino acids, about 500 amino acids to about 700 amino acids, about 500 amino acids to about 650 amino acids, about 500 amino acids to about 600 amino acids, about 500 amino acids to about 550 amino acids, about 550 amino acids to about 1000 amino acids, about 550 amino acids to about 950 amino acids, about 550 amino acids to about 900 amino acids, about 550 amino acids to about about 850 amino acids, about 550 amino acids to about 800 amino acids, about 550 amino acids to about 750 amino acids, about 550 amino acids to about 700 amino acids, about 550 amino acids to about 650 amino acids, about 550 amino acids to about 600 amino acids, about 600 amino acids to about 1000 amino acids, about 600 amino acids to about 950 amino acids, about 600 amino acids to about 900 amino acids, about 600 amino acids to about 850 amino acids, about 600 amino acids to about 800 amino acids, about 600 amino acids to about 750 amino acids, about 600 amino acids to about 700 amino acids, about 600 amino acids to about 650 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 1000 amino acids, about 700 amino acids to about 950 amino acids, about 700 amino acids to about 900 amino acids, about 700 amino acids to about 850 amino acids, about 700 amino acids to about 800 amino acids, about 700 amino acids to about 750 amino acids, about 750 amino acids to about 1000 amino acids, about 750 amino acids to about 950 amino acids, about 750 amino acids to about 900 amino acids, about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids,or about 950 amino acids to about 1000 amino acids.

[0131] Any target binding domain described herein can dissociate with an equilibrium constant (K D ) TGF-βRII binding ligand: less than 1x 10 -7 M, less than 1x 10 -8 M, less than 1x 10 -9 M, less than 1x 10 -10 M, less than 1x 10 -11 M, less than 1x 10 -12 M, or less than 1x 10 -13 M. In some embodiments, the antigen binding protein constructs provided herein can be expressed as follows: D Binding to identified antigen: approximately 1x 10 -3 M to about 1x 10 -5 M, about 1x 10 -4 M is about 1x10 -6 M, about 1x 10 -5 M to about 1x 10 -7 M, about 1x10 -6 M to about 1x 10 -8 M, about 1x 10 -7 M to about 1x 10 -9 M, about 1x 10 -8 M to about 1x 10 -10 M, or about 1x10 -9 M is about 1x10 -11 M (inclusive).

[0132] Any target binding domain described herein may be as follows: DLigand that binds TGF-βRII (e.g., TGF-β): about 1 pM to about 30 nM (e.g., about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about 650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM pM, about 1 pM to about 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM, about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM, about 1 pM to about 30 nM, about 2 pM to about 25 nM, about 2 pM to about 20 pM, about 2pM to about 15nM, about 2pM to about 10nM, about 2pM to about 5nM, about 2pM to about 2nM, about 2pM to about 1nM, about 2pM to about 950pM, about 2pM to about 900pM, about 2pM to about 850pM, about 2pM to about 800pM, about 2pM to about 750pM, about 2pM to about 700pM, about 2pM to about 650pM, about 2pM to about 600pM, about 2pM to about 550pM, about 2pM to about 500pM, about 2pM to about 450pM, about 2pM to about 400pM, about 2pM to about 350pM, about 2pM to about 300pM, about 2pM to about 250pM, about 2pM to about 200pM, about From about 2 pM to about 150 pM, from about 2 pM to about 100 pM, from about 2 pM to about 90 pM, from about 2 pM to about 80 pM, from about 2 pM to about 70 pM, from about 2 pM to about 60 pM, from about 2 pM to about 50 pM, from about 2 pM to about 40 pM, from about 2 pM to about 30 pM, from about 2 pM to about 20 pM, from about 2 pM to about 10 pM, from about 2 pM to about 5 pM, from about 2 pM to about 4 pM, from about 2 pM to about 3 pM, from about 5 pM to about 30 nM, from about 5 pM to about 25 nM, from about 5 pM to about 20 nM, from about 5 pM to about 15 nM, from about 5 pM to about 10 nM, from about 5 pM to about 5 nM, from about 5 pM to about 2 nM, from about 5 pM to about 1 nM, from about 5 pM to about 950 pM,From about 5 pM to about 900 pM, from about 5 pM to about 850 pM, from about 5 pM to about 800 pM, from about 5 pM to about 750 pM, from about 5 pM to about 700 pM, from about 5 pM to about 650 pM, from about 5 pM to about 600 pM, from about 5 pM to about 550 pM, from about 5 pM to about 500 pM, from about 5 pM to about 450 pM, from about 5 pM to about 400 pM, from about 5 pM to about 350 pM, from about 5 pM to about 300 pM, from about 5 pM to about 250 pM, from about 5 pM to about 200 pM, from about 5 pM to about 150 pM, from about 5 pM to about 100 pM, from about 5 pM to about 90 pM, from about 5 pM to about 80 pM, from about 5 pM to about 70 pM, from about 5 pM to about 60 pM, from 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 100 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 pM, about 15 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 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 pM to about 5 nM, about 30 pM to about 2 nM, about 30 pM to about 10 nM, about 30 pM to about 5 nM, about 30 pM to about 2 nM, about 30 pM to about 1 nM, about 30 pM to about 5 nM, about 30 pM to about 2 nM, about 30 pM to about 1 nM, about 30 pM to about 950 pM, about 30 pM to about 900 pM, about 30 pM to about 850 pM, about 30 pM to about 800 pM, about 30 pM to about 750 pM, about 30 pM to about 700 pM, about 30 pM to about 650 pM, about 3 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 150 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 pM, 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 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 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 to about 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 pM to about 5nM, about 80pM to about 2nM, about 80pM to about 1nM, about 80pM to about 950pM, about 80pM to about 900pM, about 80pM to about 850pM, about 80pM to about 800pM, about 80pM to about 750pM, about 80pM to about 700pM, about 80pM to about 650pM, about 80pM to about 600pM, about 80pM to about 550pM, about 80pM to about 500pM, about 80pM to about 450pM, about 80pM to about 400pM, about 80pM to about 350pM, about 80pM to about 300pM, about 80pM to about 250pM, about 80pM to about 200pM, about 80pM 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 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 25 nM, about 100 pM to about 30 nM, about 100 pM to about 15 nM, about 100 pM to about 10 nM, about 100 pM to about 5 nM, about 100 pM to about 2 nM, about 100 pM to about 1 nM, about 100 pM to about 950 pM, about 100 pM to about 900 pM, about 100 pM to about 850 pM, 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 1 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 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 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, about 200 pM to about 100 pM, about 200 pM to about 500 pM, about 200 pM to about 200 pM, about 200 pM to about 100 pM, about 200 pM to about 500 pM, about 200 pM to about 200 pM, about 200 pM to about 100 pM, about 200 pM to about 500 pM, about 200 pM to about 200 pM, about 200 pM to about 100 pM, about 200 pM to about 500 pM, about 200 pM to about 200 pMabout 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 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 400 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 500 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 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 7 50pM, about 800pM to about 30nM, about 800pM to about 25nM, about 800pM to about 30nM, about 800pM to about 15nM, about 800pM to about 10nM, about 800pM to about 5nM, about 800pM to about 2nM, about 800pM to about 1nM, about 800pM to about 950pM, about 800pM to about 900pM, about 800pM to about 850pM, about 900pM to about 30nM, about 900pM to about 25nM, about 900pM to about 30nM, about 900pM to about 15nM, about 900pM 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 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).

