Multi-chain chimeric polypeptides for treatment of circadian clock genetic disorders

Abstract

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

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 330,715, filed April 13, 2022, and U.S. Provisional Patent Application Serial No. 63 / 330,694, filed April 13, 2022.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted electronically as an XML file named 47039-0033WO1_SL_ST26.XML. This XML file was created on April 4, 2023, and is 83,483 bytes in size. The material in the XML file is hereby incorporated in its entirety by reference. Technical Field

[0005] This disclosure relates to the field of biotechnology, and more specifically, to antigen-binding molecules and their use in regulating the expression of one or more circadian rhythm clock genes. Background Technology

[0006] Tissue factor (TF) is an integrated membrane glycoprotein of approximately 46 kDa and 263 amino acids, and a trigger protein of the extrinsic coagulation pathway, serving as a major initiator of coagulation in vivo. TF normally does not come into contact with circulating blood, but initiates the coagulation cascade upon exposure to circulating coagulation serine protease factors. Vascular injury exposes subendothelial cells expressing TF, leading to the formation of a calcium-dependent, high-affinity complex with pre-existing plasma factor VIIa (FVIIa). The binding of the serine protease FVIIa to TF promotes the rapid cleavage of FX to FXa and the rapid cleavage of FIX to FIXa. The resulting FXa has insufficient proteolytic activity and an inefficient membrane surface to convert small amounts of prothrombin into thrombin. The thrombin generated from FXa initiates platelet activation and activates trace amounts of pre-cofactors (factor V (FV) and factor VIII (FVIII)) into active cofactors (factor Va (FVa) and factor VIIIa (FVIIIa)). FIXa combines with FVIIIa on the platelet surface to form an intrinsic tenase complex, leading to the rapid generation of FXa. FXa combines with FVa to form a prothrombinase complex on the activated platelet surface, causing prothrombin to rapidly cleave into thrombin.

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

[0008] Many tissue factors expressed on the cell surface are "encrypted" and must be "decrypted" to fully participate in coagulation. Currently, the mechanism of this "decryption" is unclear, but the exposure of anionic phospholipids plays a crucial role. Healthy cells actively isolate anionic phospholipids (such as phosphatidylserine (PS)) into the inner lobe of the plasma membrane. Cell damage, activation, or cytoplasmic calcium... 2+ Upon increasing levels, this bilayer asymmetry disappears, leading to increased PS exposure on the outer leaflets, thereby increasing the specific activity of the cell surface tissue factor-FVIIa complex. PS exposure is known to reduce the apparent Km of FIX and FX activated by the tissue factor-FVIIa complex, but other mechanisms may include conformational rearrangement of tissue factor or tissue factor-FVIIa and subsequent exposure to substrate binding sites.

[0009] Circadian rhythms are a series of oscillators generated by the molecular circadian clock, coordinated with the daily 24-hour cycle. The circadian clock is an internal timing system regulated at the transcriptional level, producing a network of genes that oscillates in approximately 24-hour cycles. 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, mental illness, metabolic syndrome, developmental abnormalities, aging, and cancer. Summary of the Invention

[0010] This article provides a method for regulating the expression of one or more circadian rhythm clock genes in the tissues of a subject in need, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target-binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first and second domains of the affinity domain pair; and the first and second target-binding domains each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0011] This article also provides a method for treating circadian rhythm clock gene disorders in subjects, comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target-binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first and second domains of the affinity domain pair; and the first and second target-binding domains each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0012] This article also provides a method for reducing the rate of progression of circadian rhythm gene disorder in subjects, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target-binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first and second domains of the affinity domain pair; and the first and second target-binding domains each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0013] In some embodiments, the first target-binding domain and the soluble tissue factor domain are directly adjacent to each other in the first chimeric peptide. In some embodiments, the first chimeric peptide further includes a linker sequence between the first target-binding domain and the soluble tissue factor domain in the first chimeric peptide. In some embodiments, the soluble tissue factor domain and the first domain of the affinity domain pair are directly adjacent to each other in the first chimeric peptide. In some embodiments, the first chimeric peptide further includes a linker sequence between the soluble tissue factor domain and the first domain of the affinity domain pair in the first chimeric peptide. In some embodiments, in the second chimeric peptide, the second domain of the affinity domain pair and the second target-binding domain are directly adjacent to each other. In some embodiments, the second chimeric peptide further includes a linker sequence between the second domain of the affinity domain pair and the second target-binding domain in the second chimeric peptide.

[0014] 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.

[0015] 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.

[0016] 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 having at least 80% identity with SEQ ID NO:1.

[0017] In some embodiments, the affinity domain pair is the sushi domain of the α 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: Bacillus RNase 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 protein synaptic fusion proteins, synapse-binding proteins, small synaptic vesicle proteins, and SNAP25. In some embodiments, the first chimeric polypeptide and / or the second chimeric polypeptide further includes a signal sequence at its N-terminus.

[0018] In some embodiments, the first target-binding domain comprises a first sequence having at least 80% identity with SEQ ID NO:2 and a second sequence having at least 80% identity with SEQ ID NO:2, wherein the first and second sequences are separated by a connector. In some embodiments, the first target-binding domain comprises a first sequence having at least 90% identity with SEQ ID NO:2 and a second sequence having at least 90% identity with SEQ ID NO:2. In some embodiments, the first target-binding domain comprises the first sequence of SEQ ID NO:2 and the second sequence of SEQ ID NO:2. In some embodiments, the connector comprises the sequence of SEQ ID NO:3.

[0019] In some embodiments, the first target-binding domain comprises a sequence having at least 80% identity with SEQ ID NO:4. In some embodiments, the first target-binding domain comprises a sequence having at least 90% identity with SEQ ID NO:4. In some embodiments, the first target-binding domain comprises the sequence of SEQ ID NO:4.

[0020] In some embodiments, the first chimeric polypeptide comprises a sequence having at least 80% identity with SEQ ID NO:6. In some embodiments, the first chimeric polypeptide comprises a sequence having at least 90% identity with SEQ ID NO:6. In some embodiments, the first chimeric polypeptide comprises the sequence of SEQ ID NO:6.

[0021] 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.

[0022] In some embodiments, the second target-binding domain comprises a first sequence having at least 80% identity with SEQ ID NO:2 and a second sequence having at least 80% identity with SEQ ID NO:2, wherein the first and second sequences are separated by a connector. In some embodiments, the second target-binding domain comprises a first sequence having at least 90% identity with SEQ ID NO:2 and a second sequence having at least 90% identity with SEQ ID NO:2. In some embodiments, the second target-binding domain comprises the first sequence of SEQ ID NO:2 and the second sequence of SEQ ID NO:2. In some embodiments, the connector comprises the sequence of SEQ ID NO:3.

[0023] In some embodiments, the second target-binding domain comprises a sequence having at least 80% identity with SEQ ID NO:4. In some embodiments, the second target-binding domain comprises a sequence having at least 90% identity with SEQ ID NO:4. In some embodiments, the second target-binding domain comprises the sequence of SEQ ID NO:4.

[0024] In some embodiments, the second chimeric polypeptide comprises a sequence having at least 80% identity with SEQ ID NO:5. In some embodiments, the second chimeric polypeptide comprises a sequence having at least 90% identity with 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.

[0025] In some embodiments, administration of the multi-chain chimeric peptide causes an increase in the expression levels of one or more of Per, Cry, Nr1d1, Nr1d2, Tef, and Dbp compared to the expression levels of one or more of the same subjects who have not been administered the multi-chain chimeric peptide. In some embodiments, administration of the multi-chain chimeric peptide causes a decrease in the expression levels of one or more (e.g., all) of Arntl, Npas2, and Ppard compared to the expression levels of the same of the same subjects who have not been administered the multi-chain chimeric peptide.

[0026] In some embodiments, the tissue is heart tissue, kidney tissue, spleen tissue, pancreas tissue, intestinal tissue, brain tissue, and / or liver tissue.

[0027] In some embodiments, administration of the multi-chain chimeric peptide causes an increase in the expression of one or more of the following: Per, Cry, Nr1d1, Nr1d2, Tef, and Dbp. In some embodiments, administration of the multi-chain chimeric peptide causes a decrease in the expression of one or more of the following: Arntl, Npas2, and Ppard.

[0028] In some embodiments, the subject has been diagnosed or identified as having a circadian rhythm gene disorder. In some embodiments, the subject has been identified as having an increased risk of developing a circadian rhythm gene disorder. In some embodiments, the circadian rhythm 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, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), metabolic disorders, cancer, cardiovascular disease, sleep disorders, age-related physical impairment, sarcopenia, and liver fibrosis.

[0029] As used herein, the term "circadian rhythm" or "circadian clock" refers to an internal timing system regulated at the transcriptional level that produces a network of genes that oscillates in a period of approximately 24 hours. The circadian clock controls physiological and behavioral processes over approximately 24 hours, including but not limited to the regulation of sleep, metabolism, the immune system, the cardiovascular and nervous systems, the microbiome, cancer, and aging. Circadian rhythms control physical, psychological, and behavioral processes.

[0030] As used herein, the term "circadian clock gene" refers to a gene that controls the circadian rhythms of physiology and behavior. Circadian clock genes can also refer to genes whose protein products are essential components of the generation and regulation of circadian rhythms. Circadian clock genes are known to regulate human physiological functions and have a wide range of homeostatic functions, and disturbances to these genes may be associated with sleep disorders, cancer, susceptibility to infection, 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, circadian clock genes can be clock control genes, including Usp2, Tsc22d3, and Tspan4.

[0031] As used herein, the term "circadian clock gene disorder" refers to a disease at least in part caused by dysregulation of a person's circadian rhythm. Disruptions to one or more circadian clock genes (the molecular clocks that maintain all cellular, tissue, and related molecular mechanisms) are known to affect a wide range of diseases and disorders. For example, circadian clock gene disorders can include, but are not limited to, bipolar disorder (BPD), major depressive disorder (MDD), ADHD, anxiety disorders, 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 physical impairment, and sarcopenia.

[0032] As used herein, the term "chimera" refers to a polypeptide comprising amino acid sequences (e.g., domains) originally derived from two different sources (e.g., two different naturally occurring proteins, such as those from the same or different species). For example, a chimeric polypeptide may include domains from at least two different naturally occurring human proteins. In some instances, a chimeric polypeptide may include a domain (e.g., scFv) as a synthetic sequence and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide may include at least two different domains (e.g., two different scFvs) as a synthetic sequence.

[0033] An "antigen-binding domain" is one or more protein domains (e.g., formed from amino acids from a single polypeptide, or from amino acids from two or more polypeptides, e.g., the same or different polypeptides) capable of specifically binding to one or more different antigens. In some instances, an antigen-binding domain can bind to an antigen or epitope with specificity and affinity similar to that of naturally occurring antibodies. In some embodiments, the antigen-binding domain may be an antibody or a fragment thereof. In some embodiments, the antigen-binding domain may include an alternative scaffold. Non-limiting examples of antigen-binding domains are described herein. Further examples of antigen-binding domains are known in the art.

[0034] "Soluble tissue factor domain" refers to a polypeptide that shares at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with a segment of a wild-type mammalian tissue factor protein (e.g., wild-type human tissue factor protein) lacking both transmembrane and intracellular domains. Non-limiting examples of soluble tissue factor domains are described herein.

