Multi-chain chimeric polypeptides for use in the treatment of circadian clock gene disorders - Patents.com

Abstract

Provided herein are multi-chain chimeric polypeptides and their uses in regulating circadian clock gene expression in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 330,715, filed on April 13, 2022, and U.S. Provisional Patent Application No. 63 / 330,694, filed on April 13, 2022.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file titled 47039-0033WO1_SL_ST26.XML. This XML file, created on April 4, 2023, is 83,483 bytes in size. The data within the XML file is incorporated herein by reference in its entirety.

[0003] The present invention relates to the field of biotechnology, and more specifically to antigen-binding molecules and their use to modulate the expression of one or more circadian clock genes. [Background technology]

[0004] Tissue factor (TF), a 263 amino acid integral membrane glycoprotein with a molecular weight of approximately 46 kDa and the trigger protein of the extrinsic blood coagulation pathway, is the primary initiator of coagulation in vivo. Tissue factor is not normally in contact with circulating blood and initiates the coagulation cascade upon exposure to circulating coagulation serine protease factors. Vascular injury exposes subendothelial cells that express tissue factor, leading to the formation of a calcium-dependent, high-affinity complex with preexisting 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 of FXa and active membrane surfaces then inefficiently converts small amounts of prothrombin to thrombin. Thrombin generated by FXa initiates platelet activation, activating trace amounts of pro-cofactors factor V (FV) and factor VIII (FVIII) to the active cofactors, factor Va (FVa) and factor VIIIa (FVIIIa). FIXa complexes with FVIIIa on the platelet surface to form the intrinsic tenase complex, which leads to the rapid generation of FXa. FXa complexes with FVa to form the prothrombinase complex on the activated platelet surface, which leads to the rapid cleavage of prothrombin to thrombin.

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

[0006] Much of the tissue factor expressed on the cell surface is "encoded" and must be "decoded" to fully participate in coagulation. Although the mechanism of "decoding" cell surface tissue factor is currently unknown, exposure of anionic phospholipids plays a major role in this process. Healthy cells actively sequester anionic phospholipids such as phosphatidylserine (PS) in the inner leaflet of the plasma membrane. Upon cell damage, activation, or loss of cytosolic Ca 2+ Following increased levels, this bilayer asymmetry is lost, leading to increased PS exposure on the outer leaflet, which increases the specific activity of the cell surface tissue factor-FVIIa complex. Although PS exposure is known to decrease the apparent Km for activation of FIX and FX by tissue factor-FVIIa complexes, additional mechanisms may include conformational rearrangements of tissue factor or tissue factor-FVIIa and subsequent exposure of substrate binding sites.

[0007] Circadian rhythms are a series of oscillators generated by a molecular circadian clock that cycles through a 24-hour day. The circadian clock is an internal timing system controlled at the transcriptional level, resulting in a gene network that oscillates with an approximately 24-hour cycle. The circadian clock system is a major regulator of almost all physiological activities. Disorders of circadian rhythms and the circadian clock system have serious consequences for human health, including cognitive impairment, psychiatric disorders, metabolic syndrome, malformations, aging, and cancer. Summary of the Invention [Means for solving the problem]

[0008] Provided herein is a method of modulating expression of one or more circadian clock genes in a tissue of a subject in need thereof, 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 the affinity domain pair; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of the affinity domain pair, and wherein the first target binding domain and the second target binding domain, respectively, specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0009] Also provided herein is a method of treating a circadian clock genetic disorder in a subject, 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 the affinity domain pair; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated via 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, respectively, specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0010] Also provided herein is a method of reducing the rate of progression of a circadian clock genetic disorder in a subject, 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 the affinity domain pair; and (ii) a second target binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated via 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, respectively, specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0011] In some embodiments, the first target binding domain and the soluble tissue factor domain are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments, 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 immediately 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 immediately 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.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In some aspects, the affinity domain pair is the 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.

[0016] 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, the first and second sequences being 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 a sequence of SEQ ID NO:3.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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, the first and second sequences being 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 of SEQ ID NO:2 and a second sequence of SEQ ID NO:2. In some embodiments, the linker comprises a sequence of SEQ ID NO:3.

[0021] 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.

[0022] 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.

[0023] In some embodiments, administration of the multi-chain chimeric polypeptide increases the expression level of one or more of Per, Cry, Nr1d1, Nr1d2, Tef, and Dbp, compared to the expression level in a similar subject not administered the multi-chain chimeric polypeptide. In some embodiments, administration of the multi-chain chimeric polypeptide decreases the expression level of one or more (e.g., all) of Arntl, Npas2, and Ppard, compared to the expression level in a similar subject not administered the multi-chain chimeric polypeptide.

[0024] In some embodiments, the tissue is heart tissue, kidney tissue, spleen tissue, pancreatic tissue, intestinal tissue, brain tissue, and / or liver tissue.

[0025] In some embodiments, administration of the multi-chain chimeric polypeptide increases expression of one or more of the following: Per, Cry, Nr1d1, Nr1d2, Tef, and Dbp. In some embodiments, administration of the multi-chain chimeric polypeptide decreases expression of one or more of the following: Arntl, Npas2, and Ppard.

[0026] In some embodiments, the subject has been diagnosed or identified as having a circadian clock genetic disorder. In some embodiments, the subject has been identified as being at high risk for developing a circadian clock genetic disorder. In some embodiments, the circadian clock genetic disorder is selected from the group consisting of bipolar disorder (BPD), major depressive disorder (MDD), attention deficit hyperactivity disorder (ADHD), anxiety disorder, cognitive impairment, schizophrenia, obesity, diabetes, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), metabolic disorders, cancer, cardiovascular disease, sleep disorders, age-related disabilities, sarcopenia, and liver fibrosis.

[0027] As used herein, the term "circadian rhythm" or "circadian clock" refers to an internal timing system controlled at the transcriptional level that results in a gene network that oscillates on an approximately 24-hour cycle. The circadian clock controls approximately 24-hour physiological and behavioral processes, including, but not limited to, sleep, metabolism, the immune system, the cardiovascular and nervous systems, the microbiome, cancer, and the regulation of aging. Circadian rhythms control physical, mental, and behavioral processes.

[0028] As used herein, the term "circadian clock gene" refers to a gene that controls circadian rhythms in physiology and behavior. Circadian clock gene may also refer to a gene whose protein product is a necessary element for the generation and regulation of circadian rhythms. Circadian clock genes are known to control human physiology and have various homeostatic functions, and disruption of these genes may be associated with sleep disorders, cancer, susceptibility to infections, metabolic syndrome, Alzheimer's disease, and aging. Non-limiting examples of circadian clock genes include, but are not limited to, CLOCK, BMAL1 (brain and muscle ARNT-like protein 1 (ARNTL)), Dbp, NPAS2, Per1, Per2, Per3, Cry1, Cry2, Rorαm, Rev-Erbα (Nr1d1), and Rev-Erbβ (Nr1d2). In some embodiments, the circadian clock gene may be a clock-controlled gene including Usp2, Tsc22d3, and Tspan4.

[0029] As used herein, the term "circadian clock genetic disorder" refers to a disease caused, at least in part, by dysregulation of human circadian rhythm. Disruption of circadian clock genes, which maintain the molecular clock of all cells, tissues, and related molecular mechanisms, is known to affect multiple diseases and disorders. For example, circadian clock genetic disorders include, but are not limited to, bipolar disorder (BPD), major depressive disorder (MDD), ADHD, anxiety disorder, cognitive disorder, schizophrenia, obesity, diabetes, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), metabolic disorder, cancer, cardiovascular disease, sleep disorder, age-related physical disability, and sarcopenia.

[0030] As used herein, the term "chimeric" refers to a polypeptide that includes amino acid sequences (e.g., domains) that are originally derived from two different sources (e.g., two different naturally occurring proteins, e.g., from the same species or different species). For example, a chimeric polypeptide can include domains from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide can include a domain that is a synthetic sequence (e.g., an scFv) and a domain that is derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide can include at least two different domains that are synthetic sequences (e.g., two different scFvs).

[0031] An "antigen binding domain" is one or more protein domains (e.g., formed from amino acids from a single polypeptide or formed from amino acids from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens. In some examples, an antigen binding domain can bind to an antigen or epitope with specificity and affinity similar to that of a naturally occurring antibody. In some embodiments, an antigen binding domain may be an antibody or a fragment thereof. In some embodiments, an antigen binding domain may comprise an alternative scaffold. Non-limiting examples of antigen binding domains are described herein. Further examples of antigen binding domains are known in the art.

[0032] "Soluble tissue factor domain" refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) to a segment of a wild-type mammalian tissue factor protein (e.g., a wild-type human tissue factor protein) lacking the transmembrane and intracellular domains. Non-limiting examples of soluble tissue factor domains are described herein.

[0033] The term "soluble interleukin receptor" is used in the broadest sense herein and refers to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that can bind to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate-buffered saline at room temperature). For example, a soluble interleukin receptor may include a sequence that is at least 70% identical (e.g., at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical) to the extracellular domain of a wild-type interleukin receptor and retains 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.

[0034] The term "soluble cytokine receptor" is used herein in the broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that can bind to one or more of its natural ligands (e.g., under physiological conditions, e.g., in phosphate buffered saline at room temperature). For example, a soluble 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.

[0035] The term "antibody" is used herein in its broadest sense and includes a specific type of immunoglobulin molecule that includes one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. Further examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0036] "Affinity" refers to the strength of the sum of non-covalent interactions between an antigen-binding site and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to the intrinsic binding affinity that 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 is determined by the dissociation equilibrium constant (K D ) The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Further methods for determining the affinity of antigen-binding domains and their corresponding antigens or epitopes are known in the art.

[0037] A "multi-chain polypeptide," as used herein, refers to a polypeptide that includes two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), where the two or more protein chains associate through non-covalent bonds to form a quaternary structure.

[0038] The term "affinity domain pair" refers to a pair of domains that is 1 × 10 -7 K less than M D (For example, 1×10-8 Less than M, 1×10 -9 Less than M, 1×10 -10 Less than M or 1×10 -11 The affinity domain pair is two different protein domains that specifically bind to each other with a specific affinity of less than M. In some examples, 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.

[0039] The term "epitope" refers to the portion of an antigen that specifically binds to an antigen-binding domain. An epitope may consist of, for example, surface-accessible amino acid residues and / or sugar side chains and may have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter may be lost in the presence of denaturing solvents. An epitope may 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 the epitope to which an antigen-binding domain binds are known in the art.

[0040] The term "treatment" refers to improving at least one symptom of a disorder. In some instances, the disorder being treated is cancer, and improving at least one symptom of cancer includes reducing the abnormal proliferation, gene expression, signal transduction, translation, and / or secretion of a factor. In general, the treatment method includes administering to a subject in need of such treatment, or a subject determined to be in need of such treatment, a therapeutically effective amount of a composition that reduces at least one symptom of the disorder.

[0041] The phrase "rate of progression of circadian clock genetic disorder" refers to the rate at which symptoms of circadian clock genetic disorder gradually worsen over time. For example, circadian clock genetic disorder may progress at a rapid rate (e.g., days or weeks) or at a slower rate (e.g., months or years). In some embodiments, the rate of progression of circadian clock genetic disorder can be determined by a physician evaluating sleep habits or performing diagnostic and cognitive tests. In some embodiments, the rate of progression of circadian clock genetic disorder can also be measured by imaging tests (e.g., CT scan, magnetic resonance imaging (MRI)).

[0042] Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are merely illustrative and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will govern.

[0043] 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]