[0133] Any target binding domain described herein can be expressed as follows: DBinding to a ligand for TGFβRII: 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 2 nM to about 6 nM , about 3 nM to about 5 nM, about 3 nM to about 4 nM, about 4 nM to about 10 nM, about 4 nM to about 9 nM, about 4 nM to about 8 nM, about 4 nM to about 7 nM, about 4 nM to about 6 nM, about 4 nM to about 5 nM, about 5 nM to about 10 nM, about 5 nM to about 9 nM, about 5 nM to about 8 nM, about 5 nM to about 7 nM, about 5 nM to about 6 nM, about 6 nM to about 10 nM, about 6 nM to about 9 nM, about 6 nM to about 8 nM, about 6 nM to about 7 nM, about 7 nM to 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).

[0134] The K of any of the antigen binding protein constructs described herein can be determined using a variety of different methods known in the art (e.g., electrophoretic mobility shift assays, membrane filtration binding assays, surface plasmon resonance, and biomolecular binding kinetics assays, etc.). D value.

[0135] Antigen binding domain

[0136] 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. 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.

[0137] In some embodiments of any multi-chain chimeric polypeptide 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 multi-chain chimeric polypeptide described herein, each of the first target binding domain and the second target binding domain is an antigen binding domain.

[0138] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen binding domain comprises or is a scFv or a single domain antibody (eg, a VHH or VNAR domain).

[0139] In some examples, the antigen binding domain (e.g., any antigen binding domain described herein) can specifically bind to a ligand of TGF-βRII (see, e.g., antigen binding domains that can specifically bind to TGF-β described in US2021 / 0061897, US2020 / 0399358, US2020 / 0392221, US2019 / 0315850, US2019 / 0092846, US2021 / 0403545, US 2021 / 0355204, and US2019 / 0177406, each of which is incorporated herein by reference).

[0140] The antigen binding domains present in any multi-chain chimeric polypeptide described herein are each independently selected from: a VHH domain, a VNAR domain, and a scFv. In some embodiments, any antigen binding domain 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 HSA body, a scdiabody-HSA, or a tandem-scFv. Other examples of antigen binding domains that can be used in any multi-chain chimeric polypeptide are known in the art.

[0141] The VHH domain is a single monomeric variable antibody domain found in camelids. The VNAR domain is a single monomeric variable antibody domain found in cartilaginous fish. NARNon-limiting aspects of domains are described in, for example, Cromie et al., Curr. Top. Med. Chem. [Current Topics in Medicinal Chemistry] 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. [Development and Comparative Immunology] 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.

[0142] In some embodiments, the antigen binding domains in the multi-chain chimeric polypeptides described herein are all VHH domains, or at least one antigen binding domain is a VHH domain. In some embodiments, the antigen binding domains in the multi-chain chimeric polypeptides described herein are all VNAR domains, or at least one antigen binding domain is a VNAR domain. In some embodiments, the antigen binding domains in the multi-chain chimeric polypeptides described herein are all scFv domains, or at least one antigen binding domain is a scFv domain.