[0035] The term "soluble interleukin receptor" is used herein in its broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its native ligands (e.g., under physiological conditions, such as in phosphate-buffered saline at room temperature). For example, a soluble interleukin receptor may comprise a sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with the extracellular domain of a wild-type interleukin receptor and retaining its ability to specifically bind one or more of its native ligands, but lacking its transmembrane domain (and optionally, further lacking its intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0036] The term "soluble cytokine receptor" is used herein in its broadest sense to refer to a polypeptide lacking a transmembrane domain (and optionally an intracellular domain) that is capable of binding one or more of its native ligands (e.g., under physiological conditions, such as in phosphate-buffered saline at room temperature). For example, a soluble cytokine receptor may comprise a sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity) with the extracellular domain of a wild-type cytokine receptor and retaining its ability to specifically bind one or more of its native ligands, but lacking its transmembrane domain (and optionally, further lacking its intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0037] The term "antibody" is used herein in its broadest sense and includes certain types of immunoglobulin molecules that include one or more antigen-binding domains that specifically bind to antigens or epitopes. 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. Other examples of antibodies are described herein. Further examples of antibodies are known in the art.

[0038] "Affinity" refers to the sum of the strengths of non-covalent interactions between an antigen-binding site and its binding partner (e.g., antigen or epitope). Unless otherwise stated, "affinity" as used herein refers to intrinsic binding affinity, which reflects a 1:1 interaction between a member of the antigen-binding domain and the antigen or epitope. The affinity of molecule X for its partner Y can be expressed using the dissociation equilibrium constant (K0). D The following describes in more detail the kinetic components that contribute to the dissociation equilibrium constant. Affinity can be measured by methods commonly known in the art, including those described herein. For example, surface plasmon resonance (SPR) techniques (e.g., ) or biological layer interferometry (e.g., Affinity can be determined by [method / method]. Other methods for determining the affinity of antigen-binding domains and their corresponding antigens or epitopes are known in the art.

[0039] As used herein, a "multi-chain polypeptide" refers to a polypeptide comprising two or more (e.g., three, four, five, six, seven, eight, nine, or ten) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide), wherein the two or more protein chains are linked by non-covalent bonds to form a quaternary structure.

[0040] The term "affinity domain pair" refers to two distinct protein domains that specifically bind to each other, whose K... D Less than 1x10 -7 M (for example, less than 1x10) -8 M, less than 1x10 -9 M, less than 1x10 -10 M, or less than 1x10 -11 M). In some instances, the affinity domain pair may be a pair of naturally occurring proteins. In some embodiments, the affinity domain pair may be a pair of synthetic proteins. Non-limiting examples of affinity domain pairs are described herein.

[0041] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen-binding domain. An epitope may consist, for example, of surface-accessible amino acid residues and / or sugar side chains, and may possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational epitopes and non-conformational epitopes is that binding to the former may be lost in the presence of denaturing solvents, while binding to the latter may not be lost. An epitope may contain amino acid residues that directly participate in binding and other amino acid residues that do not directly participate in binding. Methods for identifying epitopes bound to antigen-binding domains are known in the art.

[0042] The term "treatment" means improving at least one symptom of a disorder. In some instances, the disorder being treated is cancer, and improving at least one symptom of the cancer includes reducing the abnormal proliferation of factors, gene expression, signal transduction, translation, and / or secretion. Generally, treatment methods involve administering a therapeutically effective amount of a composition to a subject who needs or has been identified as needing such treatment, the composition reducing at least one symptom of the disorder.

[0043] The phrase "progression rate of circadian rhythm gene disorder" refers to the rate at which the symptoms of a circadian rhythm gene disorder gradually worsen over time. For example, a circadian rhythm gene disorder can progress at a rapid rate (e.g., days or weeks) or at a slower rate (e.g., months or years). In some embodiments, the progression rate of a circadian rhythm gene disorder can be determined by a physician assessing sleep habits or performing diagnostic and cognitive tests. In some embodiments, the progression rate of a circadian rhythm gene disorder can also be measured by imaging studies (e.g., CT scans, magnetic resonance imaging (MRI)).

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0045] Other features and advantages of the invention will become clear from the following detailed description, drawings and claims. Attached Figure Description

[0046] Figure 1 Example diagrams of multi-chain chimeric peptides are shown: (i) a first chimeric peptide comprising a first target-binding domain (A), a soluble tissue factor domain, a first domain of an affinity pair (soluble interleukin IL-15), and an additional target-binding domain (B); and (ii) a second chimeric peptide comprising a second domain of an affinity pair (IL-15 receptor αsushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). A top cartoon depicts the association of the first and second chimeric peptides through a pair of affinity domains. A bottom schematic shows the order of the domains in the first and second chimeric peptides.

[0047] Figure 2 Example diagrams of multi-chain chimeric peptides are shown: (i) a first chimeric peptide comprising a first target-binding domain (A), a soluble tissue factor domain (containing five amino acid substitutions to remove the binding of the soluble tissue factor domain to FVIIa), a first domain of an affinity domain pair (soluble interleukin IL-15, which contains D8N or D8A amino acid substitutions), and an additional target-binding domain (B); and (ii) a second chimeric peptide comprising a second domain of an affinity domain pair (IL-15 receptor αsushi domain), a second target-binding domain (C), and an additional antigen-binding domain (D). A top cartoon depicts the association of the first and second chimeric peptides through a pair of affinity domains. A bottom schematic shows the order of the domains in the first and second chimeric peptides. In other embodiments of any multi-chain chimeric peptides described herein, the soluble tissue factor domain may comprise or consist of a soluble wild-type human tissue factor domain (comprising or consisting of a continuous sequence within wild-type human tissue factor).

[0048] Figure 3 A schematic diagram of the TGFRt15-TGFR construct is shown.

[0049] Figure 4 Another schematic diagram of the TGFRt15-TGFR construct is shown.

[0050] Figure 5 The results show the inhibition of TGFβ1 by TGFRt15-TGFR and TGFR-Fc.

[0051] Figure 6 The results of 32Dβ cell proliferation assays using TGFRt15-TGFR or recombinant IL-15 are shown.

[0052] Figure 7A and 7B The results show the detection of IL-15 and TGFβRII in TGFRt15-TGFR using ELISA with the corresponding antibodies.

[0053] Figure 8 The line graph shows the chromatogram of cell culture supernatant containing TGFRt15-TGFR protein after binding and elution on anti-TF antibody resin.

[0054] Figure 9 The analytical SEC spectra of TGFRt15-TGFR are shown.

[0055] Figure 10 The images show TGFRt15-TGFR before and after deglycosylation, as analyzed by reductive SDS-PAGE.

[0056] Figure 11A and 11B The figures show the spleen weight and percentage of immune cell types in mice treated with TGFRt15-TGFR and in control mice. Figure 85A shows the spleen weight of mice treated with TGFRt15-TGFR compared to the PBS control. Figure 85B shows the percentage of CD4+ T cells, CD8+ T cells, and NK cells in mice treated with TGFRt15-TGFR compared to the PBS control.

[0057] Figure 12A and 12B The figures show spleen weight and immune stimulation in mice treated with TGFRt15-TGFR within 92 hours. Figure 86A shows spleen weight in mice treated with TGFRt15-TGFR at 16, 24, 48, 72, and 92 hours post-treatment. Figure 86B shows the percentage of immune cells in mice treated with TGFRt15-TGFR at 16, 24, 48, 72, and 92 hours post-treatment.

[0058] Figure 13A and 13BThe expression of Ki67 and granzyme B over time was shown in mice treated with TGFRt15-TGFR.

[0059] Figure 14 The study demonstrated enhanced cytotoxicity of TGFRt15-TGFR on spleen cells in C57BL / 6 mice.

[0060] Figure 15 The changes in tumor size in response to PBS treatment, chemotherapy alone, TGFRt15-TGFR alone, or a combination of chemotherapy and TGFRt15-TGFR are shown in a mouse model of pancreatic cancer.

[0061] Figure 16 The cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFR was demonstrated.

[0062] Figures 17A-17C The study showed in vivo stimulation of Treg, NK cells, and CD8+ T cells in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFR.

[0063] Figures 18A-18C Immunostimulation in C57BL / 6 mice after treatment with TGFRt15-TGFR was demonstrated.

[0064] Figure 19A and Figure 19B This study demonstrated the in vivo induction of NK cell and CD8+ T cell proliferation in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFR.

[0065] Figure 20A and 20B The study showed that NK cells exhibited enhanced cytotoxicity after treatment with TGFRt15-TGFR.

[0066] Figure 21A and 21B The study showed that ADCC activity of NK cells was enhanced after treatment with TGFRt15-TGFR.

[0067] Figures 22A-22H The study demonstrated the antitumor activity of TGFRt15-TGFR combined with anti-TRP1 antibody (TA99) and chemotherapy in a mouse model of melanoma.

[0068] Figures 23A-23C The study demonstrated that TGFRt15-TGFR improved Western diet-induced hyperglycemia in ApoE- / - mice.

[0069] Figure 24 The study showed upregulation of CD44hi memory T cells after treatment with TGFRt15-TGFR.

[0070] Figure 25A-25K The study demonstrated that TGFRt15-TGFR(HCW9218) enhances immune-mediated bioactivity in diabetic db / db mice. Figures 25A to 25E Representative flow cytometry data are presented, showing an increase in immune cell surface markers on spleen cells of mice treated with TGFRt15-TGFR(HCW9218) on day 4 compared to the control. Figures 25F to 25G Representative flow cytometry data are shown, demonstrating an increase in the number of central memory cells and effector memory cells in the spleen cell population on day 4 compared to the control when using TGFRt15-TGFR(HCW9218). Figure 25H The study demonstrated that in vivo-treated TGFRt15-TGFR(HCW9218) killed Yac1 target cells compared to control spleen cells. Figure 25I This study demonstrated an increase in interferon (IFN)-γ release from CD3+ cells compared to the control after antigen-independent stimulation with in vivo TGFRt15-TGFR (HCW9218) treatment and ex vivo stimulation of spleen cells with -CD3 / a-CD28 beads. Figure 25J Representative data on increases in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) are shown from spleen cells treated in vivo with PBS or TGFRt15-TGFR (HCW9218) or TGFRt15*-TGFR (HCW9228) and analyzed using a Seahorse XFe Bioanalyzer (Agilent Technologies). Figure 25K The ELISA data shown indicates that plasma TGFβ1 and TGFβ2 decreased after TGFRt15-TGFR(HCW9218) or TGFRt15*-TGFR(HCW9228), but TGFβ3 did not decrease.

[0071] Figures 26A-26P The study demonstrated that TGFRt15-TGFR(HCW9218) treatment reduced senescent pancreatic β-islet cells and SASP factor to improve type 2 diabetes in db / db mice. Figure 26A The treatment regimen of TGFRt15-TGFR(HCW9218) in the db / db mouse model is shown. Figure 26B This study demonstrates the analysis of the expression of senescence, SASP, and β-cell index (related genes) in pancreatic islet transcripts using quantitative PCR and normalization to the control treatment. Figure 26C-26D Immunofluorescence staining of p21+ cells (yellow) and insulin+β islet cells (green) in pancreatic tissue sections is shown. Figure 26EThe number of insulin-positive pancreatic islet cells in the tissue section is shown. Figure 26F The number of p21+ senescent cells in β pancreatic islet cells was shown in the tissue section. Figure 26G-26I The results show aging, SASP, and β-cell indices in the pancreas as determined by quantitative PCR. Figure 26J This shows fasting blood glucose levels after treatment with TGFRt15-TGFR(HCW9218). Figure 26K The HOMA-IR index after TGFRt15-TGFR(HCW9218) treatment is shown. Figure 26L A volcano plot showing RNAseq analysis of the livers of db / db mice is displayed. Figure 26M-26P A heatmap showing genes related to metabolism, aging, inflammation, and blood vessels was displayed.