[0044] [Figure 1]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 pair of domains (soluble interleukin IL-15), and an additional target binding domain (B), and (ii) a second chimeric polypeptide comprising a second domain of an affinity pair of domains (IL-15 receptor alpha sushi domain), a second target binding domain (C), and an additional antigen binding domain (D). The top schematic shows the association of the first and second chimeric polypeptides via the pair of affinity domains. The bottom schematic shows the order of the domains in the first and second chimeric polypeptides. [Diagram 2] Exemplary diagrams of multi-chain chimeric polypeptides: (i) a first chimeric polypeptide comprising a first target binding domain (A), a soluble tissue factor domain comprising five amino acid substitutions to eliminate binding of the soluble tissue factor domain to FVIIa, a first domain of an affinity pair of domains (soluble interleukin IL-15 comprising D8N or D8A amino acid substitutions), and an additional target binding domain (B), and (ii) a second chimeric polypeptide comprising a second domain of an affinity pair of domains (IL-15 receptor alpha sushi domain), a second target binding domain (C), and an additional antigen binding domain (D). The top schematic shows the association of the first and second chimeric polypeptides via the affinity domain pair. The bottom schematic 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). [Diagram 3] A schematic diagram of the TGFRt15-TGFRs construct is shown. [Figure 4] A further schematic of the TGFRt15-TGFRs construct is shown. [Diagram 5] The results of TGFβ1 inhibition by TGFRt15-TGFRs and TGFR-Fc are shown. [Figure 6]1 shows the results of a 32D β cell proliferation assay using TGFRt15-TGFRs or recombinant IL-15. [Figure 7A] The results of detecting IL-15 and TGFβRII in TGFRt15-TGFRs by ELISA using the corresponding antibodies are shown. [Figure 7B] This is a continuation of Figure 7A. [Figure 8] 1 is a line graph showing the chromatographic profile of TGFRt15-TGFRs protein-containing cell culture supernatant after binding on an anti-TF antibody resin and elution. [Figure 9] Analytical SEC profile of TGFRt15-TGFRs is shown. [Figure 10] Shown is TGFRt15-TGFRs before and after deglycosylation analyzed by reducing SDS-PAGE. [Figure 11A] Figure 85 shows spleen weight and percentage of immune cell types in TGFRt15-TGFRs-treated mice and control-treated mice. Figure 85A shows spleen weight in mice treated with TGFRt15-TGFRs compared to PBS control. Figure 85B shows percentage of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with TGFRt15-TGFRs compared to PBS control. [Figure 11B] This is a continuation of Figure 11A. [Figure 12A] Figure 86A shows spleen weight and immune stimulation over 92 hours in mice treated with TGFRt15-TGFRs. Figure 86B shows the percentage of immune cells in mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours after treatment. [Figure 12B] This is a continuation of Figure 12A. [Figure 13A] 1 shows Ki67 and Granzyme B expression over time in mice treated with TGFRt15-TGFRs. [Figure 13B] This is a continuation of Figure 13A. [Figure 14]1 shows enhanced cytotoxicity of splenocytes by TGFRt15-TGFRs in C57BL / 6 mice. [Figure 15] 1 shows the change in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or a combination of chemotherapy and TGFRt15-TGFRs in a mouse model of pancreatic cancer. [Figure 16] 1 shows the cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFRs. [Figure 17-1] FIG. 1 shows in vivo stimulation of Tregs, NK cells and CD8+ T cells in ApoE− / − mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 17-2] This is a continuation of Figure 17-1. [Figure 18-1] FIG. 1 shows immune stimulation in C57BL / 6 mice following treatment with TGFRt15-TGFRs. [Figure 18-2] This is a continuation of Figure 18-1. [Figure 19] FIG. 1 shows in vivo induction of NK cell and CD8+ T cell proliferation in ApoE− / − mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 20] 1 shows enhanced NK cell cytotoxicity following treatment of NK cells with TGFRt15-TGFRs. [Figure 21] 1 shows the enhancement of ADCC activity of NK cells following treatment of the NK cells with TGFRt15-TGFRs. [Figure 22-1] 1 shows the anti-cancer activity of TGFRt15-TGFRs+anti-TRP1(TA99) in combination with chemotherapy in a melanoma mouse model. [Figure 22-2] This is a continuation of Figure 22-1. [Figure 22-3] Continuation of Figure 22-2. [Figure 23] TGFRt15-TGFRs ameliorates Western diet-induced hyperglycemia in ApoE− / − mice. [Figure 24]Shows upregulation of CD44hi memory T cells upon treatment with TGFRt15-TGFRs. [Figure 25-1] TGFRt15-TGFRs (HCW9218) enhances immune-mediated biological activity in diabetic db / db mice. Figures 25A-25E show representative flow cytometry data showing increased immune cell surface markers on splenocytes of TGFRt15-TGFRs (HCW9218) treated mice compared to controls at day 4. Figures 25F-25G show representative flow cytometry data showing increased numbers of central and effector memory cells in splenocyte populations compared to controls with TGFRt15-TGFR (HCW9218) at day 4. Figure 25H shows killing of Yacl target cells by in vivo TGFRt15-TGFRs (HCW9218) treatment compared to control splenocytes. Figure 25I shows increased interferon (IFN)-γ release by CD3+ cells upon antigen-independent stimulation with TGFRt15-TGFRs (HCW9218)-treated in vivo and -CD3 / a-CD28 bead-stimulated splenocytes ex vivo compared to controls. Figure 25J shows representative data on increased extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) in vivo treated with PBS or TGFRt15-TGFRs (HCW9218) or TGFRt15*-TGFRs (HCW9228) splenocytes and analyzed with a Seahorse XFe Bioanalyzer (Agilent). Figure 25K shows ELISA data showing decreased TGFβ1 and TGFβ2, but not TGFβ3, in plasma after TGFRt15-TGFRs (HCW9218) or TGFRt15*-TGFRs (HCW9228). [Figure 25-2] This is a continuation of Figure 25-1. [Figure 25-3] Continuation of Figure 25-2. [Figure 25-4] Continuation of Figure 25-3. [Figure 26-1]TGFRt15-TGFRs (HCW9218) treatment reduces senescent pancreatic beta islet cells and SASP factors and ameliorates type 2 diabetes in db / db mice. Figure 26A shows a schematic diagram of TGFRt15-TGFRs (HCW9218) treatment in db / db mouse model. Figure 26B shows the expression of senescence, SASP, and beta cell index (related genes) in islet transcripts analyzed by quantitative PCR and normalized to control treatment. Figures 26C-26D show immunofluorescence staining of p21+ cells (yellow) and insulin+ beta islet cells (green) in pancreatic tissue sections. Figure 26E shows the number of insulin-positive islet cells in tissue sections. Figure 26F shows the number of p21+ senescent cells in beta islet cells in tissue sections. Figures 26G-26I show pancreatic senescence, SASP, and beta cell index by quantitative PCR. Figure 26J shows fasting blood glucose after TGFRt15-TGFRs (HCW9218) treatment. Figure 26K shows HOMA-IR index after TGFRt15-TGFRs (HCW9218) treatment. Figure 26L shows volcano plot of RNAseq analysis in liver of db / db mice. Figures 26M-26P show heatmaps of metabolic, aging, inflammatory, and vascular genes. [Figure 26-2] This is a continuation of Figure 26-1. [Figure 26-3] Continuation of Figure 26-2. [Figure 26-4] Continuation of Figure 26-3. [Figure 26-5] Continuation of Figure 26-4. [Figure 26-6] Continuation of Figure 26-5. [Figure 27-1]TGFRt15-TGFRs (HCW9218) stimulates immune cell activity and metabolic function in the liver of aged mice. Figure 27A shows a schematic of TGFRt15-TGFRs (HCW9218) and TGFRt15*-TGFRs (HCW9228) treatment in young and old mouse models at days 4 and 10. Figure 27B shows a composite unbiased t-SNE discriminating B cells, T cells, and group 1 ILCs. Figure 27C shows a representative viSNE plot color-coded by density from liver harvested at day 4. Gates are color-coded by population, and inset numbers represent the frequency of I-ILCs (ILC1 and NK cells). Figures 27D-27E show summary data from N=3 independent experiments, showing that N=6 mice / group increases the total frequency of ILC-1 cells in the liver at days 4 (Figure 27D) and 10 (Figure 27E). Figures 27F-27I show total NK cell frequency in liver at days 4 (Figure 27F) and 10 (Figure 27G) and in spleen at days 4 (Figure 27H) and 10 (Figure 27I). Figure 27J shows the positive percentage of Ki-67 proliferation marker in liver. Figures 27K-27L show total CD8+ T cell frequency in liver (Figure 27K) and spleen (Figure 27L) at days 4 and 10. Figures 27M-27N show representative data for the increase in extracellular acidification rate (ECAR) (Figure 27M) and oxygen consumption rate (OCR) (Figure 27N) of splenocytes stimulated in vivo with TGFRt15-TGFRs (HCW9218) from young and old mice compared to controls by Seahorse XFe Bioanalyzer. Figure 27O shows flow cytometric measurements of ex vivo cytotoxic activity against Yac1 target cells by in vivo TGFRt15-TGFRs (HCW9218) stimulated splenocytes from young and old mice compared to controls (PBS or TGFRt15*-TGFRs (HCW9228)). Figures 27P-27Q show increased interferon (IFN)-γ and (TNF)-α release by CD3+ cells after antigen-independent stimulation by in vivo HCW9218-treated and ex vivo α-CD3 / CD28 bead-stimulated splenocytes compared to control old and young mice measured with the MAGPIXX multiplex system.FIG. 27R shows representative flow cytometry data showing the increase in intracellular granzyme B levels as a percentage. [Figure 27-2] This is a continuation of Figure 27-1. [Figure 27-3] Continuation of Figure 27-2. [Figure 27-4] Continuation of Figure 27-3. [Figure 28-1] Figure 28 shows that TGFRt15-TGFRs (HCW9218) reduces inflammation (SASP) and cellular senescence markers in the liver of aged mice after one or two subcutaneous administrations of TGFRt15-TGFRs (HCW9218) or PBS. Figure 28A shows the difference in total RNASeq of aged mouse hepatocytes treated with TGFRt15-TGFRs (HCW9218) for 60 days compared to control treatment in a volcano plot. Figures 28B-28D show heatmaps of differentially expressed senescence and inflammation, glycogenesis and fatty acid metabolism, and circadian rhythm-related genes in the liver after treatment with TGFRt15-TGFRs (HCW9218) compared to control treatment (adjusted p-value < 0.05). Figures 28E-28F show the relative mRNA expression 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 after treatment with TGFRt15-TGFRs (HCW9218) compared to controls on days 10 and / or 60. [Figure 28-2] This is a continuation of Figure 28-1. [Figure 28-3] Continuation of Figure 28-2. [Figure 28-4] Continuation of Figure 28-3. [Figure 29-1]Figure 29A shows that two doses of TGFRt15-TGFRs(HCW9218) stimulate metabolic function and reduce inflammation (SASP) and cellular senescence markers in the liver of aged mice over a prolonged period. Figure 29A shows a schematic of two doses of TGFRt15-TGFRs(HCW9218) treatment in aged (76 weeks) female C57BL / 6J mice subcutaneously injected with 3 mg / kg TGFRt15-TGFRs(HCW9218) (n=5-8) or saline. Mice received a second dose of TGFRt15-TGFRs(HCW9218) on day 60 and were euthanized on day 120. Figures 29B-29C show representative data on the increase in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of splenocytes stimulated in vivo with two doses of TGFRt15-TGFRs (HCW9218) compared to control as measured by Seahorse XFe Bioanalyzer. Figure 29D shows the relative mRNA expression of Il1α, Cdkn1a, Pai-1, Il1b, and Il6 in liver after treatment with one or two doses of TGFRt15-TGFRs (HCW9218) compared to control group at day 120 as measured by quantitative PCR. Figures 29E-29F show ELISA data showing protein levels of IL-1α, IL-6, IL-8, PAI-1, and fibronectin in liver tissue by liver ELISA after treatment with one or two doses of TGFRt15-TGFRs (HCW9218) compared to control on day 120. Figures 29G-29H show immunofluorescence staining of liver tissue cells expressing p21+ (yellow) after treatment with two doses of TGFRt15-TGFRs (HCW9218). Figure 29I shows a heatmap of differentially expressed senescence, inflammation, and circadian rhythm-related genes in liver after treatment with TGFRt15-TGFRs (HCW9218) compared to control treatment (adjusted p-value <0.05). [Figure 29-2] This is a continuation of Figure 29-1. [Figure 29-3] Continuation of Figure 29-2. [Figure 29-4] Continuation of Figure 29-3. [Figure 29-5] Continuation of Figure 29-4. [Figure 30-1]TGFRt15-TGFRs (HCW9218) reduces neuroinflammation and affects neuronal function in naturally aged mice. Figure 30A shows the relative mRNA expression of aging-related genes Cdkn1a, Il6, Il1α, cdkn2a, Il1β, Tnfα, and Il8 in the hippocampus of young and old mice 120 measured using quantitative PCR. Figure 30B shows the relative mRNA expression of aging-related genes Cdkn1a, Il1α, Il6, Tnfα, Il1β, and Il18 in the hippocampus of old mice compared to controls after 60 days of treatment with TGFRt15-TGFRs (HCW9218) measured using quantitative PCR. 18S rRNA was used for normalization. Figure 30C shows total RNASeq volcano plots in the hippocampus of aged mice 60 days after treatment with TGFRt15-TGFRs (HCW9218) compared to control treatment. Figures 30D-30E show heatmaps of differentially expressed neuronal, circadian rhythm and neuroinflammation-related genes in the hippocampus after treatment with TGFRt15-TGFRs (HCW9218) compared to control treatment (adjusted p-value < 0.05). [Figure 30-2] This is a continuation of Figure 30-1. [Figure 30-3] Continuation of Figure 30-2. [Figure 30-4] Continuation of Figure 30-3. [Figure 31-1]Both TGFRt15-TGFRs (HCW9218) and TGFRt15*-TGFRs (HCW9228) treated aged mice show significant maintenance of physical performance over time, with TGFRt15-TGFRs (HCW9218) being well tolerated by mice and non-human primates and having no long-term adverse effects on naturally aged mice. Figure 31A shows grip strength tests performed on aged mice treated with TGFRt15-TGFRs (HCW9218) and TGFRt15*-TGFRa (HCW9228) compared to controls. Figure 31B shows the rotarod performance of mice treated with saline, TGFRt15-TGFRs (HCW9218), and TGFRt15*-TGFRs (HCW9228), and Figure 31C shows the open field test performed on the same mice to measure total distance traveled. Figures 31D-31E show flow cytometry analysis of Ki67 expression (Figure 31D) and absolute numbers of CD4, CD8, Treg, and CD16+ NK cells (Figure 31E) in blood from cynomolgus monkeys (10 monkeys / group) after two dose treatments (study days 1 and 15) with TGFRt15-TGFRs (HCW9218) or saline. Figure 31F shows survival probability monitored and analyzed using the log-rank test. Figure 31G shows body weight measured after 5 months. Figures 31H-31I show representative flow cytometry data showing the percentage of CD8+ T cells and NK cells in blood. [Figure 31-2] This is a continuation of Figure 31-1. [Figure 31-3] Continuation of Figure 31-2. [Figure 31-4] Continuation of Figure 31-3. [Figure 32-1] Metabolic activity of splenocytes from naturally aged mice on days 2 and 4. Figures 32A-32D show representative data on the increase in metabolic parameters of splenocytes from db / db mice stimulated in vivo with a single dose of TGFRt15-TGFRs (HCW9218) by Seahorse XFe Bioanalyzer compared to controls on days 2 (Figures 32A-32B) and 4 (Figures 32C-32D). [Figure 32-2] This is a continuation of Figure 32-1. [Figure 32-3] Continuation of Figure 32-2. [Diagram 33] TGFRt15-TGFRs (HCW9218) enhances immune-mediated biological activity in old mice. Figures 33A-33B show representative flow cytometry data showing increased immune cell surface markers in the blood (Figure 33A) and spleen (Figure 33B) of young and old mice after 4 days of treatment with TGFRt15-TGFRs (HCW9218) compared to controls. Figures 33C-33D show immune cell proliferation marker Ki67 in NK cells in the blood (Figure 33C) and spleen (Figure 33D) of young and old mice after 4 days of treatment with TGFRt15-TGFRs (HCW9218) compared to controls. Figures 33E-33G show representative flow cytometry data showing increased immune cell surface markers on liver immune cells after 4 days of treatment with TGFRt15-TGFRs (HCW9218) in young and old mice compared to controls. Figures 33H-33I show summary data from N=2 independent experiments, with N=6 mice / group showing increased frequencies of total CD4+ T cells in the liver 4 and 10 days after treatment with TGFRt15-TGFRs (HCW9218) compared to control treatment (Figure 33H) and increased frequencies of total CD4+ T cells in the spleen on days 4 and 10 (Figure 33I). [Figure 34-1]Figure 34A shows that TGFRt15-TGFRs (HCW9218) stimulates immune cell activity and metabolic function, and reduces inflammation (SASP) and cellular senescence markers in aged mice. Figure 34A shows a heat map of differentially expressed immune pathway-related genes in liver after treatment with TGFRt15-TGFRs (HCW9218) compared to control treatment (adjusted p-value < 0.05). Figure 34B shows a heat map of differentially expressed senescence, inflammation, and circadian rhythm-related genes in liver after treatment with TGFRt15*-TGFRs (HCW9228) compared to control treatment (adjusted p-value < 0.05). FIG. 34C shows a heatmap of differentially expressed senescence-, inflammation-, and circadian rhythm-related genes in liver after treatment with TGFRt15-TGFRs (HCW9218) compared to TGFRt15*-TGFRs (HCW9228) after 120 days (adjusted p-value < 0.05). [Figure 34-2] This is a continuation of Figure 34-1. [Figure 34-3] Continuation of Figure 34-2. [Figure 35-1] Figure 35A shows a heatmap of differentially expressed aging, inflammation, and circadian rhythm-related genes in the hippocampus after treatment with TGFRt15-TGFRs (HCW9218) compared to control treatment (adjusted p-value < 0.05). Figure 35B shows a line analysis of differentially expressed hippocampal genes in the hippocampus after treatment with TGFRt15-TGFRs (HCW9218) on day 120. [Figure 35-2] This is a continuation of Figure 35-1. [Figure 35-3] Continuation of Figure 35-2. [Diagram 36]Immunohistochemical staining of blood-brain barrier test. Figures 36A-36D show mice subcutaneously injected with either PBS or TGFRt15-TGFRs (HCW9218) (3 mg / kg) and euthanized the next day. Brain cryosections from 7-week-old (Figure 36A), 73-week-old (Figure 36B), or 105-week-old (Figure 36C) mice in either the control or treatment groups were processed for immunohistochemical staining with anti-human tissue factor (TF) specific antibody. No positive staining was detected in mouse brains. Figure 36D shows that the antibody shows specific staining for human TF in human brain sections. [Figure 37] Behavioral studies show that both TGFRt15-TGFRs (HCW9218) and TGFRt15*-TGFR (HCW9228) show minimal acute and significant performance maintenance in naturally aged mice. Figure 37A shows grip strength, Figure 37B shows rotarod test, and Figure 37C shows open field test, which were performed in aged mice treated with TGFRt15-TGFRs (HCW9218) and TGFRt15*-TGFRa (HCW9228), respectively, to measure the acute effect on peak force values ​​30 days after the first administration of each treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] Provided herein are methods for regulating expression of one or more circadian clock genes in a tissue of a subject in need thereof, 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 the affinity domain pair; and (ii) a second target binding domain; a method for treating a circadian clock genetic disorder in a subject; and a method for reducing the rate of progression of a circadian clock genetic disorder in a subject, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, wherein the first chimeric polypeptide and the second chimeric polypeptide are associated via 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, respectively, specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0046] In some examples of the multi-chain chimeric polypeptide described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide is, independently, 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, or about 50 amino acids to about 850 amino acids. acid, 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 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 ~ 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 95 ...15 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 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 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 480 amino acids, about 150 amino acids to about about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to 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 about 200 to about 3000 amino acids, about 200 to about 2500 amino acids, about 200 to about 2000 amino acids, about 200 to about 1500 amino acids, about 200 to about 1000 amino acids, about 200 to about 950 amino acids, about 200 to about 900 amino acids, about 200 to about 850 amino acids, about 200 to about 800 amino acids, about 200 to about 750 amino acids, about 200 to about 700 amino acids, about 200 to about 650 amino acids, about 200 to about 600 amino acids, about 200 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 3 000 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 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 2 40 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 acid, about 240 amino acids to about 260 amino acids, about 260 amino acids to about 3000 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 65 ...650 amino acids, about 260 amino acids to about 950 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 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 about 280 to about 3000 amino acids, about 280 to about 2500 amino acids, about 280 to about 2000 amino acids, about 280 to about 1500 amino acids, about 280 to about 1000 amino acids, about 280 to about 950 amino acids, about 280 to about 900 amino acids, about 280 to about 850 amino acids, about 280 to about 800 amino acids, about 280 to about 750 amino acids, about 280 to about 700 amino acids, about 280 to about 650 amino acids, about 280 to about 600 amino acids, about 280 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 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 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 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 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 ~ 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 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 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 85 ... from about 360 amino acids to about 800 amino acids, from about 360 amino acids to about 750 amino acids, from about 360 amino acids to about 700 amino acids, from about 360 amino acids to about 650 amino acids, from about 360 amino acids to about 600 amino acids, from about 360 amino acids to about 550 amino acids, from about 360 amino acids to about 500 amino acids, from about 360 amino acids to about 480 amino acids, from about 360 amino acids to about 460 amino acids, from about 360 amino acids to about 440 amino acids, from about 360 amino acids to about 420 amino acids, from about 360 amino acids to about 400 amino acids, from about 360 amino acids to about 380 amino acids, from about 380 amino acids to about 3000 amino acids,About 380 amino acids to about 2500 amino acids, about 380 amino acids to about 2000 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 ~ 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 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 acid, 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 400 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 ~ 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 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 ~ 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 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 ~ 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, 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 ~ 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 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 8 00 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 50 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 The length may be from 950 amino acids to about 1000 amino acids, from about 1000 amino acids to about 3000 amino acids, from about 1000 amino acids to about 2500 amino acids, from about 1000 amino acids to about 2000 amino acids, from about 1000 amino acids to about 1500 amino acids, from about 1500 amino acids to about 3000 amino acids, from about 1500 amino acids to about 2500 amino acids, from about 1500 amino acids to about 2000 amino acids, from about 2000 amino acids to about 3000 amino acids, from about 2000 amino acids to about 2500 amino acids, or from about 2500 amino acids to about 3000 amino acids. Diagrams of exemplary multi-chain chimeric polypeptides provided herein are shown in Figures 1 and 2.