[0143] In some embodiments, two or more polypeptides present in the multi-chain chimeric polypeptide can be assembled (e.g., non-covalently assembled) to form any antigen-binding domain described herein, such as an antigen-binding fragment of an antibody (e.g., any antigen-binding fragment of an antibody described herein), VHH-scAb, VHH-Fab, bi-scFab, F(ab')2, diabody, crossover Mab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common light chain, knobs-in-holes assembly, charge pair, Fab arm exchange, SEED body, LUZ-Y, Fcab, kappa lambda-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, diabody-CH3, triplet, minibody, 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. See, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, which is incorporated herein in its entirety, for a description of these elements. Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments. Additional examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG, e.g., human or humanized IgG1, IgG2, IgG3, or IgG4); antigen-binding fragments of IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., antigen-binding fragments of human or humanized IgA, e.g., human or humanized IgA1 or IgA2); antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD); antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE); or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0144] An "Fv" fragment comprises a non-covalently linked dimer of one heavy- and one light-chain variable domain.

[0145] The "Fab" fragment includes, in addition to the heavy and light chain variable domains of the Fv fragment, the light chain constant domain and the first constant domain of the heavy chain (C H1 ).

[0146] The "F(ab')2" fragment comprises two Fab fragments connected by a disulfide bond near the hinge region.

[0147] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and multispecific binding proteins as described, e.g., 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, the entire contents of each of which are incorporated by reference.

[0148] DARTs are described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014. In some embodiments, any antigen binding domain described herein can bind to an antigen selected from the group consisting of: a protein, a carbohydrate, a lipid, and combinations thereof.

[0149] Additional examples and aspects of antigen binding domains are known in the art.

[0150] Soluble receptors

[0151] In some embodiments of any multi-chain chimeric polypeptide described herein, one or both of the first target binding domain and the second target binding domain are soluble interleukin receptors, soluble cytokine receptors, or ligand receptors. In some embodiments, the soluble receptor is soluble TGF-β receptor II (TGF-βRII) (see, e.g., Yung et al., Am. J. Resp. Crit. Care Med. [American Journal of Respiratory and Critical Care Medicine] 194 (9): 1140-1151, 2016), or soluble TGF-βRIII (see, e.g., Heng et al., Placenta [Placenta] 57: 320, 2017). In some embodiments, one or both of the first target binding domain and the second target binding domain are soluble TGF-β receptor II (TGF-βRII). In some embodiments, the first target binding domain and the second target binding domain are soluble TGF-βRII.

[0152] In some embodiments, the first target binding domain comprises a first sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 2 and a second sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 2, wherein the first and second sequences are separated by a linker. In some embodiments, the first target binding domain comprises a first sequence of SEQ ID NO: 2 and a second sequence of SEQ ID NO: 2.

[0153] In some embodiments, the first target binding domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the first target binding domain comprises the sequence of SEQ ID NO: 4.

[0154] In some embodiments, the second target binding domain comprises a first sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 2, and a second sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 2, wherein the first and second sequences are separated by a linker. In some embodiments, the second target binding domain comprises the first sequence of SEQ ID NO: 2 and the second sequence of SEQ ID NO: 2.

[0155] In some embodiments, the second target binding domain comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO: 4. In some embodiments, the second target binding domain comprises the sequence of SEQ ID NO: 4.

[0156] Other examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0157] Additional target binding domains

[0158] In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any 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 exemplary soluble tissue factor domain described herein or known in the art) and the first domain of the affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide can further include a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and at least one of the one or more additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art), and / or a linker sequence (e.g., any 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 exemplary target binding domains described herein or known in the art) and the first domain of an affinity domain pair (e.g., any exemplary first domain described herein of any exemplary affinity domain pair described herein).

[0159] In some embodiments of any multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains located 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 exemplary target binding domains described herein or known in the art) is directly adjacent to the first domain of the affinity domain pair in the first chimeric polypeptide (e.g., any exemplary first domain described herein of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any 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 exemplary target binding domains described herein or known in the art) and the first domain of the affinity domain pair (e.g., any exemplary first domain described herein of any exemplary affinity domain pair described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art) is directly adjacent to the first target binding domain (e.g., any exemplary target binding domain described herein or known in the art) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any 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 exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any exemplary target binding domain described herein or known in the art).

[0160] 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 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 exemplary target binding domains described herein or known in the art) is located between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein or known in the art) and the first domain of the affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) in the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art) disposed at the N-terminus is directly adjacent to the first target binding domain (e.g., any exemplary target binding domain described herein or known in the art) or the first domain of the affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any linker sequence described herein or known in the art) disposed between at least one additional target binding domain (e.g., any exemplary target binding domain described herein or known in the art) and the first target binding domain (e.g., any exemplary target binding domain described herein or known in the art) or the first domain of an affinity domain pair (e.g., any exemplary first domain described herein of any exemplary affinity domain pair described herein). In some embodiments, at least one additional target binding domain (e.g., any exemplary target binding domain 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 exemplary target binding domain described herein or known in the art) or the first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) disposed between at least one additional target binding domain (e.g., any exemplary target binding domain described herein or known in the art) and the first target binding domain (e.g., any exemplary target binding domain described herein or known in the art) or the first domain of an affinity domain pair (e.g., any exemplary first domain described herein of any exemplary affinity domain pair described herein).In some embodiments, at least one of the one or more additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art) positioned between a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and a first domain of an affinity domain pair (e.g., any first domain described herein or any exemplary affinity domain pair described herein) is directly adjacent to the soluble tissue factor domain and / or the first domain of the affinity domain pair. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) disposed between: (i) the soluble tissue factor domain (e.g., any exemplary soluble tissue factor described herein) and at least one of the one or more additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art), which is positioned between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and the first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein), and / or (ii) the first domain of an affinity domain pair and at least one of the one or more additional target binding domains, which is positioned between the soluble tissue factor domain and the first domain of an affinity domain pair.