[0072] Figure 27A-27R The study demonstrated the activity and metabolic function of immune cells in the liver of aging mice stimulated by TGFRt15-TGFR(HCW9218) over time. Figure 27A The treatment regimens of TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) on days 4 and 10 in young and old mouse models are shown. Figure 27B The composite unbiased t-SNE was demonstrated to recognize B cells, T cells, and type 1 ILC. Figure 27C A representative viSNE plot showing density staining of livers harvested on day 4 is displayed. Gland types are stained by population, and the inset numbers represent the frequency of I-ILCs (ILC1 and NK cells). Figures 27D to 27E The summary data from N=3 independent experiments are shown, where N=6 mice / groups demonstrate that on day 4 ( Figure 27D ) and the 10th day ( Figure 27E The frequency of total ILC-1 cells in the liver increased. Figures 27F to 27I Shown on day 4 ( Figure 27F ) and the 10th day ( Figure 27G In the liver and on day 4 ( Figure 27H ) and the 10th day ( Figure 27I Total NK cell frequency in the spleen. Figure 27J The percentage of positive Ki-67 proliferation markers in the liver is shown. Figure 27K-27L The liver was shown on days 4 and 10. Figure 27K ) and spleen ( Figure 27L The total CD8+ T cell frequency in ). Figures 27M to 27N The extracellular acidification rate (ECAR) of spleen cells stimulated in vivo with TGFRt15-TGFR (HCW9218) from young and aged mice is shown by analysis using the Seahorse XFe Bioanalyzer. Figure 27M ) and oxygen consumption rate (OCR) Figure 27N In terms of data, there is an increase in representative data compared to the control group. Figure 27O The study demonstrated the in vitro cytotoxic activity of spleen cells stimulated with TGFRt15-TGFR(HCW9218) in vivo against Yac1 target cells by flow cytometry, compared with controls (PBS or TGFRt15*-TGFR(HCW9228)). Figures 27P to 27Q The study demonstrated an increase in interferon (IFN)-γ and (TNF)-α release from CD3+ cells after antigen-independent stimulation with in vivo HCW9218 treatment and ex vivo α-CD3 / CD28 bead stimulation, as measured by the MAGPIXX multiplexing system, compared to control aged and young mice. Figure 27R Representative flow cytometry data are shown, revealing a percentage increase in intracellular granzyme B levels.

[0073] Figures 28A to 2 8F showed that after one or two subcutaneous doses of TGFRt15-TGFR(HCW9218) or PBS, TGFRt15-TGFR(HCW9218) reduced inflammation (SASP) and cellular senescence markers in the livers of age-related mice. Figure 28A The volcano plot shows the 60-day difference in total RNASeq of hepatocytes in aged mice treated with TGFRt15-TGFR(HCW9218) compared to the control treatment. Figures 28B to 28D A heatmap showing differentially expressed genes related to aging and inflammation, glucose production and fatty acid metabolism, and circadian rhythms in the liver after treatment with TGFRt15-TGFR(HCW9218) compared to the control is presented (adjusted p-values ​​<0.05). Figures 28E to 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 at days 10 and / or 60 after treatment with TGFRt15-TGFR(HCW9218) compared to the control.

[0074] Figures 29A to 29I The study demonstrated that two doses of TGFRt15-TGFR (HCW9218) stimulated metabolic function and reduced inflammatory markers (SASP) and cellular senescence markers in the liver of mice over time-aged conditions for an extended period. Figure 29AThe protocol for two-dose TGFRt15-TGFR(HCW9218) treatment in age-aged female C57BL / 6J mice (76 weeks old) treated subcutaneously with 3 mg / kg TGFRt15-TGFR(HCW9218) (n = 5 to 8) or saline is shown. Mice received a second dose of TGFRt15-TGFR(HCW9218) on day 60 and were sacrificed on day 120. Figures 29B to 29C This study presents representative data showing an increase in extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of spleen cells stimulated in vivo with two doses of TGFRt15-TGFR (HCW9218) compared to the control, as measured by the Seahorse XFe Bioanalyzer. Figure 29D The relative mRNA expression of Il1α, Cdkn1a, Pai-1, Il1b, and Il6 in the liver was determined by quantitative PCR on day 120 after treatment with one or two doses of TGFRt15-TGFR (HCW9218) compared to the control. Figures 29E to 29F The data presented showed the protein levels of IL-1α, IL-6, IL-8, PAI-1, and fibronectin in liver tissue as determined by ELISA on day 120, compared with the control, after treatment with one or two doses of TGFRt15-TGFR (HCW9218). Figure 29G-29H Immunofluorescence staining of liver cells expressing p21+ (yellow) after treatment with two doses of TGFRt15-TGFR (HCW9218) is shown. Figure 29I A heatmap of differentially expressed genes related to aging, inflammation, and circadian rhythm in the liver after treatment with TGFRt15-TGFR(HCW9218) is shown compared with the control treatment (adjusted p < 0.05).

[0075] Figures 30A-30E The study showed that TGFRt15-TGFR(HCW9218) reduced neuroinflammation and affected neuronal function in naturally aging mice. Figure 30A The relative mRNA expression of aging-related genes Cdkn1a, Il6, Il1α, cdkn2a, Il1β, Tnfα, and Il8 in the hippocampus of 120 young and old mice, as determined by quantitative PCR, is shown. Figure 30B The relative mRNA expression of aging-related genes Cdkn1a, Il1α, Il6, Tnfα, Il1β, and Il18 in the hippocampus of aged mice, as determined by quantitative PCR, is shown after 60 days of treatment with TGFRt15-TGFR(HCW9218) and compared with the control. 18S rRNA was used for normalization. Figure 30CThe image shows a total RNA volcano plot of the hippocampus in aged mice 60 days after TGFRt15-TGFR(HCW9218) treatment, compared with the control treatment. Figures 30D to 30E Heatmaps of differentially expressed neuronal, circadian rhythm, and neuroinflammatory genes in the hippocampus after treatment with TGFRt15-TGFR(HCW9218) are shown compared to the control treatment (adjusted p < 0.05).

[0076] Figures 31A to 31I The study demonstrated that aged mice treated with both TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) maintained their physical condition significantly over time, and that TGFRt15-TGFR(HCW9218) was well tolerated in mice and non-human primates and had no long-term adverse effects on naturally aging mice. Figure 31A The grip strength test was shown in aged mice treated with TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) compared with the control. Figure 31B The rotarod performance of mice treated with saline, TGFRt15-TGFR (HCW9218), and TGFRt15*-TGFR (HCW9228) is shown. Figure 31C The open field test used to measure total distance traveled was shown in the same mice described above. Figures 31D to 31E The study showed the expression of Ki67 in CD4, CD8, Treg, and CD16+ NK cells in the blood of cynomolgus monkeys (n=10 / group) after two-dose treatment with TGFRt15-TGFR (HCW9218) or saline (days 1 and 15 of the study). Figure 31D ) and absolute quantity ( Figure 31E Flow cytometry analysis of ). Figure 31F The survival probabilities are shown by monitoring survival and analyzing using the log-rank test. Figure 31G The weight was measured 5 months later. Figure 31H-31I Representative flow cytometry data are shown, which display the percentage of CD8+ T cells and NK cells in the blood.

[0077] Figures 32A-32D The metabolic activity of spleen cells on days 2 and 4 in naturally aged mice was shown. Figures 32A to 32D It was shown that on the 2nd day ( Figures 32A to 32B ) and the 4th day ( Figures 32C to 32D Representative data on the increase in metabolic parameters of spleen cells from db / db mice stimulated in vivo with a single dose of TGFRt15-TGFR (HCW9218) compared to the control, analyzed using the Seahorse XFe Bioanalyzer.

[0078] Figure 33A-3 3H showed that TGFRt15-TGFR(HCW9218) enhanced immune-mediated bioactivity in aged mice. Figures 33A to 33B Representative data measured by flow cytometry are shown, illustrating the blood of young and aged mice on day 4 after treatment with TGFRt15-TGFR (HCW9218). Figure 33A ) and spleen ( Figure 33B The increase in immune cell surface markers in the liver compared to the control was shown in Figures 33C through 33D. Figures 33C and 33D show the immune cell proliferation marker Ki67 in NK cells of young and aged mice in blood (Figure 33C) and spleen (Figure 33D) as measured by flow cytometry on day 4. Figures 33E through 33G show representative flow cytometry data demonstrating an increase in immune cell surface markers on liver immune cells in young and aged mice on day 4 after treatment with TGFRt15-TGFR (HCW9218) compared to the control. Figures 33H through 33I show pooled data from N = 2 independent experiments, with N = 6 mice / group, demonstrating an increase in the frequency of total CD4+ T cells in the liver on days 4 and 10 and in the spleen on days 4 and 10 (Figure 33I) after treatment with TGFRt15-TGFR (HCW9218) compared to the control treatment (Figure 33H).

[0079] Figures 34A-34C The study demonstrated that TGFRt15-TGFR(HCW9218) stimulates immune cell activity and metabolic function and reduces inflammation (SASP) and cellular senescence markers in age-related mice. Figure 34A A heatmap of differentially expressed immune pathway-related genes in the liver after treatment with TGFRt15-TGFR(HCW9218) is shown (adjusted p-value < 0.05) compared to the control treatment. Figure 34B A heatmap of differentially expressed genes related to aging, inflammation, and circadian rhythm in the liver after treatment with TGFRt15*-TGFR(HCW9228) is shown compared with the control treatment (adjusted p-value <0.05). Figure 34C A heatmap of differentially expressed aging, inflammation, and circadian rhythm-related genes in the liver after treatment with TGFRt15-TGFR(HCW9218) was shown after 120 days, compared with TGFRt15*-TGFR(HCW9228) treatment (adjusted p-value < 0.05).

[0080] Figure 35AA heatmap of differentially expressed aging, inflammation, and circadian rhythm-related genes in the hippocampus after treatment with TGFRt15-TGFR(HCW9218) compared to control treatment is shown (adjusted p-value < 0.05). Figure 35B The string analysis of differentially expressed hippocampal genes is shown after TGFRt15-TGFR(HCW9218) treatment on day 120.

[0081] Figures 36A-36D The study showed immunohistochemical staining for blood-brain barrier research. Figures 36A-36D Mice were shown to have received subcutaneous injections of PBS or TGFRt15-TGFR (HCW9218) (3 mg / kg) followed by euthanasia the next day. This was observed in 7-week-old mice from either the control or treatment groups. Figure 36A ), 73 weeks old ( Figure 36B ) or 105 weeks of age ( Figure 36C Frozen sections of mouse brains were processed for immunohistochemical staining with an anti-human tissue factor (TF) specific antibody. No positive staining was detected in the mouse brains. Figure 36D The antibody showed specific staining of human TF in human brain slices.