[0047] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain (e.g., any of the first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein) between the first target binding domain (e.g., any of the exemplary first target binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) in the first chimeric polypeptide.

[0048] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the affinity domain pair (e.g., any of the exemplary first domains of any of the exemplary affinity domain pairs described herein) are immediately adjacent to each other in the first chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the affinity domain pair (e.g., any of the exemplary first domains of any of the exemplary affinity domain pairs described herein) in the first chimeric polypeptide.

[0049] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second domain of the affinity domain pair (e.g., any of the exemplary second domains of any of the exemplary affinity domain pairs described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein) between the second domain of the affinity domain pair (e.g., any of the exemplary second domains of any of the exemplary affinity domain pairs described herein) and the second target binding domain (e.g., any of the exemplary second target binding domains described herein) in the second chimeric polypeptide.

[0050] Non-limiting aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are described below and can be used in any combination without limitation. Additional aspects of these chimeric polypeptides, nucleic acids, vectors, cells, and methods are known in the art.

[0051] tissue factor Human tissue factor is a 263 amino acid transmembrane protein that contains three domains: (1) a 219 amino acid N-terminal extracellular domain (residues 1-219); (2) a 22 amino acid transmembrane domain (residues 220-242); and (3) a 21 amino acid cytoplasmic C-terminal tail (residues 242-263) (UniProtKB Identification Number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, and one S-palmitoylation site at Cys245. Deletions or mutations in the cytoplasmic domain were found to have no effect on tissue factor clotting activity. Tissue factor has one S-palmitoylation site in the intracellular domain of the protein at Cys245, which is located at the amino acid terminus of the intracellular domain and close to the membrane surface. The tissue factor transmembrane domain is composed of a single α-helix.

[0052] The extracellular domain of tissue factor, composed of two fibronectin type III domains, is linked to the transmembrane domain via a six amino acid linker. This linker provides conformational flexibility to separate the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module is composed of two overlapping beta sheets with an upper sheet domain containing three antiparallel beta strands and a lower sheet containing four beta strands. The beta strands are connected by beta loops between strands beta A and beta B, beta C and beta D, and beta E and beta F, all of which have conserved conformations in the two modules. There are three short alpha helical segments connecting the beta strands. A unique feature of tissue factor is the 17 amino acid beta-hairpin between the beta 10 and beta 11 strands, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12 amino acid loop between beta 6F and beta 7G, which is absent in the C-terminal domain and is unique to tissue factor. This fibronectin type III domain structure is characteristic of the immunoglobulin-like family of protein folds and is conserved among a wide variety of extracellular proteins.

[0053] Zymogen FVII, once bound to tissues to form the active tissue factor-FVIIa complex, is rapidly converted to FVIIa by limited proteolysis. FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also bind directly to tissue factor. Allosteric interactions between tissue factor and FVIIa on the tissue factor-FVIIa complex greatly increase the enzymatic activity of FVIIa: approximately a 20- to 100-fold increase in the rate of hydrolysis of small chromogenic peptidyl substrates, and an almost million-fold increase in the rate of activation of the natural polymeric substrates FIX and FX. In response to the allosteric activation of the active site of FVIIa upon binding to tissue factor, tissue factor-FVIIa complexes on phospholipid bilayers (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface) undergo Ca activation. 2+It further increases the rate of FIX or FX activation by 1,000-fold depending on the tissue factor FVIIa. The approximately 1 million-fold overall increase in FX activation by tissue factor-FVIIa-phospholipid complexes compared to free FVIIa is a key control point in the coagulation cascade.

[0054] FVII is a single polypeptide chain of approximately 50 kDa consisting of 406 amino acid residues, with an N-terminal γ-carboxyglutamic acid-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII is a single polypeptide chain consisting of Ile- in the short linker region between the EGF2 and protease domains. 154 -Arg 152 It is activated to FVIIa by specific proteolytic cleavage of the Cys bond. 135 and Cys 262 FVIIa binds to the Ca receptor through its N-terminal GLA domain. 2+ Immediately C-terminal to the GLA domain is an aromatic stack and two EGF domains. The aromatic stack binds a single Ca 2+ GLA is connected to the EGF1 domain that binds to the Ca ion. 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 A single Ca domain in the FVIIa protease domain 2+ Ion binding is important for its catalytic activity. Proteolytic activation of FVII to FVIIa occurs via Ile 153 releases the newly formed amino terminus, folds back, and inserts into the activation pocket, where it is Asp 343to generate an oxyanion hole. This salt bridge formation is important for FVIIa activity. However, in free FVIIa, oxyanion hole formation does not occur upon proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is poorly recognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

[0055] Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is important for FVIIa to its cognate substrate. Free FVIIa adopts a stable extended conformation when bound to a membrane with its active site located approximately 80 Å above the membrane surface. Upon binding of FVIIa to tissue factor, the FVa active site relocates approximately 6 Å closer to the membrane. This adjustment may aid in the proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using FVIIa without the GLA domain, it was shown that the active site is still located at a similar distance on the membrane, demonstrating that tissue factor can fully support the positioning of the FVIIa active site even in the absence of FVIIa-membrane interactions. Further data showed that tissue factor supports full FVIIa proteolytic activity as long as the tissue factor extracellular domain is somehow tethered to the membrane surface. However, elevating the active site of FVIIa above 80 Å of the membrane surface significantly reduced the ability of the tissue factor-FVIIa complex to activate FX, but did not decrease tissue factor-FVIIa amidolytic activity.

[0056] Alanine scanning mutagenesis has been used to evaluate the role of specific amino acid side chains in the tissue factor extracellular domain for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitutions identified a limited number of residue positions where alanine substitutions caused a 5- to 10-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be well exposed to solvent in the crystal structure, consistent with a macromolecular ligand interaction. The FVIIa ligand binding site is located over an extensive region of the interface between the two modules. In the C module, residues Arg, located on the protruding BC loop, 135 and Phe 140 provides independent contact with FVIIa. 133 is located at the base of the finger-like structure and is packed into the gap between the two modules. 20 , Thr 60 , Asp 58 , and Ile 22 It provides continuity to the main cluster of important binding residues consisting of Thr 60 is only partially exposed to the solvent and may play a local structural role rather than making significant contacts with the ligand. 24 and Gln 110 , as well as potentially more distant residues Val 207 The binding region extends from Asp58 to the concave side of the intermodule angle containing 48 , Lys 46 , Gln 37 , Asp 44 , and Trp 45 It spans the convex surface area formed by Trp 45 and Asp 44 does not interact with FVIIa independently, which suggests that Trp 45 The effect of a mutation at position 44 and Gln 37The interaction region further includes two surface-exposed aromatic residues, Phe, which may reflect the structural importance of this side chain for local packing of the side chains. 76 and Tyr 78 which form part of the hydrophobic cluster in the N module.