[0161] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art) at the N-terminal end and / or C-terminal end of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art) is directly adjacent to the second domain of the affinity domain pair in the second chimeric polypeptide (e.g., any exemplary second domain of any exemplary affinity domain pair described herein).

[0162] In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any 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 exemplary target binding domains described herein or known in the art) and the second domain of the affinity domain pair (e.g., any second domain described herein of any exemplary affinity domain pair described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any exemplary target binding domains described herein or known in the art) is directly adjacent to the second target binding domain (e.g., any target binding domain described herein or known in the art) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any 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 exemplary target binding domains described herein or known in the art) and the second target binding domain (e.g., any exemplary target binding domain described herein or known in the art) in the second chimeric polypeptide.

[0163] In some embodiments of any multi-chain chimeric polypeptide 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) first target binding domains, second target binding domains, and 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) first target binding domains, second target binding domains, and 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) first target binding domains, second target binding domains, and 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.

[0164] In some embodiments of any multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to different antigens. In some embodiments of any multi-chain chimeric polypeptides described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more target binding domains are antigen binding domains. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains are each an antigen binding domain (e.g., scFv or single domain antibody).

[0165] Affinity domain pairs

[0166] In some embodiments, the multi-chain chimeric polypeptide comprises: 1) a first chimeric polypeptide comprising a first domain of an affinity domain pair, and 2) a second chimeric polypeptide comprising a second domain of the affinity domain pair, such that the first chimeric polypeptide and the second chimeric polypeptide are associated through the binding of the first and second domains of the affinity domain pair. In some embodiments, the affinity domain pair is a sushi domain from the α chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. Sushi domains, also known as short consensus repeats or type 1 glycoprotein motifs, are common motifs in protein-protein interactions. Sushi domains have been found on many protein-binding molecules, including complement components C1r, C1s, factor H, and C2m, as well as non-immune 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 affinity domain pair is soluble IL-15, the soluble IL15 has a D8N or D8A amino acid substitution. In some embodiments where one member of the affinity domain pair is the α chain of the human IL-15 receptor (IL15Rα), the human IL15Rα is mature, full-length IL15Rα. In some embodiments, the affinity domain pair is barnase and barnstar. In some embodiments, the affinity domain pair is PKA and AKAP. In some embodiments, the affinity domain pairs are adaptor / docking tag modules based on mutant RNase I fragments (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 SNARE modules based on the interaction between syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492, 2003).

[0167] In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first domain of an affinity domain pair, and a second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a second domain of an affinity domain pair, wherein the first domain of the affinity domain pair and the second domain of the affinity domain pair have a dissociation equilibrium constant (K D ) combined with each other: less than 1x10 -7 M, less than 1x 10 -8 M, less than 1x 10 -9 M, less than 1x 10 -10 M, less than 1x 10 -11 M, less than 1x 10 -12 M, or less than 1x10 -13 In some embodiments, the first domain of the affinity domain pair and the second domain of the affinity domain pair are as follows: D Combined with each other: about 1x10 -4 M to about 1x 10 -6 M, about 1x 10 -5 M to about 1x 10 -7 M, about 1x 10 -6 M to about 1x 10 -8 M, about 1x 10 -7 M to about 1x 10 -9 M, about 1x 10 -8 M to about 1x 10 -10 M, about 1x 10 -9 M to about 1x 10 -11 M, about 1x 10 -10 M to about 1x 10 -12 M, about 1x 10 -11 M to about 1x 10 -13 M, about 1x 10 -4 M to about 1x 10 -5 M, about 1x10 -5 M to about 1x 10 -6 M, about 1x 10 -6 M to about 1x 10 -7 M, about 1x 10 -7 M to about 1x 10 -8 M, about 1x 10 -8 M to about 1x 10 -9 M, about 1x 10 -9 M to about 1x 10 -10 M, about 1x 10 -10 M to about 1x 10 -11 M, about 1x 10 -11 M to about 1x 10 -12M or about 1x 10 -12 M is about 1x10 -13 Any of a variety of different methods known in the art (e.g., electrophoretic mobility shift assays, membrane filtration binding assays, surface plasmon resonance, and biomolecule binding kinetics assays, etc.) can be used to determine the K for binding of the first domain of an affinity domain pair to the second domain of the affinity domain pair. D value.

[0168] In some embodiments, a first chimeric polypeptide of the multi-chain chimeric polypeptide comprises a first domain of an affinity domain pair, and a second chimeric polypeptide of the multi-chain chimeric polypeptide comprises a second domain of an affinity domain pair, wherein the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both can be about 10 to 100 amino acids in length. For example, the length of the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both can be about 10 to 100 amino acids, about 15 to 100 amino acids, about 20 to 100 amino acids, about 25 to 100 amino acids, about 30 to 100 amino acids, about 35 to 100 amino acids, about 40 to 100 amino acids, about 45 to 100 amino acids, about 50 to 100 amino acids, about 55 to 100 amino acids, about 60 to 100 amino acids, about 65 to 100 amino acids, about 70 to 100 amino acids, about 75 to 100 amino acids, about 80 to 100 amino acids, about 85 to 100 amino acids, about 90 to 100 amino acids, about 95 to 100 amino acids, about 10 to 95 amino acids, about 10 to 90 amino acids, about 10 to 85 amino acids, about 10 to or any range in between. In some embodiments, the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both, 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.