[0082] Figures 37A-37C Behavioral studies showed that TGFRt15-TGFR(HCW9218) and TGFRt15*-TGFR(HCW9228) maintained minimal acutely significant performance in naturally aging mice. Figure 37A It showed grip strength. Figure 37B The rotator test was shown, and Figure 37C Open field tests were performed in aged mice treated with TGFRt15-TGFR (HCW9218) and TGFRt15*-TGFR (HCW9228) to measure the acute effect on peak force values ​​30 days after each of the corresponding treatments with the first dose. Detailed Implementation

[0083] This article provides methods for regulating the expression of one or more circadian clock genes in the tissues of a subject in need; methods for treating circadian clock gene disorders in a subject; and methods for reducing the rate of progression of circadian clock gene disorders in a subject, the methods comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target-binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of an affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of an affinity domain pair; and (ii) a second target-binding domain, wherein: the first chimeric polypeptide and the second chimeric polypeptide are associated by binding of the first and second domains of the affinity domain pair; and the first and second target-binding domains each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

[0084] In some examples of any multi-chain chimeric polypeptides described herein, the total length of the first chimeric polypeptide and / or the second chimeric polypeptide may each independently be about 50 amino acids to about 3000 amino acids, about 50 amino acids to about 2500 amino acids, about 50 amino acids to about 2000 amino acids, about 50 amino acids to about 1500 amino acids, about 50 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 480 amino acids, about 50 amino acids to about 460 amino acids, about 50 amino acids to about 440 amino acids, about 50 amino acids to about 420 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 380 amino acids, about 50 amino acids to about 360 amino acids, about 50 amino acids to about 340 amino acids, about 50 amino acids to about 320 amino acids, about 50 amino acids to about 300 amino acids, about 50 amino acids to about 280 amino acids, about 50 Amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 150 amino acids, about 50 amino acids to about 100 amino acids, about 100 amino acids to about 3000 amino acids, about 100 amino acids to about 2500 amino acids, about 100 amino acids to about 2000 amino acids, about 100 amino acids to about 1500 amino acids, about 100 amino acids to about 1000 amino acids, about 100 amino acids to about 950 amino acids, about 100 amino acids to about 900 amino acids, about 100 amino acids to about 850 amino acids, about 1 00 amino acids to about 800 amino acids, about 100 amino acids to about 750 amino acids, about 100 amino acids to about 700 amino acids, about 100 amino acids to about 650 amino acids, about 100 amino acids to about 600 amino acids, about 100 amino acids to about 550 amino acids, about 100 amino acids to about 500 amino acids, about 100 amino acids to about 480 amino acids, about 100 amino acids to about 460 amino acids, about 100 amino acids to about 440 amino acids, about 100 amino acids to about 420 amino acids, about 100 amino acids to about 400 amino acids, about 100 amino acids to about 380 amino acids, about 100 amino acids to about 360 amino acids,Approximately 100 amino acids to approximately 340 amino acids, approximately 100 amino acids to approximately 320 amino acids, approximately 100 amino acids to approximately 300 amino acids, approximately 100 amino acids to approximately 280 amino acids, approximately 100 amino acids to approximately 260 amino acids, approximately 100 amino acids to approximately 240 amino acids, approximately 100 amino acids to approximately 220 amino acids, approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 150 amino acids, approximately 150 amino acids to approximately 3000 amino acids, approximately 150 amino acids to approximately 2500 amino acids, approximately 150 amino acids to approximately 2000 amino acids, approximately 150 amino acids to approximately 1500 amino acids, approximately 150 amino acids to approximately 10 00 amino acids, about 150 amino acids to about 950 amino acids, about 150 amino acids to about 900 amino acids, about 150 amino acids to about 850 amino acids, about 150 amino acids to about 800 amino acids, about 150 amino acids to about 750 amino acids, about 150 amino acids to about 700 amino acids, about 150 amino acids to about 650 amino acids, about 150 amino acids to about 600 amino acids, about 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 480 amino acids, about 150 amino acids to about 460 amino acids, about 150 amino acids to about 440 amino acids, about 150 amino acids to Approximately 420 amino acids, approximately 150 amino acids to approximately 400 amino acids, approximately 150 amino acids to approximately 380 amino acids, approximately 150 amino acids to approximately 360 amino acids, approximately 150 amino acids to approximately 340 amino acids, approximately 150 amino acids to approximately 320 amino acids, approximately 150 amino acids to approximately 300 amino acids, approximately 150 amino acids to approximately 280 amino acids, approximately 150 amino acids to approximately 260 amino acids, approximately 150 amino acids to approximately 240 amino acids, approximately 150 amino acids to approximately 220 amino acids, approximately 150 amino acids to approximately 200 amino acids, approximately 200 amino acids to approximately 3000 amino acids, approximately 200 amino acids to approximately 2500 amino acids, approximately 200 amino acids Amino acids to about 2000 amino acids, about 200 amino acids to about 1500 amino acids, about 200 amino acids to about 1000 amino acids, about 200 amino acids to about 950 amino acids, about 200 amino acids to about 900 amino acids, about 200 amino acids to about 850 amino acids, about 200 amino acids to about 800 amino acids, about 200 amino acids to about 750 amino acids, about 200 amino acids to about 700 amino acids, about 200 amino acids to about 650 amino acids, about 200 amino acids to about 600 amino acids, about 200 amino acids to about 550 amino acids, about 200 amino acids to about 500 amino acids, about 200 amino acids to about 480 amino acids.Approximately 200 amino acids to approximately 460 amino acids, approximately 200 amino acids to approximately 440 amino acids, approximately 200 amino acids to approximately 420 amino acids, approximately 200 amino acids to approximately 400 amino acids, approximately 200 amino acids to approximately 380 amino acids, approximately 200 amino acids to approximately 360 amino acids, approximately 200 amino acids to approximately 340 amino acids, approximately 200 amino acids to approximately 320 amino acids, approximately 200 amino acids to approximately 300 amino acids, approximately 200 amino acids to approximately 280 amino acids, approximately 200 amino acids to approximately 260 amino acids, approximately 200 amino acids to approximately 240 amino acids, approximately 200 amino acids to approximately 220 amino acids, approximately 220 amino acids to approximately 3000 amino acids. Amino acids, approximately 220 amino acids to approximately 2500 amino acids, approximately 220 amino acids to approximately 2000 amino acids, approximately 220 amino acids to approximately 1500 amino acids, approximately 220 amino acids to approximately 1000 amino acids, approximately 220 amino acids to approximately 950 amino acids, approximately 220 amino acids to approximately 900 amino acids, approximately 220 amino acids to approximately 850 amino acids, approximately 220 amino acids to approximately 800 amino acids, approximately 220 amino acids to approximately 750 amino acids, approximately 220 amino acids to approximately 700 amino acids, approximately 220 amino acids to approximately 650 amino acids, approximately 220 amino acids to approximately 600 amino acids, approximately 220 amino acids to approximately 550 amino acids, approximately 220 amino acids to Approximately 500 amino acids, approximately 220 amino acids to approximately 480 amino acids, approximately 220 amino acids to approximately 460 amino acids, approximately 220 amino acids to approximately 440 amino acids, approximately 220 amino acids to approximately 420 amino acids, approximately 220 amino acids to approximately 400 amino acids, approximately 220 amino acids to approximately 380 amino acids, approximately 220 amino acids to approximately 360 amino acids, approximately 220 amino acids to approximately 340 amino acids, approximately 220 amino acids to approximately 320 amino acids, approximately 220 amino acids to approximately 300 amino acids, approximately 220 amino acids to approximately 280 amino acids, approximately 220 amino acids to approximately 260 amino acids, approximately 220 amino acids to approximately 240 amino acids, approximately 240 amino acids Acids of approximately 3000 amino acids, approximately 240 amino acids of approximately 2500 amino acids, approximately 240 amino acids of approximately 2000 amino acids, approximately 240 amino acids of approximately 1500 amino acids, approximately 240 amino acids of approximately 1000 amino acids, approximately 240 amino acids of approximately 950 amino acids, approximately 240 amino acids of approximately 900 amino acids, approximately 240 amino acids of approximately 850 amino acids, approximately 240 amino acids of approximately 800 amino acids, approximately 240 amino acids of approximately 750 amino acids, approximately 240 amino acids of approximately 700 amino acids, approximately 240 amino acids of approximately 650 amino acids, approximately 240 amino acids of approximately 600 amino acids, approximately 240 amino acids of approximately 550 amino acids.Approximately 240 amino acids to approximately 500 amino acids, approximately 240 amino acids to approximately 480 amino acids, approximately 240 amino acids to approximately 460 amino acids, approximately 240 amino acids to approximately 440 amino acids, approximately 240 amino acids to approximately 420 amino acids, approximately 240 amino acids to approximately 400 amino acids, approximately 240 amino acids to approximately 380 amino acids, approximately 240 amino acids to approximately 360 amino acids, approximately 240 amino acids to approximately 340 amino acids, approximately 240 amino acids to approximately 320 amino acids, approximately 240 amino acids to approximately 300 amino acids, approximately 240 amino acids to approximately 280 amino acids, approximately 240 amino acids to approximately 260 amino acids, approximately 260 amino acids to approximately 3000 amino acids. Amino acids, approximately 260 amino acids to approximately 2500 amino acids, approximately 260 amino acids to approximately 2000 amino acids, approximately 260 amino acids to approximately 1500 amino acids, approximately 260 amino acids to approximately 1000 amino acids, approximately 260 amino acids to approximately 950 amino acids, approximately 260 amino acids to approximately 900 amino acids, approximately 260 amino acids to approximately 850 amino acids, approximately 260 amino acids to approximately 800 amino acids, approximately 260 amino acids to approximately 750 amino acids, approximately 260 amino acids to approximately 700 amino acids, approximately 260 amino acids to approximately 650 amino acids, approximately 260 amino acids to approximately 600 amino acids, approximately 260 amino acids to approximately 550 amino acids, approximately 260 amino acids to Approximately 500 amino acids, approximately 260 amino acids to approximately 480 amino acids, approximately 260 amino acids to approximately 460 amino acids, approximately 260 amino acids to approximately 440 amino acids, approximately 260 amino acids to approximately 420 amino acids, approximately 260 amino acids to approximately 400 amino acids, approximately 260 amino acids to approximately 380 amino acids, approximately 260 amino acids to approximately 360 amino acids, approximately 260 amino acids to approximately 340 amino acids, approximately 260 amino acids to approximately 320 amino acids, approximately 260 amino acids to approximately 300 amino acids, approximately 260 amino acids to approximately 280 amino acids, approximately 280 amino acids to approximately 3000 amino acids, approximately 280 amino acids to approximately 2500 amino acids, approximately 280 Amino acids to about 2000 amino acids, about 280 amino acids to about 1500 amino acids, about 280 amino acids to about 1000 amino acids, about 280 amino acids to about 950 amino acids, about 280 amino acids to about 900 amino acids, about 280 amino acids to about 850 amino acids, about 280 amino acids to about 800 amino acids, about 280 amino acids to about 750 amino acids, about 280 amino acids to about 700 amino acids, about 280 amino acids to about 650 amino acids, about 280 amino acids to about 600 amino acids, about 280 amino acids to about 550 amino acids, about 280 amino acids to about 500 amino acids, about 280 amino acids to about 480 amino acids.Approximately 280 amino acids to approximately 460 amino acids, approximately 280 amino acids to approximately 440 amino acids, approximately 280 amino acids to approximately 420 amino acids, approximately 280 amino acids to approximately 400 amino acids, approximately 280 amino acids to approximately 380 amino acids, approximately 280 amino acids to approximately 360 amino acids, approximately 280 amino acids to approximately 340 amino acids, approximately 280 amino acids to approximately 320 amino acids, approximately 280 amino acids to approximately 300 amino acids, approximately 300 amino acids to approximately 3000 amino acids, approximately 300 amino acids to approximately 2500 amino acids, approximately 300 amino acids to approximately 2000 amino acids, approximately 300 amino acids to approximately 1500 amino acids, approximately 300 amino acids to approximately 10 00 amino acids, approximately 300 amino acids to approximately 950 amino acids, approximately 300 amino acids to approximately 900 amino acids, approximately 300 amino acids to approximately 850 amino acids, approximately 300 amino acids to approximately 800 amino acids, approximately 300 amino acids to approximately 750 amino acids, approximately 300 amino acids to approximately 700 amino acids, approximately 300 amino acids to approximately 650 amino acids, approximately 300 amino acids to approximately 600 amino acids, approximately 300 amino acids to approximately 550 amino acids, approximately 300 amino acids to approximately 500 amino acids, approximately 300 amino acids to approximately 480 amino acids, approximately 300 amino acids to approximately 460 amino acids, approximately 300 amino acids to approximately 440 amino acids, approximately 300 amino acids to Approximately 420 amino acids, approximately 300 amino acids to approximately 400 amino acids, approximately 300 amino acids to approximately 380 amino acids, approximately 300 amino acids to approximately 360 amino acids, approximately 300 amino acids to approximately 340 amino acids, approximately 300 amino acids to approximately 320 amino acids, approximately 320 amino acids to approximately 3000 amino acids, approximately 320 amino acids to approximately 