[0057] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as certain proteinase-activated receptors. Mutational analysis has identified a number of residues that, when mutated, support full FVIIa amidolytic activity on small peptidyl substrates, but lose their ability to support macromolecular substrate (i.e., FVII, FIX, and FX) activation (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9):6375-6381, 1992; Huang et al., J Biol Chem 271(36):21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region of residues 159-165, and residues within or adjacent to this flexible loop, have been shown to be important for the proteolytic activity of the tissue factor-FVIIa complex. This defines a proposed substrate-binding exosite region of tissue factor that is quite distant from the FVIIa active site. Substitution of glycine residues by the slightly bulky residue alanine significantly impairs tissue factor-FVIIa proteolytic activity. This suggests that the flexibility conferred by glycine is important for the loop of residues 159-165 for tissue factor macromolecular substrate recognition.

[0058] Residue Lys 165 and Lys 166 Lys has also been demonstrated to be important for substrate recognition and binding. Mutation of either of these residues to alanine results in a significant decrease in tissue factor cofactor function. 165 and Lys 166 are opposite to each other, and Lys 165points towards FVIIa in most tissue factor-FVIIa structures, and Lys 166 points towards the substrate-binding exosite region in the crystal structure. Lys of FVIIa 165 and Gla 35 Putative salt bridge formation between tissue factor and the GLA domain of FVIIa supports the idea that tissue factor interaction with the GLA domain of FVIIa regulates substrate recognition. These results suggest that the C-terminal part of the tissue factor ectodomain 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.

[0059] Soluble Tissue Factor Domain In some embodiments of any of the polypeptides, compositions, or methods described herein, the soluble tissue factor domain can be a wild-type tissue factor polypeptide lacking the signal sequence, the transmembrane domain, and the intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor variant in which the wild-type tissue factor polypeptide lacks the signal sequence, the transmembrane domain, and the intracellular domain and is further modified in selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, mouse soluble tissue factor domains, rat soluble tissue factor domains, and variant soluble tissue factor domains are provided below.

[0060] Exemplary Soluble Human Tissue Factor Domain (SEQ ID NO:1) [ka] Exemplary Nucleic Acids Encoding Soluble Human Tissue Factor Domains (SEQ ID NO:9) [ka] Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO:10) [ka] Exemplary Mutant Soluble Human Tissue Factor Domain (SEQ ID NO:11) [ka] Exemplary Soluble Mouse Tissue Factor Domain (SEQ ID NO: 12) [ka] Exemplary soluble rat tissue factor domain (SEQ ID NO: 13) [ka]

[0061] 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, a soluble tissue factor domain may comprise the sequence of SEQ ID NO:1, 10, 11, 12, or 13 with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) removed from its N-terminus and / or with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) removed from its C-terminus.

[0062] As can be understood in the art, one of skill in the art will understand that mutations in amino acids that are conserved among different mammalian species are more likely to reduce the activity and / or structural stability of a protein, while mutations in amino acids that are not conserved among different mammalian species are less likely to reduce the activity and / or structural stability of a protein.

[0063] In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain cannot bind to factor VIIa. In some examples of any of the multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the multi-chain chimeric polypeptide does not stimulate blood clotting in a mammal.

[0064] In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain can be a soluble rat tissue factor domain.

[0065] In some examples, the soluble tissue factor domain does not contain 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 the mature wild-type human tissue factor protein; an isoleucine at an amino acid position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein; a tryptophan at an amino acid position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein; an aspartic acid at an amino acid position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein; a tyrosine at an amino acid position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein; an arginine at an amino acid position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein; and a phenylalanine at an amino acid position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the mutant soluble tissue factor has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

[0066] 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.

[0067] In some embodiments, the soluble tissue factor domain is from about 20 amino acids to about 220 amino acids, from about 20 amino acids to about 215 amino acids, from about 20 amino acids to about 210 amino acids, from about 20 amino acids to about 205 amino acids, from about 20 amino acids to about 200 amino acids, from about 20 amino acids to about 195 amino acids, from about 20 amino acids to about 190 amino acids, from about 20 amino acids to about 185 amino acids, from about 20 amino acids to about 180 amino acids, from about 20 amino acids to about 175 amino acids, from about 20 amino acids to about 170 amino acids, from about 20 amino acids to about 165 amino acids, from about 20 amino acids to about 16 ... 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 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 21 ...10 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 2 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 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 50 to about 70 amino acids, about 50 to about 60 amino acids, about 60 to about 220 amino acids, about 60 to about 215 amino acids, about 60 to about 210 amino acids, about 60 to about 205 amino acids, about 60 to about 200 amino acids, about 60 to about 195 amino acids, about 60 to about 190 amino acids, about 60 to about 185 amino acids, about 60 to about 180 amino acids, about 60 to about 175 amino acids, about 60 to about 170 amino acids, about 60 to about 165 amino acids, about 60 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 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 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 ...5 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 195 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 1 20 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 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 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 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 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 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 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 1 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 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 220 ... 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 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.

[0068] Linker sequence In some embodiments, the linker sequence may be a flexible linker sequence.Non-limiting examples of linker sequences that can be used are described in Klein et al., Protein Engineering, Design & Selection 27(10):325-330, 2014; Priyanka et al., Protein Sci. 22(2):153-167, 2013.In some examples, the linker sequence is a synthetic linker sequence.

[0069] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

[0070] In some embodiments, the linker sequence may be from 1 to about 100 amino acids, from 1 to about 90 amino acids, from 1 to about 80 amino acids, from 1 to about 70 amino acids, from 1 to about 60 amino acids, from 1 to about 50 amino acids, from 1 to about 45 amino acids, from 1 to about 40 amino acids, from 1 to about 35 amino acids, from 1 to about 30 amino acids, from 1 to about 25 amino acids, from 1 to about 24 amino acids, from 1 to about 22 amino acids, from 1 to about 20 amino acids, from 1 to about 18 amino acids, from 1 to about 16 amino acids, from 1 to about 20 ... 2 to about 14 amino acids, 1 to about 12 amino acids, 1 to about 10 amino acids, 1 to about 8 amino acids, 1 to about 6 amino acids, 1 to about 4 amino acids, about 2 to about 100 amino acids, about 2 to about 90 amino acids, about 2 to about 80 amino acids, about 2 to about 70 amino acids, about 2 to about 60 amino acids, about 2 to about 50 amino acids, about 2 to about 45 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 to about 14 amino acids, about 1 to about 12 amino acids, about 1 to about 10 amino acids, about 1 to about 8 amino acids, about 1 to about 6 amino acids, about 1 to about 4 amino acids, about 2 to about 100 amino acids, about 2 to about 90 amino acids, about 2 to about 80 amino acids, about 2 to about 70 amino acids, about 2 to about 60 amino acids, about 2 to about 50 amino acids, about 2 to about 45 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 30 amino acids, about 2 to about 25 amino acids, about 2 to about 14 amino acids, about 1 to about 12 amino acids, about 1 to about 10 amino acids, about 2 to about 8 amino acids, about 2 to about 100 amino acids, about 2 to about 90 amino acids, about 2 to about 80 amino acids, about 2 to about 70 amino acids, about 2 to about 60 amino acids, about 2 to about 50 amino acids, about 2 to about 45 amino acids, about 2 to about 40 amino acids, about 2 to about 35 amino acids, about 2 to about 24 amino acids, about 2 to about 22 amino acids, about 2 to about 20 amino acids, about 2 to about 18 amino acids, about 2 to about 16 amino acids, about 2 to about 14 amino acids, about 2 to about 12 amino acids, about 2 to about 10 amino acids, about 2 to about 8 amino acids, about 2 to about 6 amino acids, about 2 to about 4 amino acids, about 4 to about 100 amino acids, about 4 to about 90 amino acids, about 4 to about 80 amino acids, about 4 to about 70 amino acids, about 4 to about 60 amino acids, about 4 to about 50 amino acids, about 4 from about 4 amino acids to about 45 amino acids, from about 4 amino acids to about 40 amino acids, from about 4 amino acids to about 35 amino acids, from about 4 amino acids to about 30 amino acids, from about 4 amino acids to about 25 amino acids, from about 4 amino acids to about 24 amino acids, from about 4 amino acids to about 22 amino acids, from about 4 amino acids to about 20 amino acids, from about 4 amino acids to about 18 amino acids, from about 4 amino acids to about 16 amino acids, from about 4 amino acids to about 14 amino acids, from about 4 amino acids to about 12 amino acids, from about 4 amino acids to about 10 amino acids, from about 4 amino acids to about 8 amino acids, from about 4 amino acids to about 6 amino acids, from about 6 amino acids to about 100 amino acids, from about 6 amino acids to about 90 amino acids,about 6 amino acids to about 80 amino acids, about 6 amino acids to about 70 amino acids, about 6 amino acids to about 60 amino acids, about 6 amino acids to about 50 amino acids, about 6 amino acids to about 45 amino acids, about 6 amino acids to about 40 amino acids, about 6 amino acids to about 35 amino acids, about 6 amino acids to about 30 amino acids, about 6 amino acids to about 25 amino acids, about 6 amino acids to about 24 amino acids, about 6 amino acids to about 22 amino acids, about 6 amino acids to about 20 amino acids, about 6 amino acids to about 18 amino acids, about 6 amino acids to about 16 amino acids, about 6 amino acids to about 14 amino acids, about 6 amino acids to about 12 amino acids, about 6 amino acids to about 1 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,about 12 amino acids to about 30 amino acids, about 12 amino acids to about 25 amino acids, about 12 amino acids to about 24 amino acids, about 12 amino acids to about 22 amino acids, about 12 amino acids to about 20 amino acids, about 12 amino acids to about 18 amino acids, about 12 amino acids to about 16 amino acids, about 12 amino acids to about 14 amino acids, about 14 amino acids to about 100 amino acids, about 14 amino acids to about 90 amino acids, about 14 amino acids to about 80 amino acids, about 14 amino acids to about 70 amino acids, about 14 amino acids to about 60 amino acids, about 14 amino acids to about 50 amino acids, about 14 amino acids to about 45 amino acids, 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, about 16 amino acids to about 40 amino acids, about 16 amino acids to about 35 amino acids, about 16 amino acids to about 30 amino acids, about 16 amino acids to about 25 amino acids, about 16 amino acids to about 24 amino acids, about 16 amino acids to about 22 amino acids, about 16 amino acids to about 20 amino acids, about 16 amino acids to about 18 amino acids, about 18 amino acids to about 100 amino acids, about 18 amino acids to about 90 amino acids, about 18 amino acids to about 80 amino acids, about 18 amino acids to about 70 amino acids, about 18 amino acids to about 60 amino acids, about 18 amino acids to about 50 amino acids, about 18 amino acids to about 45 amino acids, about 18 amino acids to about 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, 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,The linker may have a total length of about 60 to about 90 amino acids, about 60 to about 80 amino acids, about 60 to about 70 amino acids, about 70 to about 100 amino acids, about 70 to about 90 amino acids, about 70 to about 80 amino acids, about 80 to about 100 amino acids, about 80 to about 90 amino acids, or about 90 to about 100 amino acids. In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some aspects, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

[0071] In some embodiments, the linker sequence may comprise or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 3). In some aspects, the linker sequence may be encoded by a nucleic acid that comprises or consists of GGCGGTGGAGGATCCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 14). In some embodiments, the linker sequence may comprise or consist of GGGSGGGS (SEQ ID NO: 15).

[0072] Target Binding Domain In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the additional target binding domain or domains can be an antigen binding domain that specifically binds to a ligand of TGF-βRII (e.g., any of the exemplary antigen binding domains 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 of the exemplary soluble interleukin receptors or soluble cytokine receptors described herein).