[0169] In some embodiments, any of the first and / or second domains of the affinity domain pairs disclosed herein may include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at their N-terminus and / or C-terminus, as long as the function of the first and / or second domains of the affinity domain pair remains intact. For example, the sushi domain of the α chain from the human IL-15 receptor (IL15Rα) may include one or more additional amino acids at the N-terminus and / or C-terminus while still retaining the ability to bind to soluble IL-15. Additionally or alternatively, soluble IL-15 may include one or more additional amino acids at the N-terminus and / or C-terminus while still retaining the ability to bind to the sushi domain of the α chain from the human IL-15 receptor (IL15Rα).

[0170] Non-limiting examples of sushi domains from the alpha chain of IL-15 receptor alpha (IL15Rα) can include sequences that are 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: 16). In some embodiments, the sushi domain from the alpha chain of IL15Rα can be encoded by a nucleic acid comprising ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 17).

[0171] 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 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 18). In some embodiments, soluble IL-15 can be encoded by a nucleic acid comprising the sequence: AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO: 19).

[0172] In some embodiments, the soluble IL-15 may include a D8N amino acid substitution. In some embodiments, the soluble IL-15 with the D8N mutant (IL15D8N) 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 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 62). In some embodiments, a soluble IL-15 with a D8N mutant (IL15D8N) (e.g., SEQ ID NO: 62) can be encoded by a nucleic acid comprising the following sequence: AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT (SEQ ID NO: 63).

[0173] signal sequence

[0174] In some embodiments, the multi-chain chimeric polypeptide includes a first chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, the multi-chain chimeric polypeptide includes a second chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide and the second chimeric polypeptide of the multi-chain chimeric polypeptide both comprise a signal sequence. One of ordinary skill in the art will appreciate that a signal sequence is an amino acid sequence present at the N-terminus of many endogenously produced proteins that guides the protein into the secretory pathway (e.g., guiding the protein to reside in certain intracellular organelles, reside in the cell membrane, or be secreted from the cell). Signal sequences are heterogeneous and their primary amino acid sequences vary greatly. However, signal sequences are typically 16 to 30 amino acids in length and include a hydrophilic, generally positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region comprising a signal peptidase cleavage site.

[0175] 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 having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 20). 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: 21), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO: 22), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 23).

[0176] 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 having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 24). 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 having the amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVS FLFLAGRSCG (SEQ ID NO: 25). 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 having the amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLA LRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLK TSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 26). 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 having the amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 27). One of ordinary skill in the art will appreciate other suitable signal sequences that can be used in the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide described herein.

[0177] 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 of about 10 to 100 amino acids in length. For example, the signal sequence can be about 10 to 100 amino acids, about 15 to 100 amino acids, about 20 to 100 amino acids, about 25 to 100 amino acids, about 30 to 100 amino acids, about 35 to 100 amino acids, about 40 to 100 amino acids, about 45 to 100 amino acids, about 50 to 100 amino acids, about 55 to 100 amino acids, about 60 to 100 amino acids, about 65 to 100 amino acids, about 70 to 100 amino acids, about 75 to 100 amino acids, about 80 to 100 amino acids, about 85 to 100 amino acids, about 90 to 100 amino acids, about 95 to 100 amino acids, about 10 to 95 amino acids, about 10 to 90 amino acids, about 10 to 85 amino acids, about 10 to 80 amino acids, about 10 to 75 amino acids, about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range in between. 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.

[0178] In some embodiments, any signal sequence disclosed herein may comprise 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 acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 28) may comprise one or more additional amino acids at the N-terminus or C-terminus while still retaining the ability to direct the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, into the secretory pathway.

[0179] 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 directs the multi-chain chimeric polypeptide into the extracellular space. Such embodiments can be used to produce multi-chain chimeric polypeptides that are relatively easy to isolate and / or purify.

[0180] Peptide tags

[0181] In some embodiments, the 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, the 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.

[0182] Exemplary peptide tags that can be included in the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both include, but are not limited to, an Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO:29), a calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO:30), a polyglutamate tag (EEEEEE; SEQ ID NO:31), an E-tag (GAPVPYPDPLEPR; SEQ ID NO:32), a FLAG-tag (DYKDDDDK; SEQ ID NO:33), an HA-tag (peptide from hemagglutinin) (YPYDVPDYA; SEQ ID NO:34), a his-tag (HHHHH (SEQ ID NO:35); HHHHHH (SEQ ID NO:36); HHHHHHH (SEQ ID NO:37); HHHHHHHH (SEQ ID NO:38); HHHHHHHHH (SEQ ID NO:39); NO:39; or HHHHHHHHHHH (SEQ ID NO:40)), myc-tag (EQKLISEEDL; SEQ ID NO:41), NE-tag (TKENPRSNQEESYDDNES; SEQ ID NO:42), S-tag (KETAAAKFERQHMDS; SEQ ID NO:43), SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP; SEQ ID NO:44), Sof-tag 1 (SLAELLNAGLGGS; SEQ ID NO:45), Sof-tag 3 (TQDPSRVG; SEQ ID NO:46), Spot-tag (PDRVRAVSHWSS; SEQ ID NO:47), Strep-tag (WSHPQFEK; SEQ ID NO:48), TC-tag (CCPGCC; SEQ ID NO:49), Ty-tag (EVHTNQDPLD; SEQ ID NO:50), NO:50), V5 tag (GKPIPNPLLGLDST; SEQ ID NO:51), VSV-tag (YTDIEMNRLGK; SEQ ID NO:52), and Xpress tag (DLYDDDDK; SEQ ID NO:53). In some embodiments, the tissue factor protein is a peptide tag.