2500 amino acids, approximately 320 amino acids to approximately 2000 amino acids, approximately 320 amino acids to approximately 1500 amino acids, approximately 320 amino acids to approximately 1000 amino acids, approximately 320 amino acids to approximately 950 amino acids, approximately 320 amino acids to approximately 900 amino acids, approximately 320 amino acids to approximately 850 amino acids, approximately 3 20 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.Approximately 320 amino acids to approximately 340 amino acids, approximately 340 amino acids to approximately 3000 amino acids, approximately 340 amino acids to approximately 2500 amino acids, approximately 340 amino acids to approximately 2000 amino acids, approximately 340 amino acids to approximately 1500 amino acids, approximately 340 amino acids to approximately 1000 amino acids, approximately 340 amino acids to approximately 950 amino acids, approximately 340 amino acids to approximately 900 amino acids, approximately 340 amino acids to approximately 850 amino acids, approximately 340 amino acids to approximately 800 amino acids, approximately 340 amino acids to approximately 750 amino acids, approximately 340 amino acids to approximately 700 amino acids, approximately 340 amino acids to approximately 650 amino acids, approximately 340 amino acids 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. Acid, approximately 360 amino acids to approximately 1000 amino acids, approximately 360 amino acids to approximately 950 amino acids, approximately 360 amino acids to approximately 900 amino acids, approximately 360 amino acids to approximately 850 amino acids, approximately 360 amino acids to approximately 800 amino acids, approximately 360 amino acids to approximately 750 amino acids, approximately 360 amino acids to approximately 700 amino acids, approximately 360 amino acids to approximately 650 amino acids, approximately 360 amino acids to approximately 600 amino acids, approximately 360 amino acids to approximately 550 amino acids, approximately 360 amino acids to approximately 500 amino acids, approximately 360 amino acids to approximately 480 amino acids, approximately 360 amino acids to approximately 460 amino acids, approximately 360 amino acids to approximately 440 amino acids, approximately 360 amino acids to approximately 420 amino acids, approximately 360 amino acids to approximately 400 amino acids, approximately 360 amino acids to approximately 380 amino acids, approximately 380 amino acids to approximately 3000 amino acids, approximately 380 amino acids to approximately 2500 amino acids, approximately 380 amino acids to approximately 2000 amino acids, approximately 380 amino acids to approximately 1500 amino acids, approximately 380 amino acids to approximately 1000 amino acids, approximately 380 amino acids to approximately 950 amino acids, approximately 380 amino acids to approximately 900 amino acids, approximately 380 amino acids to approximately 850 amino acids, approximately 380 amino acids to approximately 800 amino acids, approximately 380 amino acids to approximately 750 amino acids.Approximately 380 amino acids to approximately 700 amino acids, approximately 380 amino acids to approximately 650 amino acids, approximately 380 amino acids to approximately 600 amino acids, approximately 380 amino acids to approximately 550 amino acids, approximately 380 amino acids to approximately 500 amino acids, approximately 380 amino acids to approximately 480 amino acids, approximately 380 amino acids to approximately 460 amino acids, approximately 380 amino acids to approximately 440 amino acids, approximately 380 amino acids to approximately 420 amino acids, approximately 380 amino acids to approximately 400 amino acids, approximately 400 amino acids to approximately 3000 amino acids, approximately 400 amino acids to approximately 2500 amino acids, approximately 400 amino acids to approximately 2000 amino acids, approximately 400 amino acids to... Approximately 1500 amino acids, approximately 400 amino acids to approximately 1000 amino acids, approximately 400 amino acids to approximately 950 amino acids, approximately 400 amino acids to approximately 900 amino acids, approximately 400 amino acids to approximately 850 amino acids, approximately 400 amino acids to approximately 800 amino acids, approximately 400 amino acids to approximately 750 amino acids, approximately 400 amino acids to approximately 700 amino acids, approximately 400 amino acids to approximately 650 amino acids, approximately 400 amino acids to approximately 600 amino acids, approximately 400 amino acids to approximately 550 amino acids, approximately 400 amino acids to approximately 500 amino acids, approximately 400 amino acids to approximately 480 amino acids, approximately 400 amino acids to approximately 460 amino acids, approximately 4 00 amino acids to about 440 amino acids, about 400 amino acids to about 420 amino acids, about 420 amino acids to about 3000 amino acids, about 420 amino acids to about 2500 amino acids, about 420 amino acids to about 2000 amino acids, about 420 amino acids to about 1500 amino acids, about 420 amino acids to about 1000 amino acids, about 420 amino acids to about 950 amino acids, about 420 amino acids to about 900 amino acids, about 420 amino acids to about 850 amino acids, about 420 amino acids to about 800 amino acids, about 420 amino acids to about 750 amino acids, about 420 amino acids to about 700 amino acids, about 420 amino acids to about 650 amino acids, approximately 420 amino acids to approximately 600 amino acids, approximately 420 amino acids to approximately 550 amino acids, approximately 420 amino acids to approximately 500 amino acids, approximately 420 amino acids to approximately 480 amino acids, approximately 420 amino acids to approximately 460 amino acids, approximately 420 amino acids to approximately 440 amino acids, approximately 440 amino acids to approximately 3000 amino acids, approximately 440 amino acids to approximately 2500 amino acids, approximately 440 amino acids to approximately 2000 amino acids, approximately 440 amino acids to approximately 1500 amino acids, approximately 440 amino acids to approximately 1000 amino acids, approximately 440 amino acids to approximately 950 amino acids, approximately 440 amino acids to approximately 900 amino acids.Approximately 440 amino acids to approximately 850 amino acids, approximately 440 amino acids to approximately 800 amino acids, approximately 440 amino acids to approximately 750 amino acids, approximately 440 amino acids to approximately 700 amino acids, approximately 440 amino acids to approximately 650 amino acids, approximately 440 amino acids to approximately 600 amino acids, approximately 440 amino acids to approximately 550 amino acids, approximately 440 amino acids to approximately 500 amino acids, approximately 440 amino acids to approximately 480 amino acids, approximately 440 amino acids to approximately 460 amino acids, approximately 460 amino acids to approximately 3000 amino acids, approximately 460 amino acids to approximately 2500 amino acids, approximately 460 amino acids to approximately 2000 amino acids, approximately 460 amino acids to Approximately 1500 amino acids, approximately 460 amino acids to approximately 1000 amino acids, approximately 460 amino acids to approximately 950 amino acids, approximately 460 amino acids to approximately 900 amino acids, approximately 460 amino acids to approximately 850 amino acids, approximately 460 amino acids to approximately 800 amino acids, approximately 460 amino acids to approximately 750 amino acids, approximately 460 amino acids to approximately 700 amino acids, approximately 460 amino acids to approximately 650 amino acids, approximately 460 amino acids to approximately 600 amino acids, approximately 460 amino acids to approximately 550 amino acids, approximately 460 amino acids to approximately 500 amino acids, approximately 460 amino acids to approximately 480 amino acids, approximately 480 amino acids to approximately 3000 amino acids, approximately 480 amino acids to about 2500 amino acids, about 480 amino acids to about 2000 amino acids, about 480 amino acids to about 1500 amino acids, about 480 amino acids to about 1000 amino acids, about 480 amino acids to about 950 amino acids, about 480 amino acids to about 900 amino acids, about 480 amino acids to about 850 amino acids, about 480 amino acids to about 800 amino acids, about 480 amino acids to about 750 amino acids, about 480 amino acids to about 700 amino acids, about 480 amino acids to about 650 amino acids, about 480 amino acids to about 600 amino acids, about 480 amino acids to about 550 amino acids, about 480 amino acids to about 500 amino acids, approximately 500 amino acids to approximately 3000 amino acids, approximately 500 amino acids to approximately 2500 amino acids, approximately 500 amino acids to approximately 2000 amino acids, approximately 500 amino acids to approximately 1500 amino acids, approximately 500 amino acids to approximately 1000 amino acids, approximately 500 amino acids to approximately 950 amino acids, approximately 500 amino acids to approximately 900 amino acids, approximately 500 amino acids to approximately 850 amino acids, approximately 500 amino acids to approximately 800 amino acids, approximately 500 amino acids to approximately 750 amino acids, approximately 500 amino acids to approximately 700 amino acids, approximately 500 amino acids to approximately 650 amino acids, approximately 500 amino acids to approximately 600 amino acids.Approximately 500 amino acids to approximately 550 amino acids, approximately 550 amino acids to approximately 3000 amino acids, approximately 550 amino acids to approximately 2500 amino acids, approximately 550 amino acids to approximately 2000 amino acids, approximately 550 amino acids to approximately 1500 amino acids, approximately 550 amino acids to approximately 1000 amino acids, approximately 550 amino acids to approximately 950 amino acids, approximately 550 amino acids to approximately 900 amino acids, approximately 550 amino acids to approximately 850 amino acids, approximately 550 amino acids to approximately 800 amino acids, approximately 550 amino acids to approximately 750 amino acids, approximately 550 amino acids to approximately 700 amino acids, approximately 550 amino acids to approximately 650 amino acids, approximately 550 amino acids to approximately... 600 amino acids, approximately 600 amino acids to approximately 3000 amino acids, approximately 600 amino acids to approximately 2500 amino acids, approximately 600 amino acids to approximately 2000 amino acids, approximately 600 amino acids to approximately 1500 amino acids, approximately 600 amino acids to approximately 1000 amino acids, approximately 600 amino acids to approximately 950 amino acids, approximately 600 amino acids to approximately 900 amino acids, approximately 600 amino acids to approximately 850 amino acids, approximately 600 amino acids to approximately 800 amino acids, approximately 600 amino acids to approximately 750 amino acids, approximately 600 amino acids to approximately 700 amino acids, approximately 600 amino acids to approximately 650 amino acids, approximately 650 amino acids to approximately 3000 amino acids, approximately 650 amino acids to about 2500 amino acids, about 650 amino acids to about 2000 amino acids, about 650 amino acids to about 1500 amino acids, about 650 amino acids to about 1000 amino acids, about 650 amino acids to about 950 amino acids, about 650 amino acids to about 900 amino acids, about 650 amino acids to about 850 amino acids, about 650 amino acids to about 800 amino acids, about 650 amino acids to about 750 amino acids, about 650 amino acids to about 700 amino acids, about 700 amino acids to about 3000 amino acids, about 700 amino acids to about 2500 amino acids, about 700 amino acids to about 2000 amino acids, about 700 amino acids to Approximately 1500 amino acids, approximately 700 amino acids to approximately 1000 amino acids, approximately 700 amino acids to approximately 950 amino acids, approximately 700 amino acids to approximately 900 amino acids, approximately 700 amino acids to approximately 850 amino acids, approximately 700 amino acids to approximately 800 amino acids, approximately 700 amino acids to approximately 750 amino acids, approximately 750 amino acids to approximately 3000 amino acids, approximately 750 amino acids to approximately 2500 amino acids, approximately 750 amino acids to approximately 2000 amino acids, approximately 750 amino acids to approximately 1500 amino acids, approximately 750 amino acids to approximately 1000 amino acids, approximately 750 amino acids to approximately 950 amino acids, approximately 750 amino acids to approximately 900 amino acids,Approximately 750 amino acids to approximately 850 amino acids, approximately 750 amino acids to approximately 800 amino acids, approximately 800 amino acids to approximately 3000 amino acids, approximately 800 amino acids to approximately 2500 amino acids, approximately 800 amino acids to approximately 2000 amino acids, approximately 800 amino acids to approximately 1500 amino acids, approximately 800 amino acids to approximately 1000 amino acids, approximately 800 amino acids to approximately 950 amino acids, approximately 800 amino acids to approximately 900 amino acids, approximately 800 amino acids to approximately 850 amino acids. Amino acids, approximately 850 amino acids to approximately 3000 amino acids, approximately 850 amino acids to approximately 2500 amino acids, approximately 850 amino acids to approximately 2000 amino acids, approximately 850 amino acids to approximately 1500 amino acids, approximately 850 amino acids to approximately 1000 amino acids, approximately 850 amino acids to approximately 950 amino acids, approximately 850 amino acids to approximately 900 amino acids, approximately 900 amino acids to approximately 3000 amino acids, approximately 900 amino acids to approximately 2500 amino acids, approximately 900 amino acids Approximately 2000 amino acids, approximately 900 amino acids to approximately 1500 amino acids, approximately 900 amino acids to approximately 1000 amino acids, approximately 900 amino acids to approximately 950 amino acids, approximately 950 amino acids to approximately 3000 amino acids, approximately 950 amino acids to approximately 2500 amino acids, approximately 950 amino acids to approximately 2000 amino acids, approximately 950 amino acids to approximately 1500 amino acids, approximately 950 amino acids to approximately 1000 amino acids, approximately 1000 amino acids to approximately 3000 amino acids Approximately 1000 amino acids to approximately 2500 amino acids, approximately 1000 amino acids to approximately 2000 amino acids, approximately 1000 amino acids to approximately 1500 amino acids, approximately 1500 amino acids to approximately 3000 amino acids, approximately 1500 amino acids to approximately 2500 amino acids, approximately 1500 amino acids to approximately 2000 amino acids, approximately 2000 amino acids to approximately 3000 amino acids, approximately 2000 amino acids to approximately 2500 amino acids, or approximately 2500 amino acids to approximately 3000 amino acids. The exemplary multi-chain chimeric polypeptides provided herein are illustrated in [the relevant context]. Figure 1 and 2 middle.