[0073] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and / or the one or more additional target binding domains are each independently from about 5 amino acids to about 1000 amino acids, from about 5 amino acids to about 950 amino acids, from about 5 amino acids to about 900 amino acids, from about 5 amino acids to about 850 amino acids, from about 5 amino acids to about 800 amino acids, from about 5 amino acids to about 750 amino acids, from about 5 amino acids to about 700 amino acids, from about 5 amino acids to about 650 amino acids, from about 5 amino acids to about 600 amino acids, from about 5 amino acids to about 550 amino acids, about 5 amino acids to about 500 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 10 ... about 95 amino acids, about 5 amino acids to about 90 amino acids, about 5 amino acids to about 85 amino acids, about 5 amino acids to about 80 amino acids, about 5 amino acids to about 75 amino acids, about 5 amino acids to about 70 amino acids, about 5 amino acids to about 65 amino acids, about 5 amino acids to about 60 amino acids, about 5 amino acids to about 55 amino acids, about 5 amino acids to about 50 amino acids, about 5 amino acids to about 45 amino acids, about 5 amino acids to about 40 amino acids, about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids,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 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 acid, 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 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 25 amino acids to about 1000 amino acids, about 25 amino acids to about 950 amino acids, about 25 amino acids to about 900 amino acids, about 25 amino acids to about 850 amino acids, about 25 amino acids to about 800 amino acids, about 25 amino acids to about 750 amino acids, about 25 amino acids to about 700 amino acids, about 25 amino acids to about 650 amino acids,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 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 1 40 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 25 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 8 0 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 to about 30 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 3 0 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, 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 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 1 85 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 125 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 Amino acids up 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 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 to about 400 amino acids, about 40 amino acids to about 350 amino acids, about 40 amino acids to about 300 amino acids,about 40 amino acids to about 280 amino acids, about 40 amino acids to about 260 amino acids, about 40 amino acids to about 240 amino acids, about 40 amino acids to about 220 amino acids, about 40 amino acids to about 200 amino acids, about 40 amino acids to about 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 1 50 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 ~ 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 65 ... from about 45 amino acids to about 600 amino acids, from about 45 amino acids to about 550 amino acids, from about 45 amino acids to about 500 amino acids, from about 45 amino acids to about 450 amino acids, from about 45 amino acids to about 400 amino acids, from about 45 amino acids to about 350 amino acids, from about 45 amino acids to about 300 amino acids, from about 45 amino acids to about 280 amino acids, from about 45 amino acids to about 260 amino acids, from about 45 amino acids to about 240 amino acids, from about 45 amino acids to about 220 amino acids, from about 45 amino acids to about 200 amino acids, from about 45 amino acids to about 195 amino acids, from about 45 amino acids to about 190 amino acids, from 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 1 10 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 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 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 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 26 ...00 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 500 amino from about 240 amino acids, from about 50 amino acids to about 220 amino acids, from about 50 amino acids to about 200 amino acids, from about 50 amino acids to about 195 amino acids, from about 50 amino acids to about 190 amino acids, from about 50 amino acids to about 185 amino acids, from about 50 amino acids to about 180 amino acids, from about 50 amino acids to about 175 amino acids, from about 50 amino acids to about 170 amino acids, from about 50 amino acids to about 165 amino acids, from about 50 amino acids to about 160 amino acids, from about 50 amino acids to about 155 amino acids, from about 50 amino acids to about 150 amino acids, from about 50 amino acids to about 145 amino acids, from 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, 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 22 0 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 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 ... 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 ~ 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 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 15 0 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 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 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 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 50 ... from about 65 amino acids to about 400 amino acids, from about 65 amino acids to about 350 amino acids, from about 65 amino acids to about 300 amino acids, from about 65 amino acids to about 280 amino acids, from about 65 amino acids to about 260 amino acids, from about 65 amino acids to about 240 amino acids, from about 65 amino acids to about 220 amino acids, from about 65 amino acids to about 200 amino acids, from about 65 amino acids to about 195 amino acids, from about 65 amino acids to about 190 amino acids, from about 65 amino acids to about 185 amino acids, from about 65 amino acids to about 180 amino acids, from about 65 amino acids to about 175 amino acids, from about 65 amino acids to about 170 amino acids, from 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 135 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 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 950 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 50 0 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 260 amino acids, about 70 amino acids to about 240 amino acids, about 70 amino acids to about 220 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 ...95 amino acids, about 70 amino acids to about 185 amino acids, about 70 amino acids to about 18 from about 170 amino acids, from about 70 amino acids to about 165 amino acids, from about 70 amino acids to about 160 amino acids, from about 70 amino acids to about 155 amino acids, from about 70 amino acids to about 150 amino acids, from about 70 amino acids to about 145 amino acids, from about 70 amino acids to about 140 amino acids, from about 70 amino acids to about 135 amino acids, from about 70 amino acids to about 130 amino acids, from about 70 amino acids to about 125 amino acids, from about 70 amino acids to about 120 amino acids, from about 70 amino acids to about 115 amino acids, from about 70 amino acids to about 110 amino acids, from about 70 amino acids to about 105 amino acids, from 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, 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 acid, 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 ~ 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 65 ... 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 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 12 ... 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 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 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 700 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 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 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 from about 1000 amino acids, from about 100 amino acids to about 950 amino acids, from about 100 amino acids to about 900 amino acids, from about 100 amino acids to about 850 amino acids, from about 100 amino acids to about 800 amino acids, from about 100 amino acids to about 750 amino acids, from about 100 amino acids to about 700 amino acids, from about 100 amino acids to about 650 amino acids, from about 100 amino acids to about 600 amino acids, from about 100 amino acids to about 550 amino acids, from about 100 amino acids to about 500 amino acids, from about 100 amino acids to about 450 amino acids, from about 100 amino acids to about 400 amino acids, from 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 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 ~ 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, 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 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 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 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 ... 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 up 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 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 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 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 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 ~ 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 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 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 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 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 up 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 24 ... 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 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 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 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 24 ...2 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 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 up 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 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, 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 ~ 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 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 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, 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 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 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 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 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 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 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 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 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 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,The total number of amino acids may be about 750 to about 800 amino acids, about 800 to about 1000 amino acids, about 800 to about 950 amino acids, about 800 to about 900 amino acids, about 800 to about 850 amino acids, about 850 to about 1000 amino acids, about 850 to about 950 amino acids, about 850 to about 900 amino acids, about 900 to about 1000 amino acids, about 900 to about 950 amino acids, or about 950 to about 1000 amino acids.

[0074] Any target binding domain described herein may be present in a concentration of 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M, 1×10 -10 Less than M, 1×10 -11 Less than M, 1×10 -12 Less than M or 1×10 -13 The dissociation equilibrium constant (K D In some embodiments, the antigen binding protein constructs provided herein can bind to a ligand of TGF-βRII at about 1×10 -3 M ~ approx. 1×10 -5 M, about 1 x 10 -4 M ~ approx. 1×10 -6 M, about 1 x 10 -5 M ~ approx. 1×10 -7 M, about 1 x 10 -6 M ~ approx. 1×10 -8 M, about 1 x 10 -7 M ~ approx. 1×10 -9 M, about 1 x 10 -8 M ~ approx. 1×10 -10 M, or approximately 1 x 10 -9 M ~ approx. 1×10 -11 K of M (inclusive) D The antibody can bind to the identified antigen.