[0183] The peptide tag that can be included in the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be used in any of a variety of applications related to multi-chain chimeric polypeptides. For example, the peptide tag can be used to purify the multi-chain chimeric polypeptide. As a non-limiting example, the first chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., the recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., the recombinantly expressed second chimeric polypeptide), or both can include a myc tag; antibodies that recognize one or more myc tags can be used to purify the multi-chain chimeric polypeptide including the first chimeric polypeptide with a myc tag, the second chimeric polypeptide with a myc tag, or both. A non-limiting example of an antibody that recognizes a myc tag is 9E10, which is available from the non-commercial Developmental Research Hybridoma Bank. As another non-limiting example, the first chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., the recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., the recombinantly expressed second chimeric polypeptide), or both can comprise a histidine tag; the multi-chain chimeric polypeptide comprising the first chimeric polypeptide with the histidine tag, the second chimeric polypeptide with the histidine tag, or both can be purified using nickel or cobalt chelates. One of ordinary skill in the art will recognize other suitable tags and agents that bind to these tags for purification of 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.

[0184] The peptide tag that can be included in the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can be used, for example, in immunoprecipitation of the multi-chain chimeric polypeptide, imaging of the multi-chain chimeric polypeptide (e.g., via Western blot, ELISA, flow cytometry and / or immunocytochemistry), and / or solubilization of the multi-chain chimeric polypeptide. 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 long, about 15 to 100 amino acids long, about 20 to 100 amino acids long, about 25 to 100 amino acids long, about 30 to 100 amino acids long, about 35 to 100 amino acids long, about 40 to 100 amino acids long, about 45 to 100 amino acids long, about 50 to 100 amino acids long, about 55 to 100 amino acids long, about 60 to 100 amino acids long. long, about 65 to 100 amino acids long, about 70 to 100 amino acids long, about 75 to 100 amino acids long, about 80 to 100 amino acids long, about 85 to 100 amino acids long, about 90 to 100 amino acids long, about 95 to 100 amino acids long, about 10 to 95 amino acids long, about 10 to 90 amino acids long, about 10 to 85 amino acids long, about 10 to 80 amino acids long, about 10 to 75 amino acids long amino acids long, about 10 to 70 amino acids long, about 10 to 65 amino acids long, about 10 to 60 amino acids long, about 10 to 55 amino acids long, about 10 to 50 amino acids long, about 10 to 45 amino acids long, about 10 to 40 amino acids long, about 10 to 35 amino acids long, about 10 to 30 amino acids long, about 10 to 25 amino acids long, about 10 to 20 amino acids long, about 10 to 15 amino acids long, about 20 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.

[0185] The peptide tag included in the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both can have any suitable length. For example, the length of the 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 embodiments where the multi-chain chimeric polypeptide comprises two or more peptide tags, the two or more peptide tags can have the same or different lengths. In some embodiments, any peptide tag 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 the C-terminus, as long as the function of the peptide tag remains intact. For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO: 54) can include one or more additional amino acids (e.g., at the N-terminus and / or the C-terminus of the peptide tag) while still retaining the ability to be bound by an antibody.

[0186] Exemplary multi-chain chimeric polypeptides

[0187] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently and specifically bind to TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide. In some examples of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

[0188] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the affinity domain pair are directly adjacent to each other in the first chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linkers described herein) between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric polypeptide.

[0189] In some embodiments of these multi-chain chimeric polypeptides, the second domain of the affinity domain pair and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of these multi-chain chimeric polypeptides, the second chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker described herein) between the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide.

[0190] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any exemplary soluble tissue factor domain described herein. In some embodiments of these multi-chain chimeric polypeptides, the affinity domain pair can be any exemplary affinity domain pair described herein.

[0191] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0192] In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain are soluble TGF-β receptors (e.g., soluble TGFβRII receptors, e.g., soluble human TGFβRII). In some embodiments of these multi-chain chimeric polypeptides, the soluble human TGFRβRII comprises a first 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 GGGGSGGGGSGGGGS (SEQ ID NO: 3).

[0193] In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFRβRII receptor comprises 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:

[0194] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 2).

[0195] In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFRβRII receptor comprises 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:

[0196] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD (SEQ ID NO: 2).

[0197] In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFRβRII receptor is 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:

[0198] ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGA CGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCTCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGAT(SEQ ID NO:55).

[0199] In some embodiments of these multi-chain chimeric polypeptides, the second sequence of the soluble human TGFRβRII receptor is 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:

[0200] ATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGA CGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:56).

[0201] In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-beta receptor comprises 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:

[0202] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGG SGGGGSIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD(SEQ IDNO:4).