[0085] In some embodiments of any multi-chain chimeric peptide described herein, a first target-binding domain (e.g., any first target-binding domain described herein) and a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) are directly adjacent to each other in the first chimeric peptide. In some embodiments of any multi-chain chimeric peptide described herein, the first chimeric peptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the first target-binding domain (e.g., any exemplary first target-binding domain described herein) and the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) in the first chimeric peptide.

[0086] In some embodiments of any multi-chain chimeric peptides described herein, a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) are directly adjacent to each other in the first chimeric peptide. In some embodiments of any multi-chain chimeric peptides described herein, the first chimeric peptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and the first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein) in the first chimeric peptide.

[0087] In some embodiments of any multi-chain chimeric polypeptide described herein, the second domain of an affinity pair (e.g., any exemplary second domain of any exemplary affinity pair described herein) and the second target-binding domain (e.g., any exemplary second target-binding domain described herein) are directly adjacent to each other in the second chimeric polypeptide. In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between the second domain of the affinity pair (e.g., any exemplary second domain of any exemplary affinity pair described herein) and the second target-binding domain (e.g., any exemplary second target-binding domain described herein).

[0088] The non-limiting aspects of these chimeric peptides, nucleic acids, vectors, cells, and methods are described below and can be used in any combination without limitation. Other aspects of these chimeric peptides, nucleic acids, vectors, cells, and methods are known in the art.

[0089] Organization Factor

[0090] Human tissue factor (TGF) is a 263-amino acid transmembrane protein containing three domains: (1) a 219-amino acid N-terminal extracellular domain (residues 1-219); (2) a 22-amino acid transmembrane domain (residues 220-242); and (3) a 21-amino acid cytoplasmic C-terminal tail (residues 242-263) (UniProtKB identifier: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, and one S-palmitoylation site at Cys245. No deletions or mutations of the cytoplasmic domains were found to affect TGF's coagulation activity. TGF has an S-palmitoylation site at Cys245 in its intracellular domain. Cys245 is located at the amino acid terminus of the intracellular domain and is close to the membrane surface. The TGF transmembrane domain consists of a single α-helix.

[0091] The extracellular domain of tissue factor, composed of two type III fibronectin domains, is linked to the transmembrane domain via a six-amino acid linker. This linker provides conformational flexibility, decoupling the extracellular domain of tissue factor from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module consists of two overlapping β-sheets, with the top fold containing three antiparallel β-chains and the bottom fold containing four β-chains. The β-chains are linked by β-loops between chains βA and βB, βC and βD, and βE and βF, all of which are conformationally conserved in both modules. Three short α-helical segments connect the β-chains. A unique feature of tissue factor is the 17-amino acid β-hairpin between the β10 and β11 chains, which is not a common element in the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is not present in the C-terminal domain and is unique to tissue factor. This fibronectin type III domain structure is characteristic of the immunoglobulin-like protein folding family and is conserved in a variety of extracellular proteins.

[0092] Once the proenzyme FVII binds to tissue to form the active tissue factor-FVIIa complex, it is rapidly converted to FVIIa through limited proteolytic activity. FVIIa cycles at a concentration of approximately 0.1 nM (1% of plasma FVII) and can also bind directly to tissue factor. Allosteric interactions between tissue factor and FVIIa on the tissue factor-FVIIa complex significantly increase the enzymatic activity of FVIIa: the hydrolysis rate of small chromophore substrates increases by approximately 20 to 100 times, and the activation rate of natural macromolecular substrates FIX and FX increases by nearly a million times. Consistent with the allosteric activation of the FVIIa active site after tissue factor binding, the formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., after exposure of phosphatidyl-L-serine on the membrane surface) increases the activation rate of FIX or FX, with Ca 2+ In a dependent manner, it increases by an additional 1,000 times. Compared to free FVIIa, the overall FX activation of the tissue factor-FVIIa-phospholipid complex increases by approximately one million times, which is a key regulator of the coagulation cascade.

[0093] FVII is a single-chain polypeptide of approximately 50 kDa, composed of 406 amino acid residues, featuring an N-terminal γ-carboxyglutamate-rich (GLA) domain, two epidermal growth factor-like domains (EGF1 and EFG2), and a C-terminal serine protease domain. FVII communicates via an Ile- molecule in the short linker region between the EGF2 and protease domains. 154 -Arg 152 The specific protein hydrolysis of the bond activates it to become FVIIa. This cleavage leads to the light and heavy chains passing through Cys... 135 and Cys 262 The individual disulfide bonds hold them together. FVIIa is held together by its N-terminal GLA domain via Ca 2+ It binds to the phospholipid membrane in a dependent manner. Immediately adjacent to the C-terminus of the GLA domain are an aromatic stack and two EGF domains. The aromatic stack connects the GLA to the binding of a single Ca²⁺ molecule. 2+ The EGF1 domain of the ion. This Ca 2+ Occupancy of the binding site increases the amide degradation activity of FVIIa and its association with tissue factor. The catalytic triad is composed of His 193 Asp 242 and Ser 344 Composition, and with a single Ca within the FVIIa protease domain 2+ The binding of ions is crucial to its catalytic activity. FVII protein hydrolysis activates it to FVIIa, which is released into Ile. 153 The newly formed amino terminus is folded back and inserted into the activation bag, along with Asp 343The carboxylate forms a salt bridge, thereby creating an oxyanion pore. This salt bridge formation is crucial for FVIIa activity. However, oxyanion pore formation does not occur in free FVIIa after proteolytic activation. Therefore, FVIIa cycles in its enzyme-as-an enzyme state, which is difficult for plasma protease inhibitors to recognize, resulting in a cycling half-life of approximately 90 minutes.

[0094] The tissue factor-mediated localization of the FVIIa active site above the membrane surface is important for FVIIa orientation toward homologous substrates. Free FVIIa, upon membrane binding, employs a stable extended structure, with its active site located approximately above the membrane surface. After FVIIa binds to tissue factor, the active site of FVa is relocated, moving closer to the membrane. This modulation may facilitate the proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using FVIIa without the GLA domain, it has been shown that the active site remains at a similar distance above the membrane, demonstrating that tissue factor can fully support the localization of the FVIIa active site even in the absence of FVIIa-membrane interactions. Other data suggest that tissue factor supports full FVIIa proteolytic activity as long as its extracellular domain is somehow bound to the membrane surface. However, increasing the distance above the membrane surface of the FVIIa active site to greater than [a certain distance]... It will greatly reduce the ability of the tissue factor-FVIIa complex to activate FX, but will not weaken the amide decomposition activity of tissue factor-FVIIa.

[0095] Alanine scanning mutagenesis has been used to assess the role of specific amino acid side chains in the extracellular domain of tissue factor in their interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitution identified a limited number of residue sites at which alanine substitution resulted in a 5- to 10-fold decrease in FVIIa binding affinity. Most of these residue side chains were found to be well exposed to the solvent in the crystal structure, consistent with macromolecular ligand interactions. The FVIIa ligand binding sites were located in a wide region at the boundary between the two modules. In the C-module, the Arg residue located on the protruding BC ring... 135 and Phe 140 Provides independent contact with FVIIa. Leu 133 Located at the bottom of the finger-like structure and filling the crack between the two modules. This is for Lys 20 Thr 60 Asp 58 and Ile22 The major clusters of key binding residues provide continuity. 60 Partial exposure to the solvent may result in a localized structural effect rather than significant contact with the ligand. The binding site extends to the concave side of the inter-module corner, involving Glu. 24 and Gln 110 And possibly even further residues Val 207 The combined region extends from Asp58 to Lys 48 Lys 46 、Gln 37 Asp 44 and Trp 45 Trp 45 and Asp 44 The interaction with FVIIa is not independent, indicating that Trp 45 The abrupt change in position may reflect the effect of this side chain on adjacent Asp. 44 and Gln 37 The structural importance of the local packaging of the side chains. The interaction region further includes two surface-exposed aromatic residues, Phe 76 and Tyr 78 They form part of the hydrophobic clusters in the N-module.

[0096] The known physiological substrates of tissue factor-FVIIa are FVII, FIX, and FX, as well as certain protease-activated receptors. Mutation analysis has identified many residues that, when mutated, support the intact FVIIa amide degradation activity against small peptide substrates but lack its ability to support activation against macromolecular substrates (i.e., FVII, FIX, and FX) (Ruf et al., J Biol Chem 267(31):22206-22210, 1992; Ruf et al., J Biol Chem 267(9):6375-6381, 1992; Huang et al., J Biol Chem 271(36):21752-21757, 1996; Kirchhofer et al., Biochemistry 39(25):7380-7387, 2000). The tissue factor loop region at residues 159-165, and residues in or adjacent to this flexible loop, have been shown to be crucial for the proteolytic activity of the tissue factor-FVIIa complex. This defines the proposed substrate-binding extra-site region of tissue factor, which is quite far from the FVIIa active site. Replacing glycine residues with slightly larger alanine residues significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the flexibility provided by glycine is essential for the 159-165 residue loop for large molecular substrate recognition by tissue factor.

[0097] residue Lys 165 and Lys 166 They have also been shown to be important for substrate recognition and binding. Mutations of any of these residues to alanine result in a significant reduction in tissue factor cofactor function. Lys 165 and Lys 166 They are facing away from each other, with Lys among them. 165 In most tissue factor-FVIIa structures, it points to FVIIa, while Lys 166 Pointing to the substrate-binding site region in the crystal structure. Lys of FVIIa 165 and Gla 35 The hypothetical salt bridge formation between them would support the idea that the interaction between tissue factor and the GLA domain of FVIIa regulates substrate recognition. These results suggest that the C-terminal portion of the extracellular domain of tissue factor directly interacts with the GLA domains of FIX and FX (possibly adjacent to the EGF1 domain), and that the presence of the FVIIa GLA domain may directly or indirectly regulate these interactions.