[0075] Any of the target binding domains described herein may have a concentration of from about 1 pM to about 30 nM (e.g., from about 1 pM to about 25 nM, from about 1 pM to about 20 nM, from about 1 pM to about 15 nM, from about 1 pM to about 10 nM, from about 1 pM to about 5 nM, from about 1 pM to about 2 nM, from about 1 pM to about 1 nM, from about 1 pM to about 950 pM, from about 1 pM to about 900 pM, from about 1 pM to about 850 pM, from about 1 pM to about 800 pM, from about 1 pM to about 750 pM, from about 1 pM to about 700 pM, from about 1 pM to about 650 pM, from about 1 pM to about 600 pM, from about 1 pM to about 550 pM, from about 1 pM to about 500 pM, from about 1 pM to about 450 pM, from about 1 pM to about 5 nM, Approximately 400pM, approximately 1pM to approximately 350pM, approximately 1pM to approximately 300pM, approximately 1pM to approximately 250pM, approximately 1pM to approximately 200pM, approximately 1pM to approximately 1 50pM, about 1pM to about 100pM, about 1pM to about 90pM, about 1pM to about 80pM, about 1pM to about 70pM, about 1pM to about 60pM, about 1pM to about 50pM, about 1pM to about 40pM, about 1pM to about 30pM, about 1pM to about 20pM, about 1pM to about 10pM, about 1pM to about 5p M, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM, about 2 pM to about 30 nM, about 2 pM to about 25 nM, about 2 pM to about 20 nM, 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 9 50pM, about 2pM to about 900pM, about 2pM to about 850pM, about 2pM to about 800pM, about 2pM to about 750pM, about 2pM to about 700 pM, about 2 pM to about 650 pM, about 2 pM to about 600 pM, about 2 pM to about 550 pM, about 2 pM to about 500 pM, about 2 pM to about 450 pM , about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2pM to about 150pM, about 2pM to about 100pM, about 2pM to about 90pM, about 2pM to about 80pM, about 2pM to about 70pM, about 2pM to about 60pM, about 2pM to about 50pM, about 2pM to about 40pM, about 2pM to about 30pM, about 2pM to about 20pM, about 2pM to about 10pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5pM to about 15nM, about 5pM to about 10nM, about 5pM to about 5nM, about 5pM to about 2nM, about 5pM to about 1nM, about 5pM to about 950pM,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, from about 5 pM to about 40 pM, from about 5 pM to about 30 pM, from about 5 pM to about 20 pM, from about 5 pM to about 10 pM, from about 10 pM to about 30 nM, from about 10 pM to about 25 nM, from about 10 pM to about 20 nM, from about 10 pM to about 15 nM, from about 10 pM to about 10 nM, from about 10 pM to about 5 nM, from about 10 pM to about 2 nM, from about 10 pM to about 1 nM, from about 10 pM to about 950 pM, from about 10 pM to about 900 pM, from about 10 pM to about 850 pM, from about 10 pM to about 800 pM, from about 10 pM to about 750 pM, from about 10 pM to about 700 pM, from about 10 pM to about 650 pM, from about 10 pM to about 600 pM, from about 10 pM to about 550 pM, from about 10 pM to about 500 pM, from about 10 pM to about 450 pM, from about 10 pM to about 400 pM, from about 10 pM to about 350 pM, from about 10 pM to about 300 pM, from about 10 pM to about 250 pM, from about 10 pM to about 200 pM, from about 10 pM to about 150 pM, from about 10 pM to about 100 pM, from about 10 pM to about 90 pM, from about 10 pM to about 80 pM, from about 10 pM to about 70 pM, from about 10 pM to about 60 pM, from about 10 pM to about 50 pM, from about 10 pM to about 40 pM, from about 10 pM to about 30 pM, from about 10 pM to about 20 pM, from about 15 pM to about 30 nM, from about 15 pM to about 25 nM, from about 15 pM to about 20 nM, from about 15 pM to about 15 nM, from about 15 pM to about 10 nM, from about 15 pM to about 5 nM, from about 15 pM to about 2 nM, from about 15 pM to about 1 nM, from about 15 pM to about 950 pM, from about 15 pM to about 900 pM, from about 15 pM to about 850 pM, from about 15 pM to about 800 pM, from about 15 pM to about 750 pM, from about 15 pM to about 700 pM, from about 15 pM to about 650 pM, from about 15 pM to about 600 pM, from about 15 pM to about 550 pM, from about 15 pM to about 500 pM, from about 15 pM to about 450 pM, from about 15 pM to about 400 pM, from about 15 pM to about 350 pM, from about 15 pM to about 300 pMfrom about 15 pM to about 250 pM, from about 15 pM to about 200 pM, from about 15 pM to about 150 pM, from about 15 pM to about 100 pM, from about 15 pM to about 90 pM, from about 15 pM to about 80 pM, from about 15 pM to about 70 pM, from about 15 pM to about 60 pM, from about 15 pM to about 50 pM, from about 15 pM to about 40 pM, from about 15 pM to about 30 pM, from about 15 pM to about 20 pM, from about 20 pM to about 30 nM, from about 20 pM to about 25 nM, from about 20 pM to about 20 nM, from about 20 pM to about 15 nM, from about 20 pM to about 10 nM, from about 20 pM to about 5 nM, from about 20 pM to about 2 nM, from about 20 pM to about 1 nM, from about 20 pM to about 950 pM, from about 20 pM to about 900 pM, from about 20 pM to about 850 pM, from about 20 pM to about 800 pM, from about 20 pM to about 750 pM, from about 20 pM to about 700 pM, from about 20 pM to about 650 pM, from about 20 pM to about 600 pM, from about 20 pM to about 550 pM, from about 20 pM to about 500 pM, from about 20 pM to about 450 pM, from about 20 pM to about 400 pM, from about 20 pM to about 350 pM, from about 20 pM to about 300 pM, from about 20 pM to about 250 pM, from about 20 pM to about 20 pM, from about 200 pM to about 150 pM, from about 20 pM to about 100 pM, from about 20 pM to about 90 pM, from about 20 pM to about 80 pM, from about 20 pM to about 70 pM, from about 20 pM to about 60 pM, from about 20 pM to about 50 pM, from about 20 pM to about 40 pM, from about 20 pM to about 30 pM, from about 30 pM to about 30 nM, from about 30 pM to about 25 nM, from about 30 pM to about 30 nM, from about 30 pM to about 15 nM, from about 30 pM to about 10 nM, from about 30 pM to about 5 nM, from about 30 pM to about 2 nM, from about 30 pM to about 1 nM, from about 30 pM to about 950 pM, from about 30 pM to about 900 pM, from about 30 pM to about 850 pM, from about 30 pM to about 800 pM, from about 30 pM to about 750 pM, from about 30 pM to about 700 pM, from about 30 pM to about 650 pM, from about 30 pM to about 600 pM, from about 30 pM to about 550 pM, from about 30 pM to about 500 pM, from about 30 pM to about 450 pM, from about 30 pM to about 400 pM, from about 30 pM to about 350 pM, from about 30 pM to about 300 pM, from about 30 pM to about 250 pM, from about 30 pM to about 200 pM, from about 30 pM to about 150 pM, from about 30 pM to about 100 pM, from about 30 pM to about 90 pM, from about 30 pM to about 80 pM, from about 30 pM to about 70 pM, from about 30 pM to about 60 pM, from about 30 pM to about 50 pM, from about 30 pM to about 40 pM, from about 40 pM to about 30 nM, from about 40 pM to about 25 nM, from about 40 pM to about 30 nMFrom about 40 pM to about 15 nM, from about 40 pM to about 10 nM, from about 40 pM to about 5 nM, from about 40 pM to about 2 nM, from about 40 pM to about 1 nM, from about 40 pM to about 950 pM, from about 40 pM to about 900 pM, from about 40 pM to about 850 pM, from about 40 pM to about 800 pM, from about 40 pM to about 750 pM, from about 40 pM to about 700 pM, from about 40 pM to about 650 pM, from about 40 pM to about 600 pM, from about 40 pM to about 550 pM, from about 40 pM to about 500 pM, from about 40 pM to about 450 pM, from about 40 pM to about 400 pM, from about 40 pM to about 350 pM, from about 40 pM to about 300 pM, from about 40 pM to about 250 pM, from about 40 pM to about 200 pM, from about 40 pM to about 150 pM, from about 40 pM to about 100 pM, from about 40 pM to about 90 pM, from about 40 pM to about 80 pM, from about 40 pM to about 70 pM, from about 40 pM to about 60 pM, from about 40 pM to about 50 pM, from about 50 pM to about 30 nM, from about 50 pM to about 25 nM, from about 50 pM to about 30 nM, from about 50 pM to about 15 nM, from about 50 pM to about 10 nM, from about 50 pM to about 5 nM, from about 50 pM to about 2 nM, from about 50 pM to about 1 nM, from about 50 pM to about 950 pM, from about 50 pM to about 900 pM, from about 50 pM to about 850 pM, from about 50 pM to about 800 pM, from about 50 pM to about 750 pM, from about 50 pM to about 700 pM, from about 50 pM to about 650 pM, from about 50 pM to about 600 pM, from about 50 pM to about 550 pM, from about 50 pM to about 500 pM, from about 50 pM to about 450 pM, from about 50 pM to about 400 pM, from about 50 pM to about 350 pM, from about 50 pM to about 300 pM, from about 50 pM to about 250 pM, from about 50 pM to about 200 pM, from about 50 pM to about 150 pM, from about 50 pM to about 100 pM, from about 50 pM to about 90 pM, from about 50 pM to about 80 pM, from about 50 pM to about 70 pM, from about 50 pM to about 60 pM, from about 60 pM to about 30 nM, from about 60 pM to about 25 nM, from about 60 pM to about 30 nM, from about 60 pM to about 15 nM, from about 60 pM to about 10 nM, from about 60 pM to about 5 nM, from about 60 pM to about 2 nM, from about 60 pM to about 1 nM, from about 60 pM to about 950 pM, from about 60 pM to about 900 pM, from about 60 pM to about 850 pM, from about 60 pM to about 800 pM, from about 60 pM to about 750 pM, from about 60 pM to about 700 pM, from about 60 pM to about 650 pM, from about 60 pM to about 600 pM, from about 60 pM to about 550 pM, from about 60 pM to about 500 pM, from about 60 pM to about 450 pM, from about 60 pM to about 400 pM, from about 60 pM to about 350 pM, from about 60 pM to about 300 pMAbout 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 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, from about 80 pM to about 10 nM, from about 80 pM to about 5 nM, from about 80 pM to about 2 nM, from about 80 pM to about 1 nM, from about 80 pM to about 950 pM, from about 80 pM to about 900 pM, from about 80 pM to about 850 pM, from about 80 pM to about 800 pM, from about 80 pM to about 750 pM, from about 80 pM to about 700 pM, from about 80 pM to about 650 pM, from about 80 pM to about 600 pM, from about 80 pM to about 550 pM, from about 80 pM to about 500 pM, from about 80 pM to about 450 pM, from about 80 pM to about 400 pM, from about 80 pM to about 350 pM, from about 80 pM to about 300 pM, from about 80 pM to about 250 pM, from about 80 pM to about 200 pM, from about 80 pM to about 150 pM, from about 80 pM to about 100 pM, from about 80 pM to about 90 pM, from about 90 pM to about 30 nM, from about 90 pM to about 25 nM, from about 90 pM to about 30 nM, from about 90 pM to about 15 nM, from about 90 pM to about 10 nM, from about 90 pM to about 5 nM, from about 90 pM to about 2 nM, from about 90 pM to about 1 nM, from about 90 pM to about 950 pM, from about 90 pM to about 900 pM, from about 90 pM to about 850 pM, from about 90 pM to about 800 pM, from about 90 pM to about 750 pM, from about 90 pM to about 700 pM, from about 90 pM to about 650 pM, from about 90 pM to about 600 pM, from about 90 pM to about 550 pM, from about 90 pM to about 500 pM, from about 90 pM to about 450 pM, from about 90 pM to about 400 pM, from about 90 pM to about 350 pM, from about 90 pM to about 300 pM, from about 90 pM to about 250 pM, from about 90 pM to about 200 pM, from about 90 pM to about 150 pM, from about 90 pM to about 100 pM, from about 100 pM to about 30 nM, from about 100 pM to about 25 nM, from about 100 pM to about 30 nM, from about 100 pM to about 15 nM, from about 100 pM to about 10 nM, from about 100 pM to about 5 nM, from about 100 pM to about 2 nM, from about 100 pM to about 1 nM, from about 100 pM to about 950 pM, from about 100 pM to about 900 pM, from about 100 pM to about 850 pM, from about 100 pM to about 800 pM, from about 100 pM to about 750 pM, from about 100 pM to about 700 pM, from about 100 pM to about 650 pM, from about 100 pM to about 600 pM, from about 100 pM to about 550 pM, from about 100 pM to about 500 pM, from about 100 pM to about 450 pM, from about 100 pM to about 400 pM, from about 100 pM to about 350 pM, from about 100 pM to about 300 pM, from about 100 pM to about 250 pM, from about 100 pM to about 200 pM, from about 100 pM to about 150 pM, from about 150 pM to about 30 nM, from about 150 pM to about 25 nM, from about 150 pM to about 30 nMFrom about 150 pM to about 15 nM, from about 150 pM to about 10 nM, from about 150 pM to about 5 nM, from about 150 pM to about 2 nM, from about 150 pM to about 1 nM, from about 150 pM to about 950 pM, from about 150 pM to about 900 pM, from about 150 pM to about 850 pM, from about 150 pM to about 800 pM, from about 150 pM to about 750 pM, from about 150 pM to about 700 pM, from about 150 pM to about 650 pM, from about 150 pM to about 600 pM, from about 150 pM to about 550 pM, from about 150 pM to about 500 pM, from about 150 pM to about 450 pM, from about 150 pM to about 400 pM, from about 150 pM to about 350 pM, from about 150 pM to about 300 pM, from about 150 pM to about 250 pM, from about 200 pM to about 30 nM, from about 200 pM to about 25 nM, from about 200 pM to about 30 nM, from about 200 pM to about 15 nM, from about 200 pM to about 10 nM, from about 200 pM to about 5 nM, from about 200 pM to about 2 nM, from about 200 pM to about 1 nM, from about 200 pM to about 950 pM, from about 200 pM to about 900 pM, from about 200 pM to about 850 pM, from about 200 pM to about 800 pM, from about 200 pM to about 750 pM, from about 200 pM to about 700 pM, from about 200 pM to about 650 pM, from about 200 pM to about 600 pM, from about 200 pM to about 550 pM, from about 200 pM to about 500 pM, from about 200 pM to about 450 pM, from about 200 pM to about 400 pM, from about 200 pM to about 350 pM, from about 200 pM to about 300 pM, from about 200 pM to about 250 pM, from about 300 pM to about 30 nM, from about 300 pM to about 25 nM, from about 300 pM to about 30 nM, from about 300 pM to about 15 nM, from about 300 pM to about 10 nM, from about 300 pM to about 5 nM, from about 300 pM to about 2 nM, from about 300 pM to about 1 nM, from about 300 pM to about 950 pM, from about 300 pM to about 900 pM, from about 300 pM to about 850 pM, from about 300 pM to about 800 pM, from about 300 pM to about 750 pM, from about 300 pM to about 700 pM, from about 300 pM to about 650 pM, from about 300 pM to about 600 pM, from about 300 pM to about 550 pM, from about 300 pM to about 500 pM, from about 300 pM to about 450 pM, from about 300 pM to about 400 pM, from about 300 pM to about 350 pM, from about 400 pM to about 30 nM, from about 400 pM to about 25 nM, from about 400 pM to about 30 nM, from about 400 pM to about 15 nM, from about 400 pM to about 10 nM, from about 400 pM to about 5 nM, from about 400 pM to about 2 nM, from about 400 pM to about 1 nM, from about 400 pM to about 950 pM,from about 400 pM to about 900 pM, from about 400 pM to about 850 pM, from about 400 pM to about 800 pM, from about 400 pM to about 750 pM, from about 400 pM to about 700 pM, from about 400 pM to about 650 pM, from about 400 pM to about 600 pM, from about 400 pM to about 550 pM, from about 400 pM to about 500 pM, from about 500 pM to about 30 nM, from about 500 pM to about 25 nM, from about 500 pM to about 30 nM, from about 500 pM to about 15 nM, from about 500 pM to about 10 nM, from about 500 pM to about 5 nM, from about 500 pM to about 2 nM, from about 500 pM to about 1 nM, from about 500 pM to about 950 pM, from about 500 pM to about 900 pM, from about 500 pM to about 850 pM, from about 500 pM to about 800 pM, from about 500 pM to about 750 pM, from about 500 pM to about 700 pM, from about 500 pM to about 650 pM, from about 500 pM to about 600 pM, from about 500 pM to about 550 pM, from about 600 pM to about 30 nM, from about 600 pM to about 25 nM, from about 600 pM to about 30 nM, from about 600 pM to about 15 nM, from about 600 pM to about 10 nM, from about 600 pM to about 5 nM, from about 600 pM to about 2 nM, from about 600 pM to about 1 nM, from about 600 pM to about 950 pM, from about 600 pM to about 900 pM, from about 600 pM to about 850 pM, from about 600 pM to about 800 pM, from about 600 pM to about 750 pM, from about 600 pM to about 700 pM, from about 600 pM to about 650 pM, from about 700 pM to about 30 nM, from about 700 pM to about 25 nM, from about 700 pM to about 30 nM, from about 700 pM to about 15 nM, from about 700 pM to about 10 nM, from about 700 pM to about 5 nM, from about 700 pM to about 2 nM, from about 700 pM to about 1 nM, from about 700 pM to about 950 pM, from about 700 pM to about 900 pM, from about 700 pM to about 850 pM, from about 700 pM to about 800 pM, from about 700 pM to about 750 pM, from about 800 pM to about 30 nM, from about 800 pM to about 25 nM, from about 800 pM to about 30 nM, from about 800 pM to about 15 nM, from about 800 pM to about 10 nM, from about 800 pM to about 5 nM, from about 800 pM to about 2 nM, from about 800 pM to about 1 nM, from about 800 pM to about 950 pM, from about 800 pM to about 900 pM, from about 800 pM to about 850 pM, from about 900 pM to about 30 nM, from about 900 pM to about 25 nM, from about 900 pM to about 30 nM, from about 900 pM to about 15 nM, from about 900 pM to about 10 nM, from about 900 pM to about 5 nM, from about 900 pM to about 2 nM, from about 900 pM to about 1 nM, from about 900 pM to about 950 pM, from about 1 nM to about 30 nMK of about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4 nM to about 20 nM, about 4 nM to about 15 nM, about 4 nM to about 10 nM, about 4 nM to about 5 nM, about 5 nM to about 30 nM, about 5 nM to about 25 nM, about 5 nM to about 20 nM, about 5 nM to about 15 nM, about 5 nM to about 10 nM, about 10 nM to about 30 nM, about 10 nM to about 25 nM, about 10 nM to about 20 nM, about 10 nM to about 15 nM, about 15 nM to about 30 nM, about 15 nM to about 25 nM, about 15 nM to about 20 nM, about 20 nM to about 30 nM, and about 20 nM to about 25 nM), D can bind to a ligand of TGF-βRII (e.g., TGF-β) having D .

[0076] Any of the target binding domains described herein has a K of about 1 nM to about 10 nM (e.g., about 1 nM to about 9 nM, about 1 nM to about 8 nM, about 1 nM to about 7 nM, about 1 nM to about 6 nM, about 1 nM to about 5 nM, about 1 nM to about 4 nM, about 1 nM to about 3 nM, about 1 nM to about 2 nM, about 2 nM to about 10 nM, about 2 nM to about 9 nM, about 2 nM to about 8 nM, about 2 nM to about 7 nM, about 2 nM to about 6 nM, about 2 nM to about 5 nM, about 2 nM to about 4 nM, about 2 nM to about 3 nM, about 3 nM to about 10 nM, about 3 nM to about 9 nM, about 3 nM to about 8 nM, about 3 nM to about 7 nM, about 3 nM to about 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) D can bind to a ligand of TGFβRII having D .

[0077] A variety of different methods known in the art can be used to quantify the K of any of the antigen binding protein constructs described herein. D Values ​​can be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, biomolecular binding kinetics assays, etc.).

[0078] Antigen-binding domain In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain specifically bind to the same antigen. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain specifically bind to the same epitope. In some embodiments of these multi-chain chimeric polypeptides, the first target binding domain and the second target binding domain comprise the same amino acid sequence.

[0079] 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.

[0080] In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain are antigen binding domains. In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain are each antigen binding domains.

[0081] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the antigen-binding domain comprises or is an scFv or a single domain antibody (e.g., a VHH or VNAR domain).

[0082] In some examples, the antigen binding domain (e.g., any of the antigen binding domains described herein) can specifically bind to a ligand of TGF-βRII (see, e.g., the antigen binding domains capable of specifically binding to TGF-β described in U.S. Patent Application Publication No. 2021 / 0061897, U.S. Patent Application Publication No. 2020 / 0399358, U.S. Patent Application Publication No. 2020 / 0392221, U.S. Patent Application Publication No. 2019 / 0315850, U.S. Patent Application Publication No. 2019 / 0092846, U.S. Patent Application Publication No. 2021 / 0403545, U.S. Patent Application Publication No. 2021 / 0355204, and U.S. Patent Application Publication No. 2019 / 0177406, each of which is incorporated herein by reference).

[0083] Each antigen-binding domain present in any of the multi-chain chimeric polypeptides described herein is independently selected from the group consisting of a VHH domain, a VNAR domain, and an scFv. In some embodiments, any of the antigen-binding domains described herein are selected from the group consisting of BiTe, (scFv) 2 , Nanobody, Nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or Tandem-scFv. Further examples of antigen binding domains that can be used in any of the multi-chain chimeric polypeptides are known in the art.