[0203] In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-beta receptor is 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:

[0204] ATCCCCCCCCATGTGCAAAAGAGCGTGAACAACGATATGATCGTGACCGACAACAACGGCGCCGTGAAGTTTCCCCAGCTCTGCAAGTTCTGCGATGTCAGGTTCAGCACCTGCGATAATCAGAAGTCCTGCATGTCCAACTGCAGCATCACCTCCATCTGCGAGAAGCCCCAAGAAGTGTGCGTGGCCGTGTGGCGGAAAAATGACGAGAACATCACCCTGGAGACCGTGTGTCACGACCCCAAGCTCCCTTATCACGACTTCATTCTGGAGGACGCTGCCTCCCCCAAATGCATCATGAAGGAGAAGAAGAAGCCCGGAGAGACCTTCTTTATGTGTTCCTGTAGCAGCGACGAGTGTAACGACAACATCATCTTCAGCGAAGAGTACAACACCAGCAACCCTGATGGAGGTGGCGGATCCGGAGGTGGAGGTTCTGGTGGAGGTGGGAGTATTCCTCCCCACGTGCAGAAGAGCGTGAATAATGACATGATCGTGACCGATAACAATGGCGCCGTGAAATTTCCCCAGCTGTGCAAATTCTGCGATGTGAGGTTTTCCACCTGCGACAACCAGAAGTCCTGTATGAGCAACTGCTCCATCACCTCCATCTGTGAGAAGCCTCAGGAGGTGTGCGTGGCTGTCTGGCGGAAGAATGACGAGAATATCACCCTGGAAACCGTCTGCCACGATCCCAAGCTGCCCTACCACGATTTCATCCTGGAAGACGCCGCCAGCCCTAAGTGCATCATGAAAGAGAAAAAGAAGCCTGGCGAGACCTTTTTCATGTGCTCCTGCAGCAGCGACGAATGCAACGACAATATCATCTTTAGCGAGGAATACAATACCAGCAACCCCGAC(SEQ ID NO:57)。

[0205] In some embodiments, the first chimeric polypeptide can 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:

[0206] (SEQ ID NO:6).

[0207] In some embodiments, the first chimeric polypeptide is 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:

[0208]

[0209] In some embodiments, the first chimeric polypeptide can 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:

[0210] (SEQ ID NO:7).

[0211] In some embodiments, the first chimeric polypeptide is 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:

[0212]

[0213] In some embodiments, the second chimeric polypeptide can 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:

[0214] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:5).

[0215] In some embodiments, the second chimeric polypeptide is 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:

[0216]

[0217] In some embodiments, the second chimeric polypeptide can 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:

[0218] MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDN NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:8).

[0219] In some embodiments, the second chimeric polypeptide is 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:

[0220]

[0221] In some embodiments, the second chimeric polypeptide can comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0222] IPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDNNGAVKFPQL CKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:64).

[0223] In some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0224]

[0225] In some embodiments, the second chimeric polypeptide can comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0226] MKWVTFISLLFLFSSAYSIPPHVQKSVNNDMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDGGGGSGGGGSGGGGSIPPHVQKSVNNDMIVTDN NGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR(SEQ ID NO:66).

[0227] In some embodiments, the second chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0228]

[0229] In some embodiments, the first chimeric polypeptide can comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0230] (SEQ ID NO:68).

[0231] In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0232]

[0233] In some embodiments, the first chimeric polypeptide can comprise a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0234] (SEQ ID NO:70).

[0235] In some embodiments, the first chimeric polypeptide is encoded by a sequence that is at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to:

[0236]

[0237] Composition / Kit

[0238] Also provided herein are compositions (e.g., pharmaceutical compositions) comprising at least one of any of the multi-chain chimeric polypeptides, any cells, or any nucleic acids described herein. In some embodiments, the compositions comprise at least one of any of the multi-chain chimeric polypeptides described herein. In some embodiments, the compositions comprise any immune cell (e.g., any immune cell described herein, e.g., any immune cell generated using any of the methods described herein). [023...

Claims

1. A method for reducing the level of neuroinflammation in a tissue of a subject in need thereof, the method 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 the affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of the affinity domain pair; and (ii) a second target binding domain, in: The first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the affinity domain pair; and The first target binding domain and the second target binding domain each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

2. A method of treating a neuroinflammatory disorder in a subject, the method 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 the affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of the affinity domain pair; and (ii) a second target binding domain, in: The first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the affinity domain pair; and The first target binding domain and the second target binding domain each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

3. A method of reducing the rate of progression of a neuroinflammatory disorder in a subject, the method 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 the affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of the affinity domain pair; and (ii) a second target binding domain, in: The first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the affinity domain pair; and The first target binding domain and the second target binding domain each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

4. The method of any one of claims 1-3, wherein the first target binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric polypeptide.

5. The method of any one of claims 1-3, 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.

6. The method of any one of claims 1-5, wherein the soluble tissue factor domain and the first domain of the affinity domain pair are directly adjacent to each other in the first chimeric polypeptide.

7. The method of any one of claims 1-5, 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 affinity domain pair.

8. The method of any one of claims 1-7, wherein the second domain of the affinity domain pair and the second target binding domain are directly adjacent to each other in the second chimeric polypeptide.

9. The method of any one of claims 1-7, wherein the second chimeric polypeptide further comprises a linker sequence between the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide.