[0098] Soluble tissue factor domain

[0099] In some embodiments of any peptide, composition, or method described herein, the soluble tissue factor domain may be a wild-type tissue factor peptide lacking a signal sequence, transmembrane domain, and intracellular domain. In some instances, the soluble tissue factor domain may be a tissue factor mutant, wherein the wild-type tissue factor peptide lacks a signal sequence, transmembrane domain, and intracellular domain, and has been further modified at selected amino acids. In some instances, the soluble tissue factor domain may be a soluble human tissue factor domain. In some instances, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some instances, the soluble tissue factor domain may be a soluble rat tissue factor domain. Non-limiting examples of soluble human tissue factor domains, mouse soluble tissue factor domains, rat soluble tissue factor domains, and mutant soluble tissue factor domains are shown below.

[0100] Exemplary soluble human tissue factor domain (SEQ ID NO:1)

[0101] SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0102] Exemplary nucleic acid encoding soluble human tissue factor domain (SEQ ID NO:9)

[0103] AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACACCGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGACAGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGCAAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG

[0104] Exemplary mutant soluble human tissue factor domain (SEQ ID NO:10)

[0105] SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE

[0106] Exemplary mutant soluble human tissue factor domain (SEQ ID NO:11) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLGQPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary soluble mouse tissue factor domain (SEQ ID NO:12)

[0107] agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtn lgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge

[0108] Exemplary soluble rat tissue factor domain (SEQ ID NO:13)

[0109] agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

[0110] In some embodiments, the soluble tissue factor domain may include a sequence having at least 70% identity, at least 72% identity, at least 74% identity, at least 76% identity, at least 78% identity, at least 80% identity, at least 82% identity, at least 84% identity, at least 86% identity, at least 88% identity, at least 90% identity, at least 92% identity, at least 94% identity, at least 96% identity, at least 98% identity, at least 99% identity, or 100% identity with SEQ ID NO:1, 10, 11, 12, or 13. In some embodiments, the soluble tissue factor domain may comprise the sequence SEQ ID NO:1, 10, 11, 12 or 13, wherein one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids) are removed from its N-terminus, and / or one to twenty amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids) are removed from its C-terminus.

[0111] As understood in the art, those skilled in the art will recognize that conserved amino acid mutations across different mammalian species are more likely to reduce protein activity and / or structural stability, while non-conserved amino acid mutations across different mammalian species are less likely to reduce protein activity and / or structural stability.

[0112] In some examples of any multi-chain chimeric peptides described herein, the soluble tissue factor domain cannot bind factor VIIa. In some examples of any multi-chain chimeric peptides described herein, the soluble tissue factor domain does not convert inactive factor X into factor Xa. In some embodiments of any multi-chain chimeric peptides described herein, the multi-chain chimeric peptide does not stimulate blood clotting in mammals.

[0113] In some instances, the soluble tissue factor domain may be a soluble human tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble mouse tissue factor domain. In some embodiments, the soluble tissue factor domain may be a soluble rat tissue factor domain.

[0114] In some instances, the soluble tissue factor domain does not include one or more of the following (e.g., two, three, four, five, six, or seven): lysine at amino acid position 20 of the mature wild-type human tissue factor protein; isoleucine at amino acid position 22 of the mature wild-type human tissue factor protein; tryptophan at amino acid position 45 of the mature wild-type human tissue factor protein; aspartic acid at amino acid position 58 of the mature wild-type human tissue factor protein; tyrosine at amino acid position 94 of the mature wild-type human tissue factor protein; arginine at amino acid position 135 of the mature wild-type human tissue factor protein; and phenylalanine at 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.

[0115] In some instances, the soluble tissue factor domain may be encoded by a nucleic acid comprising a sequence having at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:9.