[0084] VHH domains are single monomeric variable antibody domains that can be found in camelids. VNAR domains are single monomeric variable antibody domains that can be found in cartilaginous fish. VHH domains and V NARNon-limiting aspects of the domain are described, for example, in Cromie et al., Curr. Top. Med. Chem. 15:2543-2557, 2016; De Genst et al., Dev. Comp. Immunol. 30:187-198, 2006; De Meyer et al., Trends Biotechnol. 32:263-270, 2014; Kijanka et al., Nanomedicine 10:161-174, 2015; Kovaleva et al., Expert. Opin. Biol. Ther. 14:1527-1539, 2014; Krah et al., Immunopharmacol. Immunotoxicol. 38:21-28, 2016; Mujic-Delic et al., Trends Pharmacol. Sci. 35:247-255, 2014; Muyldermans, J. Biotechnol. 74:277-302, 2001; Muyldermans et al., Trends Biochem. Sci. 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. 40:299-338, 2011; Van Audenhove et al., EBioMedicine 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. 911:15-26, 2012, and Wesolowski et al., Med. Microbiol. Immunol. 198:157-174, 2009.

[0085] 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 an scFv domain.

[0086] In some embodiments, two or more of the polypeptides present in the multi-chain chimeric polypeptide are any of the antigen-binding domains described herein, such as an antigen-binding fragment of an antibody (e.g., any of the antigen-binding fragments of an antibody described herein), a VHH-scAb, a VHH-Fab, a Dual scFab, a F(ab') 2 , diabody, crossMab, DAF (two in one), DAF (four in one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2 scFv, scFv 4-Ig, Zybody, DVI-IgG, diabody-CH3, triple body, mini antibody, mini body, TriBi mini body, scFv-CH3 KIH, Fab-scFv, F(ab') 2 -scFv 2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, tandem scFv-Fc, intrabody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body and scFv1-PEG-scFv 2 The antibody can assemble (e.g., non-covalently assemble) to form a Fab fragment. See, e.g., Spiess et al., Mol. Immunol. 67:95-106, 2015, which are incorporated herein in their entirety for the description of these elements. Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab') fragments, and F(ab') fragments. 2 Examples of antigen-binding fragments of antibodies include IgG1, IgG2, IgG3, or IgG4 antigen-binding fragments (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 IgE or humanized IgE); or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0087] An "Fv" fragment comprises a non-covalent dimer of one heavy- and one light-chain variable domain.

[0088] A "Fab" fragment contains the heavy and light chain variable domains of the Fv fragment as well as the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ).

[0089] "F(ab') 2 The " fragment contains two Fab fragments linked by a disulfide bond near the hinge region.

[0090] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and multispecific binding proteins as described, for example, in DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012; Jakob et al., MABs 5:358-363, 2013; and in U.S. Pat. Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8822645, each of which is incorporated by reference in its entirety.

[0091] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014. In some embodiments, any of the antigen binding domains described herein are capable of binding to an antigen selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

[0092] Further examples and embodiments of antigen binding domains are known in the art.

[0093] Soluble receptors In some embodiments of any of the multi-chain chimeric polypeptides described herein, one or both of the first target binding domain and the second target binding domain 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, for example, those described in Yung et al., Am. J. Resp. Crit. Care Med. 194(9):1140-1151, 2016), or soluble TGF-βRIII (see, for example, those described in Heng et al., 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.

[0094] In some embodiments, the first target binding domain comprises a first sequence 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 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 sequence and the second sequence 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.

[0095] 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.

[0096] In some embodiments, the second target binding domain comprises a first sequence 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 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 sequence and the second sequence are separated by a linker. In some embodiments, the second target binding domain comprises a first sequence of SEQ ID NO:2 and a second sequence of SEQ ID NO:2.

[0097] 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.

[0098] Further examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0099] Additional target binding domains In some embodiments of any of the multi-chain chimeric polypeptides, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art), where at least one of the one or more additional antigen binding domains is positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and the first domain of the affinity domain pair (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide may further comprise a linker sequence (e.g., any of the exemplary linker sequences described herein) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein), and / or a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and a first domain of an affinity domain pair (e.g., any of the exemplary first domains described herein of an exemplary affinity domain pair described herein).

[0100] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is directly adjacent to the first domain of the affinity domain pair in the first chimeric polypeptide (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first domain of the affinity domain pair (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0101] In some embodiments of any of the multi-chain chimeric polypeptides described herein, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is disposed at the N- and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is positioned between a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and a first domain of an affinity domain pair (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the N-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of the affinity domain pair (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of the affinity domain pair (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein).In some embodiments, at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) of the one or more additional target binding domains disposed at the C-terminus is immediately adjacent to the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of the affinity domain pair (e.g., any of the exemplary first domains of any of the exemplary pairs of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any of the linker sequences described herein or known in the art) disposed between the at least one additional target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) in the first chimeric polypeptide and the first target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art) or the first domain of the affinity domain pair (e.g., any of the exemplary first domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) located between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the affinity domain pair (e.g., any of the first domains described herein or any of the exemplary affinity domain pairs described herein) is immediately 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 of the exemplary soluble tissue factor domains described herein) disposed between (i) the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and at least one of one or more additional target binding domains (e.g., exemplary target binding domains described herein or known in the art) located between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and the first domain of the affinity domain pair (e.g., any of the exemplary first domain of any of the exemplary affinity domain pairs described herein), and / or (ii) between the first domain of the affinity domain pair and at least one of the one or more additional target binding domains located between the first domain of the affinity domain pair and the soluble tissue factor domain and the first domain of the affinity domain pair.

[0102] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional target binding domains (e.g., any of the exemplary target binding domains described herein) at the N-terminus and / or C-terminus of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is immediately adjacent to the second domain of the affinity domain pair in the second chimeric polypeptide (e.g., any of the exemplary second domains of any of the exemplary pairs of affinity domains described herein).

[0103] In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second domain of the affinity domain pair (e.g., any of the second domains described herein of any of the exemplary pairs of affinity domains described herein). In some embodiments, at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) is directly adjacent to the second target binding domain (e.g., any of the target binding domains described herein or known in the art) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further comprises a linker sequence (e.g., any of the exemplary target binding domains described herein or known in the art) between at least one of the one or more additional target binding domains (e.g., any of the exemplary target binding domains described herein or known in the art) in the second chimeric polypeptide and the second target binding domain (e.g., any of the exemplary target binding domains described herein or known in the art).

[0104] In some embodiments of any of the multi-chain chimeric polypeptides described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target binding domain, the second target binding domain, and the one or more additional target binding domains comprise the same amino acid sequence. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same antigen. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each specifically bind to the same epitope. In some embodiments, the first target binding domain, the second target binding domain, and the one or more additional target binding domains each comprise the same amino acid sequence.

[0105] In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain, the second target binding domain, and one or more additional target binding domains specifically bind to different antigens. In some aspects of any of the multi-chain chimeric polypeptides described herein, one or more of the first target binding domain, the second target binding domain, and one or more target binding domains (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) are antigen binding domains. In some embodiments, each of the first target binding domain, the second target binding domain, and one or more additional target binding domains is an antigen binding domain (e.g., an scFv or a single domain antibody).

[0106] Affinity domain pairing 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 an affinity domain pair, such that the first chimeric polypeptide and the second chimeric polypeptide associate through binding of the first domain and the second domain of the affinity domain pair. 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. The sushi domain, also known as the short consensus repeat or type 1 glycoprotein motif, is a common motif in protein-protein interactions. Sushi domains have been identified on many protein-binding molecules, including complement components C1r, C1s, factor H, and C2m, as well as the non-immunological molecules factor XIII and β2-glycoprotein. A typical sushi domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp Biocomput. 2000:155-67). The first cysteine ​​can form a disulfide bond with the third cysteine, and the second cysteine ​​can form a disulfide bridge with the fourth cysteine. In some embodiments where one member of the 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 alpha 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 pair is an adaptor / docking tag module based on mutated 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 a SNARE module based on the interactions of the proteins syntaxin, synaptotagmin, synaptobrevin, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492, 2003).

[0107] 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 the 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 ) is 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M, 1×10 -10 Less than M, 1×10 -11 Less than M, 1×10 -12 Less than M or 1×10 -13 In some embodiments, the first domain of the affinity domain pair and the second domain of the affinity domain pair bind to each other with less than K D But about 1x10 -4 M ~ approx. 1x10 -6 M, about 1x10 -5 ~ approx. 1x10 -7 M, about 1x10 -6 M ~ approx. 1x10 -8 M, about 1x10 -7 M ~ approx. 1x10 -9 M, about 1x10 -8 M ~ approx. 1x10 -10 M, about 1x10 -9 M ~ approx. 1x10 -11 M, about 1x10 -10M ~ approx. 1x10 -12 M, about 1x10 -11 M ~ approx. 1x10 -13 M, about 1x10 -4 M ~ approx. 1x10 -5 M, about 1x10 -5 M ~ approx. 1x10 -6 M, about 1x10 -6 M ~ approx. 1x10 -7 M, about 1x10 -7 M ~ approx. 1x10 -8 M, about 1x10 -8 M ~ approx. 1x10 -9 M, about 1x10 -9 M ~ approx. 1x10 -10 M, about 1x10 -10 M ~ approx. 1x10 -11 M, about 1x10 -11 M ~ approx. 1x10 -12 M, or about 1x10 -12 M ~ approx. 1x10 -13 The K of binding between a first domain of an affinity domain pair and a second domain of an affinity domain pair can be determined using any of a variety of different methods known in the art. D Values ​​can be determined (eg, electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance and biomolecular binding kinetics assays, etc.).

[0108] 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 the affinity domain pair, and the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both, are about 10 to 100 amino acids in length. For example, the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both, may be about 10 to 100 amino acids in length, about 15 to 100 amino acids in length, about 20 to 100 amino acids in length, about 25 to 100 amino acids in length, about 30 to 100 amino acids in length, about 35 to 100 amino acids in length, about 40 to 100 amino acids in length, about 45 to 100 amino acids in length, about 50 to 100 amino acids in length, about 55 to 100 amino acids in length, about 60 to 100 amino acids in length, about 65 to 100 amino acids in length, about 70 to 100 amino acids in length, about 75 to 100 amino acids in length, about 80 to 100 amino acids in length, about 85 to 100 amino acids in length, about 90 to 100 amino acids in length, about 95 to 100 amino acids in length, about 10 to 95 amino acids in length, about 10 to 90 amino acids in length, about 10 to 85 amino acids in length, about 10 to 80 ... The amino acid length may be about 10 to 75 amino acids, about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both, are about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length.

[0109] 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 its N-terminus and / or C-terminus, so long as the function of the first and / or second domains of the affinity domain pair remains intact. For example, the sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα) may include one or more additional amino acids at its N-terminus and / or C-terminus while still retaining the ability to bind soluble IL-15. Additionally or alternatively, soluble IL-15 may include one or more additional amino acids at its N-terminus and / or C-terminus while still retaining the ability to bind to the sushi domain from the alpha chain of the human IL-15 receptor (IL15Rα).

[0110] Non-limiting examples of a sushi domain from the alpha chain of IL-15 receptor alpha (IL15Rα) can include a sequence that is at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 16). In some embodiments, the sushi domain from the alpha chain of IL15Rα can be encoded by a nucleic acid that includes: [ka]

[0111] 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, the soluble IL-15 may be encoded by a nucleic acid comprising the following sequence: [ka]

[0112] In some embodiments, the soluble IL-15 may comprise a D8N amino acid substitution. In some embodiments, the soluble IL-15 with D8N mutation (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, the soluble IL-15 with D8N mutation (IL15D8N) (e.g., SEQ ID NO: 62) may be encoded by a nucleic acid comprising the following sequence: [ka]

[0113] Signal sequence In some embodiments, the multi-chain chimeric polypeptide comprises a first chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, the multi-chain chimeric polypeptide comprises a second chimeric polypeptide comprising a signal sequence at its N-terminus. 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 signal sequence. As will be appreciated by those skilled in the art, a signal sequence is an amino acid sequence present at the N-terminus of many endogenously produced proteins that directs the protein into the secretory pathway (e.g., directing the protein to reside in a specific intracellular organelle, to reside at the cell membrane, or to be secreted from the cell). Signal sequences are heterogeneous and vary widely in their primary amino acid sequence. However, signal sequences are typically 16-30 amino acids in length and comprise a hydrophilic, usually positively charged, N-terminal region, a central hydrophobic domain, and a C-terminal region that includes a cleavage site for a signal peptidase.

[0114] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprises a signal sequence having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 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, comprises ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 21), ATGAAGTGGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCCTACAGC (SEQ ID NO: 22), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO: 23) The signal sequence is encoded by

[0115] 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 single 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 single sequence having the amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (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 single sequence having the amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (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 single sequence having the amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 27). One of skill in the art will recognize other suitable signal sequences for use in the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide described herein.

[0116] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, comprise a signal sequence that is about 10-100 amino acids in length. For example, the single sequence may 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, 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, The length may be about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the 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.

[0117] In some embodiments, any of the signal sequences disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at its N-terminus and / or C-terminus, so long as the function of the signal sequence remains intact. For example, a signal sequence having the amino acids MKCLLYLAFLFLGVNC (SEQ ID NO:28) can include one or more additional amino acids at the N-terminus or C-terminus while still retaining the function of directing a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both, into the secretory pathway.

[0118] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both, contain a signal sequence that directs the multi-chain chimeric polypeptide to the extracellular space. Such embodiments are useful for producing multi-chain chimeric polypeptides that are relatively easy to isolate and / or purify.

[0119] Peptide tags In some embodiments, a multi-chain chimeric polypeptide comprises a first chimeric polypeptide that comprises a peptide tag (e.g., at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, a multi-chain chimeric polypeptide comprises a second chimeric polypeptide that comprises 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.