10. The method of any one of claims 1-9, wherein the first target binding domain and the second target binding domain specifically bind to the same antigen.

11. The method of any one of claims 1-9, wherein the first target binding domain and the second target binding domain specifically bind different antigens.

12. The method of any one of claims 1-11, wherein one or both of the first target binding domain and the second target binding domain are antigen binding domains.

13. The method of any one of claims 1-12, wherein one or both of the first target binding domain and the second target binding domain is soluble TGF-β receptor II (TGF-βRII).

14. The method of claim 13, wherein the first target binding domain and the second target binding domain are soluble TGF-βRII.

15. The method of any one of claims 1-14, wherein the first chimeric polypeptide further comprises one or more additional target binding domains.

16. The method of any one of claims 1-15, wherein the second chimeric polypeptide further comprises one or more additional target binding domains.

17. The method of any one of claims 1-16, wherein the soluble tissue factor domain is a soluble human tissue factor domain.

18. The method of claim 17, wherein the soluble human tissue factor domain comprises a sequence that is at least 80% identical to SEQ ID NO:

1.

19. The method of any one of claims 1-18, wherein the affinity domain pair is a sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα), and soluble IL-15.

20. The method of any one of claims 1-18, wherein the affinity domain pair is selected from the group consisting of barnase and barnstar, PKA and AKAP, an adaptor / docking tag module based on mutant RNase I fragments, and a SNARE module based on the interaction of proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25.

21. The method of any one of claims 1-20, wherein the first chimeric polypeptide and / or the second chimeric polypeptide further comprises a signal sequence at its N-terminus.

22. The method of any one of claims 1-9 and 13-21, wherein the first target binding domain comprises a first sequence at least 80% identical to SEQ ID NO: 2 and a second sequence at least 80% identical to SEQ ID NO: 2, wherein the first sequence and the second sequence are separated by a linker.

23. The method of claim 22, wherein the first target binding domain comprises a first sequence that is at least 90% identical to SEQ ID NO: 2 and a second sequence that is at least 90% identical to SEQ ID NO:

2.

24. The method of claim 23, wherein the first target binding domain comprises a first sequence of SEQ ID NO: 2 and a second sequence of SEQ ID NO:

2.

25. The method of any one of claims 22-24, wherein the linker comprises the sequence of SEQ ID NO:

3.

26. The method of any one of claims 1-9 and 13-21, wherein the first target binding domain comprises a sequence at least 80% identical to SEQ ID NO:

4.

27. The method of claim 26, wherein the first target binding domain comprises a sequence that is at least 90% identical to SEQ ID NO:

4.

28. The method of claim 27, wherein the first target binding domain comprises the sequence of SEQ ID NO:

4.

29. The method of any one of claims 1-9, 13, 14, and 17-20, wherein the first chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO:

6.

30. The method of claim 29, wherein the first chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

6.

31. The method of claim 30, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

6.

32. The method of claim 31 , wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

7.

33. The method of claim 30, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

68.

34. The method of claim 33, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

70.

35. The method of any one of claims 1-9 and 13-34, wherein the second target binding domain comprises a first sequence at least 80% identical to SEQ ID NO: 2 and a second sequence at least 80% identical to SEQ ID NO: 2, wherein the first sequence and the second sequence are separated by a linker.

36. The method of claim 35, wherein the second target binding domain comprises a first sequence at least 90% identical to SEQ ID NO:2 and a second sequence at least 90% identical to SEQ ID NO:

2.

37. The method of claim 36, wherein the second target binding domain comprises a first sequence of SEQ ID NO: 2 and a second sequence of SEQ ID NO:

2.

38. The method of any one of claims 35-37, wherein the linker comprises the sequence of SEQ ID NO:

3.

39. The method of any one of claims 1-9 and 13-34, wherein the second target binding domain comprises a sequence at least 80% identical to SEQ ID NO:

4.

40. The method of claim 39, wherein the second target binding domain comprises a sequence at least 90% identical to SEQ ID NO:

4.

41. The method of claim 40, wherein the second target binding domain comprises the sequence of SEQ ID NO:

4.

42. The method of any one of claims 1-9, 13, 14, 17-20, and 22-34, wherein the second chimeric polypeptide comprises a sequence at least 80% identical to SEQ ID NO:

5.

43. The method of claim 42, wherein the second chimeric polypeptide comprises a sequence at least 90% identical to SEQ ID NO:

5.

44. The method of claim 43, wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO:

5.

45. The method of claim 44, wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO:

8.

46. ​​The method of any one of claims 1-45, wherein the tissue is one or more of brain, spinal cord, skeletal muscle, or optic nerve.

47. The method of any one of claims 1-46, wherein the subject has been diagnosed or identified as having a neuroinflammatory disease.

48. The method of any one of claims 1-46, wherein the subject has been identified as having an increased risk of developing a neuroinflammatory disease.

49. The method of claim 47 or 48, wherein the neuroinflammatory disease is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), schizophrenia, autism, depression, major depressive disorder (MDD), dysthymia, bipolar disorder, mood disorders, anxiety, fibromyalgia, Huntington's disease, amyotrophic lateral sclerosis (ALS), acute disseminated encephalomyelitis (ADEM), acute optic neuritis (AON), transverse myelitis, neuromyelitis optica (NMO), Lewy body dementia (LBD), and sarcopenia.

Images