[0120] Exemplary peptide tags that may be included in a first chimeric polypeptide of a multi-chain chimeric polypeptide, a second chimeric polypeptide of a multi-chain chimeric polypeptide, or both include an Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO:29), a calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO:30), a polyglutamic acid 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, a peptide derived from hemagglutinin (YPYDVPDYA; SEQ ID NO:34), a his-tag (HHHHH (SEQ ID NO:35); HHHHHHH (SEQ ID NO:36); HHHHHHHH (SEQ ID NO:37); HHHHHHHHH (SEQ ID NO:38); HHHHHHHHHH (SEQ ID NO:39); or HHHHHHHHHHH (SEQ ID NO:40)), a myc tag, a ribozyme ... -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), Soft-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), 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.

[0121] The peptide tag may 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, and may be used in any of a variety of applications related to the multi-chain chimeric polypeptide. For example, the peptide tag may be used in the purification of 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, may include a myc tag, and the multi-chain chimeric polypeptide including the myc-tagged first chimeric polypeptide, the myc-tagged second chimeric polypeptide, or both, may be purified using an antibody that recognizes the myc tag. One non-limiting example of an antibody that recognizes a myc tag is 9E10, which is available from Developmental Studies Hybridoma Bank, which is not commercially available. As another non-limiting example, the first chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a recombinantly expressed first chimeric polypeptide), the second chimeric polypeptide of the multi-chain chimeric polypeptide (e.g., a recombinantly expressed second chimeric polypeptide), or both can include a histidine tag, and the multi-chain chimeric polypeptide including the histidine-tagged first chimeric polypeptide, the histidine-tagged second chimeric polypeptide, or both can be purified using nickel or cobalt chelates. Those skilled in the art will be aware of other suitable tags and agents that bind to these tags for use in purifying the multi-chain chimeric polypeptide. 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.

[0122] A peptide tag may 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, and may be used, for example, in immunoprecipitation of the multi-chain chimeric polypeptide, imaging of the multi-chain chimeric polypeptide (e.g., via Western blotting, ELISA, flow cytometry, and / or 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, include a peptide tag that is about 10 to 100 amino acids in length. For example, the peptide tag may 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, 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, The length may be about 10 to 70 amino acids, about 10 to 65 amino acids, about 10 to 60 amino acids, about 10 to 55 amino acids, about 10 to 50 amino acids, about 10 to 45 amino acids, about 10 to 40 amino acids, about 10 to 35 amino acids, about 10 to 30 amino acids, about 10 to 25 amino acids, about 10 to 20 amino acids, about 10 to 15 amino acids, about 20 to 30 amino acids, about 30 to 40 amino acids, about 40 to 50 amino acids, about 50 to 60 amino acids, about 60 to 70 amino acids, about 70 to 80 amino acids, about 80 to 90 amino acids, about 90 to 100 amino acids, about 20 to 90 amino acids, about 30 to 80 amino acids, about 40 to 70 amino acids, about 50 to 60 amino acids, or any range therebetween. In some embodiments, the 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.

[0123] 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 be any suitable length. For example, 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 length. In embodiments in which the multi-chain chimeric polypeptide includes more than one peptide tag, the two or more peptide tags can be the same or different lengths. In some embodiments, any of the peptide tags disclosed herein can include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at the N-terminus and / or C-terminus, so long as the function of the peptide tag remains intact. For example, a myc tag having the amino acid sequence EQKLISEEDL (SEQ ID NO:54) can include one or more additional amino acids (e.g., at the N-terminus and / or C-terminus of the peptide tag) while still retaining the ability to be bound by an antibody.

[0124] Exemplary Multi-Chain Chimeric Polypeptides In some embodiments of any of the multi-chain chimeric polypeptides described herein, the first target binding domain and the second target binding domain each independently specifically bind to TGF-β. In some examples of these multi-chain chimeric polypeptides, the first target binding domain and the soluble tissue factor domain are directly adjacent to each other 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 of the exemplary linkers described herein) between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

[0125] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain and the first domain of the affinity domain pair are immediately 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.

[0126] 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 of the exemplary linkers described herein) between the second domain of the affinity domain pair and the second target binding domain in the second chimeric polypeptide.

[0127] In some embodiments of these multi-chain chimeric polypeptides, the soluble tissue factor domain can be any of the exemplary soluble tissue factor domains described herein. In some embodiments of these multi-chain chimeric polypeptides, the affinity domain pair can be any of the exemplary affinity domain pairs described herein.

[0128] 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.

[0129] 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 GGGGSGGGSGGGGGS (SEQ ID NO: 3).

[0130] In some embodiments of these multi-chain chimeric polypeptides, the first sequence of the soluble human TGFRβRII receptor comprises a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka]

[0131] In some embodiments of these multi-chain chimeric polypeptides, the second sequence of a soluble human TGFRβRII receptor comprises a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka]

[0132] 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: [ka]

[0133] 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: [ka]

[0134] In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β receptor comprises a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to the following: [ka]

[0135] In some embodiments of these multi-chain chimeric polypeptides, the soluble TGF-β 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 the following: [ka]

[0136] In some embodiments, the first chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka]

[0137] In some embodiments, the first chimeric polypeptide is encoded by a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka] [ka]

[0138] In some embodiments, the first chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka]

[0139] In some embodiments, the first chimeric polypeptide is encoded by a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka] [ka]

[0140] In some embodiments, the second chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka]

[0141] 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: [ka]

[0142] In some embodiments, the second chimeric polypeptide can comprise a sequence at least 80% identical (e.g., at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical) to: [ka]

[0143] 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: [ka]

[0144] In some embodiments, the second chimeric polypeptide can comprise a sequence 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: [ka]

[0145] 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: [ka]

[0146] In some embodiments, the second chimeric polypeptide can comprise a sequence 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: [ka]

[0147] 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: [ka]

[0148] In some embodiments, the first chimeric polypeptide can comprise a sequence 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: [ka]

[0149] In some embodiments, the first chimeric polypeptide is encoded by a sequence 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: [ka] [ka]

[0150] In some embodiments, the first chimeric polypeptide can comprise a sequence 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: [ka]

[0151] In some embodiments, the first chimeric polypeptide is encoded by a sequence 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: [ka] [ka]

[0152] Compositions / kits Also provided herein are compositions (e.g., pharmaceutical compositions) comprising at least one of any of the multi-chain chimeric polypeptides, any of the cells, or any of the 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 of the immune cells (e.g., any of the immune cells described herein, e.g., any of the immune cells produced using any of the methods described herein).

[0153] In some embodiments, the pharmaceutical compositions are formulated for different routes of administration (e.g., intravenous, subcutaneous). In some embodiments, the pharmaceutical compositions may include a pharma- ceutically acceptable carrier (e.g., phosphate buffered saline).

[0154] Single or multiple administrations of the pharmaceutical composition can be given to a subject in need thereof, depending, for example, on the dosage and frequency required and tolerated by the subject. The formulation should provide a sufficient amount of the active agent to effectively treat, prevent or ameliorate the condition, disease or symptom.

[0155] Also provided herein are kits that include any of the multi-chain chimeric polypeptides, compositions, nucleic acids, or cells (e.g., immune cells) described herein. In some aspects, the kits can include instructions for carrying out any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the pharmaceutical compositions described herein.

[0156] Nucleic Acid / Vector Also provided herein are nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first nucleic acid can encode a first chimeric polypeptide and a second nucleic acid can encode a second chimeric polypeptide. In some embodiments, a single nucleic acid can encode both the first chimeric polypeptide and the second chimeric polypeptide.

[0157] Also provided herein is a vector comprising any of the nucleic acids encoding any of the multi-chain chimeric polypeptides described herein. In some embodiments, a first vector can comprise a nucleic acid encoding a first chimeric polypeptide, and a second vector can comprise a nucleic acid encoding a second chimeric polypeptide. In some embodiments, a single vector can comprise a first nucleic acid encoding a first chimeric polypeptide and a second nucleic acid encoding a second chimeric polypeptide.

[0158] Any of the vectors described herein can be an expression vector. For example, the expression vector can include a promoter sequence operably linked to a sequence encoding a first chimeric polypeptide and a second chimeric polypeptide.

[0159] Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral vectors (e.g., any adenoviral vector (e.g., pSV or pCMV vectors), adeno-associated viral (AAV) vectors, lentiviral vectors, and retroviral vectors), as well as any Gateway® vectors. A vector can, for example, contain sufficient cis-acting e...

Claims

1. A pharmaceutical composition for regulating the expression of one or more circadian clock genes in a target tissue requiring such regulation, (a) A first chimeric polypeptide comprising the following: (i) First target binding domain; (ii) A soluble tissue factor domain containing a sequence that is at least 90% identical to sequence number 1; and (iii) The first domain of a pair of affinity domains containing a sequence that is at least 90% identical to sequence number 18; (b) A second chimeric polypeptide comprising the following: (i) the second domain of a pair of affinity domains containing a sequence that is at least 90% identical to sequence number 16; and (ii) Second target binding domain It contains a therapeutically effective amount of multi-chain chimeric polypeptide, The first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first and second domains of the affinity domain pair, A pharmaceutical composition in which 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 pharmaceutical composition for treating a target circadian clock gene disorder, (a) A first chimeric polypeptide comprising the following: (i) First target binding domain; (ii) A soluble tissue factor domain containing a sequence that is at least 90% identical to sequence number 1; and (iii) The first domain of a pair of affinity domains containing a sequence that is at least 90% identical to sequence number 18; (b) A second chimeric polypeptide comprising the following: (i) the second domain of a pair of affinity domains containing a sequence that is at least 90% identical to sequence number 16; and (ii) Second target binding domain It contains a therapeutically effective amount of multi-chain chimeric polypeptide, The first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first and second domains of the affinity domain pair, A pharmaceutical composition in which 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 pharmaceutical composition for reducing the progression rate of a target circadian clock gene disorder, (a) A first chimeric polypeptide comprising the following: (i) First target binding domain; (ii) A soluble tissue factor domain containing a sequence that is at least 90% identical to sequence number 1; and (iii) The first domain of a pair of affinity domains containing a sequence that is at least 90% identical to sequence number 18; (b) A second chimeric polypeptide comprising the following: (i) the second domain of a pair of affinity domains containing a sequence that is at least 90% identical to sequence number 16; and (ii) Second target binding domain It contains a therapeutically effective amount of multi-chain chimeric polypeptide, The first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first and second domains of the affinity domain pair, A pharmaceutical composition in which 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 pharmaceutical composition according to any one of claims 1 to 3, wherein the first target-binding domain comprises a first sequence which is at least 90% identical to SEQ ID NO: 2, and a second sequence which is at least 90% identical to SEQ ID NO: 2, and the first and second sequences are separated by a linker.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the first target-binding domain comprises a sequence that is at least 90% identical to sequence number 4.

6. The pharmaceutical composition according to any one of claims 1 to 3, wherein the first chimeric polypeptide comprises a sequence that is at least 80% identical to SEQ ID NO:

6.

7. The pharmaceutical composition according to claim 6, wherein the first chimeric polypeptide comprises the sequence of SEQ ID NO:

7.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the second target-binding domain comprises a first sequence which is at least 90% identical to SEQ ID NO: 2, and a second sequence which is at least 90% identical to SEQ ID NO: 2, and the first and second sequences are separated by a linker.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein the second target-binding domain comprises a sequence that is at least 90% identical to sequence number 4.

10. The pharmaceutical composition according to any one of claims 1 to 3, wherein the second chimeric polypeptide comprises a sequence that is at least 80% identical to sequence number 5.

11. The pharmaceutical composition according to claim 10, wherein the second chimeric polypeptide comprises the sequence of SEQ ID NO:

8.

12. The pharmaceutical composition according to claim 1, wherein administration of a multi-chain chimeric polypeptide increases the expression level of one or more of Per, Cry, Nr1d1, Nr1d2, Tef, and Dbp compared to the expression levels in a similar subject that has not been administered the multi-chain chimeric polypeptide.

13. The pharmaceutical composition according to claim 1, wherein administration of a multi-chain chimeric polypeptide reduces the expression levels of one or all of Arntl, Npas2, and Ppard compared to the expression levels in a similar subject that has not been administered the multi-chain chimeric polypeptide.

14. The pharmaceutical composition according to claim 1, wherein the tissue is heart tissue, kidney tissue, spleen tissue, pancreatic tissue, intestinal tissue, brain tissue, and / or liver tissue.

15. The pharmaceutical composition according to claim 1, wherein the expression of one or more of Per, Cry, Nr1d1, Nr1d2, Tef, and Dbp is increased upon administration of a multi-chain chimeric polypeptide.

16. The pharmaceutical composition according to claim 1, wherein the expression of one or more Arntl, Npas2, and Ppard is reduced upon administration of a multi-chain chimeric polypeptide.

17. The pharmaceutical composition according to any one of claims 1 to 3, wherein the subject is diagnosed or identified as having a circadian clock gene disorder.

18. The pharmaceutical composition according to claim 17, wherein the circadian clock gene disorder is selected from the group consisting of bipolar disorder (BPD), major depressive disorder (MDD), attention deficit hyperactivity disorder (ADHD), anxiety disorder, cognitive impairment, schizophrenia, obesity, diabetes mellitus, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), metabolic disorders, cancer, cardiovascular disease, and sleep disorders, age-related physical disabilities, and sarcopenia.

19. The pharmaceutical composition according to any one of claims 1 to 3, wherein the subject is identified as being at high risk of developing circadian clock gene disorders.

20. The pharmaceutical composition according to claim 19, wherein the circadian clock gene disorder is selected from the group consisting of bipolar disorder (BPD), major depressive disorder (MDD), attention deficit hyperactivity disorder (ADHD), anxiety disorder, cognitive impairment, schizophrenia, obesity, diabetes mellitus, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), metabolic disorders, cancer, cardiovascular disease, and sleep disorders, age-related physical disabilities, and sarcopenia.

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