[0116] In some embodiments, the total length of the soluble tissue factor domain can be about 20 amino acids to about 220 amino acids, about 20 amino acids to about 215 amino acids, about 20 amino acids to about 210 amino acids, about 20 amino acids to about 205 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... Amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 2 0 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 50 amino acids, about 20 amino acids to about 40 amino acids, about 20 amino acids to about 30 amino acids, about 30 amino acids to about 220 amino acids, about 30 amino acids to about 215 amino acids, about 30 amino acids to about 210 amino acids, about 30 amino acids to about 205 amino acids, about 30 amino acids to about 200 amino acids, about 30 amino acids to about 195 amino acids, about 30 amino acids to about 190 amino acids, about 30 amino acids to about 185 amino acids, about 30 Amino acids to about 180 amino acids, about 30 amino acids to about 175 amino acids, about 30 amino acids to about 170 amino acids, about 30 amino acids to about 165 amino acids, about 30 amino acids to about 160 amino acids, about 30 amino acids to about 155 amino acids, about 30 amino acids to about 150 amino acids, about 30 amino acids to about 145 amino acids, about 30 amino acids to about 140 amino acids, about 30 amino acids to about 135 amino acids, about 30 amino acids to about 130 amino acids, about 30 amino acids to about 125 amino acids, about 30 amino acids to about 120 amino acids, about 30 amino acids to about 115 amino acids, about 30 amino acids to about 110 amino acids.Approximately 30 amino acids to approximately 105 amino acids, approximately 30 amino acids to approximately 100 amino acids, approximately 30 amino acids to approximately 95 amino acids, approximately 30 amino acids to approximately 90 amino acids, approximately 30 amino acids to approximately 85 amino acids, approximately 30 amino acids to approximately 80 amino acids, approximately 30 amino acids to approximately 75 amino acids, approximately 30 amino acids to approximately 70 amino acids, approximately 30 amino acids to approximately 60 amino acids, approximately 30 amino acids to approximately 50 amino acids, approximately 30 amino acids to approximately 40 amino acids, approximately 40 amino acids to approximately 220 amino acids, approximately 40 amino acids to approximately 215 amino acids, approximately 40 amino acids to approximately 210 amino acids, approximately 40 amino acids to approximately 205 amino acids. Approximately 40 amino acids to approximately 200 amino acids, approximately 40 amino acids to approximately 195 amino acids, approximately 40 amino acids to approximately 190 amino acids, approximately 40 amino acids to approximately 185 amino acids, approximately 40 amino acids to approximately 180 amino acids, approximately 40 amino acids to approximately 175 amino acids, approximately 40 amino acids to approximately 170 amino acids, approximately 40 amino acids to approximately 165 amino acids, approximately 40 amino acids to approximately 160 amino acids, approximately 40 amino acids to approximately 155 amino acids, approximately 40 amino acids to approximately 150 amino acids, approximately 40 amino acids to approximately 145 amino acids, approximately 40 amino acids to approximately 140 amino acids, approximately 40 amino acids to approximately 135 amino acids, approximately 40 amino acids to approximately 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 Approximately 220 amino acids, approximately 50 amino acids to approximately 215 amino acids, approximately 50 amino acids to approximately 210 amino acids, approximately 50 amino acids to approximately 205 amino acids, approximately 50 amino acids to approximately 200 amino acids, approximately 50 amino acids to approximately 195 amino acids, approximately 50 amino acids to approximately 190 amino acids, approximately 50 amino acids to approximately 185 amino acids, approximately 50 amino acids to approximately 180 amino acids, approximately 50 amino acids to approximately 175 amino acids, approximately 50 amino acids to approximately 170 amino acids, approximately 50 amino acids to approximately 165 amino acids, approximately 50 amino acids to approximately 160 amino acids, approximately 50 amino acids to approximately 155 amino acids, approximately 50 amino acids to approximately 150 amino acids.Approximately 50 amino acids to approximately 145 amino acids, approximately 50 amino acids to approximately 140 amino acids, approximately 50 amino acids to approximately 135 amino acids, approximately 50 amino acids to approximately 130 amino acids, approximately 50 amino acids to approximately 125 amino acids, approximately 50 amino acids to approximately 120 amino acids, approximately 50 amino acids to approximately 115 amino acids, approximately 50 amino acids to approximately 110 amino acids, approximately 50 amino acids to approximately 105 amino acids, approximately 50 amino acids to approximately 100 amino acids, approximately 50 amino acids to approximately 95 amino acids, approximately 50 amino acids to approximately 90 amino acids, approximately 50 amino acids to approximately 85 amino acids, approximately 50 amino acids to approximately 80 amino acids, approximately 50 amino acids to approximately 75 amino acids. Amino acids, approximately 50 amino acids to approximately 70 amino acids, approximately 50 amino acids to approximately 60 amino acids, approximately 60 amino acids to approximately 220 amino acids, approximately 60 amino acids to approximately 215 amino acids, approximately 60 amino acids to approximately 210 amino acids, approximately 60 amino acids to approximately 205 amino acids, approximately 60 amino acids to approximately 200 amino acids, approximately 60 amino acids to approximately 195 amino acids, approximately 60 amino acids to approximately 190 amino acids, approximately 60 amino acids to approximately 185 amino acids, approximately 60 amino acids to approximately 180 amino acids, approximately 60 amino acids to approximately 175 amino acids, approximately 60 amino acids to approximately 170 amino acids, approximately 60 amino acids to approximately 165 amino acids, approximately 60 amino acids to approximately 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 The sequence of amino acids is: approximately 60 amino acids to approximately 80 amino acids, approximately 60 amino acids to approximately 75 amino acids, approximately 60 amino acids to approximately 70 amino acids, approximately 70 amino acids to approximately 220 amino acids, approximately 70 amino acids to approximately 215 amino acids, approximately 70 amino acids to approximately 210 amino acids, approximately 70 amino acids to approximately 205 amino acids, approximately 70 amino acids to approximately 200 amino acids, approximately 70 amino acids to approximately 195 amino acids, approximately 70 amino acids to approximately 190 amino acids, approximately 70 amino acids to approximately 185 amino acids, approximately 70 amino acids to approximately 180 amino acids, approximately 70 amino acids to approximately 175 amino acids, approximately 70 amino acids to approximately 170 amino acids.Approximately 70 amino acids to approximately 165 amino acids, approximately 70 amino acids to approximately 160 amino acids, approximately 70 amino acids to approximately 155 amino acids, approximately 70 amino acids to approximately 150 amino acids, approximately 70 amino acids to approximately 145 amino acids, approximately 70 amino acids to approximately 140 amino acids, approximately 70 amino acids to approximately 135 amino acids, approximately 70 amino acids to approximately 130 amino acids, approximately 70 amino acids to approximately 125 amino acids, approximately 70 amino acids to approximately 120 amino acids, approximately 70 amino acids to approximately 115 amino acids, approximately 70 amino acids to approximately 110 amino acids, approximately 70 amino acids to approximately 105 amino acids, approximately 70 amino acids to approximately 100 amino acids, approximately 70 amino acids to approximately 95 amino acids. 1 amino acid, about 70 amino acids to about 90 amino acids, about 70 amino acids to about 85 amino acids, about 70 amino acids to about 80 amino acids, about 80 amino acids to about 220 amino acids, about 80 amino acids to about 215 amino acids, about 80 amino acids to about 210 amino acids, about 80 amino acids to about 205 amino acids, about 80 amino acids to about 200 amino acids, about 80 amino acids to about 195 amino acids, about 80 amino acids to about 190 amino acids, about 80 amino acids to about 185 amino acids, about 80 amino acids to about 180 amino acids, about 80 amino acids to about 175 amino acids, about 80 amino acids to about 170 amino acids, about 80 amino acids to about 165 amino acids, about 80 amino acids to about 160 amino acids, about 80 amino acids to about 155 amino acids, about 80 amino acids to about 150 amino acids, about 80 amino acids to about 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 Amino acids to about 90 amino acids, about 90 amino acids to about 220 amino acids, about 90 amino acids to about 215 amino acids, about 90 amino acids to about 210 amino acids, about 90 amino acids to about 205 amino acids, about 90 amino acids to about 200 amino acids, about 90 amino acids to about 195 amino acids, about 90 amino acids to about 190 amino acids, about 90 amino acids to about 185 amino acids, about 90 amino acids to about 180 amino acids, about 90 amino acids to about 175 amino acids, about 90 amino acids to about 170 amino acids, about 90 amino acids to about 165 amino acids, about 90 amino acids to about 160 amino acids, about 90 amino acids to about 155 amino acids.Approximately 90 amino acids to approximately 150 amino acids, approximately 90 amino acids to approximately 145 amino acids, approximately 90 amino acids to approximately 140 amino acids, approximately 90 amino acids to approximately 135 amino acids, approximately 90 amino acids to approximately 130 amino acids, approximately 90 amino acids to approximately 125 amino acids, approximately 90 amino acids to approximately 120 amino acids, approximately 90 amino acids to approximately 115 amino acids, approximately 90 amino acids to approximately 110 amino acids, approximately 90 amino acids to approximately 105 amino acids, approximately 90 amino acids to approximately 100 amino acids, approximately 100 amino acids to approximately 220 amino acids, approximately 100 amino acids to approximately 215 amino acids, approximately 100 amino acids to approximately 210 amino acids, approximately 100 amino acids About 205 amino acids, about 100 amino acids to about 200 amino acids, about 100 amino acids to about 195 amino acids, about 100 amino acids to about 190 amino acids, about 100 amino acids to about 185 amino acids, about 100 amino acids to about 180 amino acids, about 100 amino acids to about 175 amino acids, about 100 amino acids to about 170 amino acids, about 100 amino acids to about 165 amino acids, about 100 amino acids to about 160 amino acids, about 100 amino acids to about 155 amino acids, about 100 amino acids to about 150 amino acids, about 100 amino acids to about 145 amino acids, about 100 amino acids to about 140 amino acids, about 100 Amino acids to about 135 amino acids, about 100 amino acids to about 130 amino acids, about 100 amino acids to about 125 amino acids, about 100 amino acids to about 120 amino acids, about 100 amino acids to about 115 amino acids, about 100 amino acids to about 110 amino acids, about 110 amino acids to about 220 amino acids, about 110 amino acids to about 215 amino acids, about 110 amino acids to about 210 amino acids, about 110 amino acids to about 205 amino acids, about 110 amino acids to about 200 amino acids, about 110 amino acids to about 195 amino acids, about 110 amino acids to about 190 amino acids, about 110 amino acids to about 185 amino acids, about 1 10 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.Approximately 115 amino acids to approximately 220 amino acids, approximately 115 amino acids to approximately 215 amino acids, approximately 115 amino acids to approximately 210 amino acids, approximately 115 amino acids to approximately 205 amino acids, approximately 115 amino acids to approximately 200 amino acids, approximately 115 amino acids to approximately 195 amino acids, approximately 115 amino acids to approximately 190 amino acids, approximately 115 amino acids to approximately 185 amino acids, approximately 115 amino acids to approximately 180 amino acids, approximately 115 amino acids to approximately 175 amino acids, approximately 115 amino acids to approximately 170 amino acids, approximately 115 amino acids to approximately 165 amino acids, approximately 115 amino acids to approximately 160 amino acids, approximately 115 amino acids to approximately 155 amino acids. 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 Approximately 190 amino acids, approximately 120 amino acids to approximately 185 amino acids, approximately 120 amino acids to approximately 180 amino acids, approximately 120 amino acids to approximately 175 amino acids, approximately 120 amino acids to approximately 170 amino acids, approximately 120 amino acids to approximately 165 amino acids, approximately 120 amino acids to approximately 160 amino acids, approximately 120 amino acids to approximately 155 amino acids, approximately 120 amino acids to approximately 150 amino acids, approximately 120 amino acids to approximately 145 amino acids, approximately 120 amino acids to approximately 140 amino acids, approximately 120 amino acids to approximately 135 amino acids, approximately 120 amino acids to approximately 130 amino acids, approximately 120 amino acids to approximately 125 amino acids, approximately 125 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their components. A direct translation wouldn't be meaningful without further context.Approximately 125 amino acids to approximately 150 amino acids, approximately 125 amino acids to approximately 145 amino acids, approximately 125 amino acids to approximately 140 amino acids, approximately 125 amino acids to approximately 135 amino acids, approximately 125 amino acids to approximately 130 amino acids, approximately 130 amino acids to approximately 220 amino acids, approximately 130 amino acids to approximately 215 amino acids, approximately 130 amino acids to approximately 210 amino acids, approximately 130 amino acids to approximately 205 amino acids, approximately 130 amino acids to approximately 200 amino acids, approximately 130 amino acids to approximately 195 amino acids, approximately 130 amino acids to approximately 190 amino acids, approximately 130 amino acids to approximately 185 amino acids, approximately 130 amino acids to approximately 180 130 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 Approximately 200 amino acids, approximately 135 amino acids to approximately 195 amino acids, approximately 135 amino acids to approximately 190 amino acids, approximately 135 amino acids to approximately 185 amino acids, approximately 135 amino acids to approximately 180 amino acids, approximately 135 amino acids to approximately 175 amino acids, approximately 135 amino acids to approximately 170 amino acids, approximately 135 amino acids to approximately 165 amino acids, approximately 135 amino acids to approximately 160 amino acids, approximately 135 amino acids to approximately 155 amino acids, approximately 135 amino acids to approximately 150 amino acids, approximately 135 amino acids to approximately 145 amino acids, approximately 135 amino acids to approximately 140 amino acids, approximately 140 amino acids to approximately 220 amino acids, approximately 140 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their components. A direct translation wouldn't be meaningful without further context.Approximately 140 amino acids to approximately 145 amino acids, approximately 145 amino acids to approximately 220 amino acids, approximately 145 amino acids to approximately 215 amino acids, approximately 145 amino acids to approximately 210 amino acids, approximately 145 amino acids to approximately 205 amino acids, approximately 145 amino acids to approximately 200 amino acids, approximately 145 amino acids to approximately 195 amino acids, approximately 145 amino acids to approximately 190 amino acids, approximately 145 amino acids to approximately 185 amino acids, approximately 145 amino acids to approximately 180 amino acids, approximately 145 amino acids to approximately 175 amino acids, approximately 145 amino acids to approximately 170 amino acids, approximately 145 amino acids to approximately 165 amino acids, approximately 145 amino acids to approximately 160 amino acids. 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 Approximately 165 amino acids, approximately 150 amino acids to approximately 160 amino acids, approximately 150 amino acids to approximately 155 amino acids, approximately 155 amino acids to approximately 220 amino acids, approximately 155 amino acids to approximately 215 amino acids, approximately 155 amino acids to approximately 210 amino acids, approximately 155 amino acids to approximately 205 amino acids, approximately 155 amino acids to approximately 200 amino acids, approximately 155 amino acids to approximately 195 amino acids, approximately 155 amino acids to approximately 190 amino acids, approximately 155 amino acids to approximately 185 amino acids, approximately 155 amino acids to approximately 180 amino acids, approximately 155 amino acids to approximately 175 amino acids, approximately 155 amino acids to approximately 170 amino acids, approximately 155 The sequence of amino acids is: approximately 165 amino acids, approximately 155 amino acids, approximately 160 amino acids, approximately 220 amino acids, approximately 160 amino acids, approximately 215 amino acids, approximately 160 amino acids, approximately 210 amino acids, approximately 160 amino acids, approximately 205 amino acids, approximately 160 amino acids, approximately 200 amino acids, approximately 160 amino acids, approximately 195 amino acids, approximately 190 amino acids, approximately 185 amino acids, approximately 180 amino acids, approximately 175 amino acids, approximately 170 amino acids, approximately 160 amino acids, approximately 165 amino acids.Approximately 165 amino acids to approximately 220 amino acids, approximately 165 amino acids to approximately 215 amino acids, approximately 165 amino acids to approximately 210 amino acids, approximately 165 amino acids to approximately 205 amino acids, approximately 165 amino acids to approximately 200 amino acids, approximately 165 amino acids to approximately 195 amino acids, approximately 165 amino acids to approximately 190 amino acids, approximately 165 amino acids to approximately 185 amino acids, approximately 165 amino acids to approximately 180 amino acids, approximately 165 amino acids to approximately 175 amino acids, approximately 165 amino acids to approximately 170 amino acids, approximately 170 amino acids to approximately 220 amino acids, approximately 170 amino acids to approximately 215 amino acids, approximately 170 amino acids to approximately 210 170 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 Approximately 190 amino acids, approximately 175 amino acids to approximately 185 amino acids, approximately 175 amino acids to approximately 180 amino acids, approximately 180 amino acids to approximately 220 amino acids, approximately 180 amino acids to approximately 215 amino acids, approximately 180 amino acids to approximately 210 amino acids, approximately 180 amino acids to approximately 205 amino acids, approximately 180 amino acids to approximately 200 amino acids, approximately 180 amino acids to approximately 195 amino acids, approximately 180 amino acids to approximately 190 amino acids, approximately 180 amino acids to approximately 185 amino acids, approximately 185 amino acids to approximately 220 amino acids, approximately 185 amino acids to approximately 215 amino acids, approximately 185 amino acids to approximately 210 amino acids, approximately 185 The sequence of amino acids is: approximately 185 amino acids to approximately 200 amino acids, approximately 185 amino acids to approximately 195 amino acids, approximately 185 amino acids to approximately 190 amino acids, approximately 190 amino acids to approximately 220 amino acids, approximately 190 amino acids to approximately 215 amino acids, approximately 190 amino acids to approximately 210 amino acids, approximately 190 amino acids to approximately 205 amino acids, approximately 190 amino acids to approximately 200 amino acids, approximately 190 amino acids to approximately 195 amino acids, approximately 195 amino acids to approximately 220 amino acids, approximately 195 amino acids to approximately 215 amino acids, approximately 195 amino acids to approximately 210 amino acids, approximately 195 amino acids to approximately 205 amino acids.Approximately 195 amino acids to approximately 200 amino acids, approximately 200 amino acids to approximately 220 amino acids, approximately 200 amino acids to approximately 215 amino acids, approximately 200 amino acids to approximately 210 amino acids, approximately 200 amino acids to approximately 205 amino acids, approximately 205 amino acids to approximately 220 amino acids, approximately 205 amino acids to approximately 215 amino acids, approximately 205 amino acids to approximately 210 amino acids, approximately 210 amino acids to approximately 220 amino acids, approximately 210 amino acids to approximately 215 amino acids, or approximately 215 amino acids to approximately 220 amino acids.

[0117] Connector sequence

[0118] In some embodiments, the adapter sequence may be a flexible adapter sequence. Non-limiting examples of adapter sequences that may be used are described in Klein et al., Protein Engineering, Design & Selection, 27(10):325–330, 2014; Priyanka et al., Protein Sci., 22(2):153–167, 2013. In some instances, the adapter sequence is a synthetic adapter sequence.

[0119] In some embodiments of any multi-chain chimeric polypeptide described herein, a first chimeric polypeptide may include one, two, three, four, five, six, seven, eight, nine, or ten adapter sequences (e.g., the same or different adapter sequences, such as any exemplary adapter sequences described herein or known in the art). In some embodiments of any multi-chain chimeric polypeptide described herein, a second chimeric polypeptide may include one, two, three, four, five, six, seven, eight, nine, or ten adapter sequences (e.g., the same or different adapter sequences, such as any exemplary adapter sequences described herein or known in the art).

[0120] In some embodiments, the total length of the connector can be from 1 amino acid to about 100 amino acids, from 1 amino acid to about 90 amino acids, from 1 amino acid to about 80 amino acids, from 1 amino acid to about 70 amino acids, from 1 amino acid to about 60 amino acids, from 1 amino acid to about 50 amino acids, from 1 amino acid to about 45 amino acids, from 1 amino acid to about 40 amino acids, from 1 amino acid to about 35 amino acids, from 1 amino acid to about 30 amino acids, from 1 amino acid to about 25 amino acids, from 1 amino acid to about 24 amino acids, from 1 amino acid to about 22 amino acids, from 1 amino acid to about 20 amino acids, from 1 amino acid to about 18 amino acids, from 1 amino acid to about 16 amino acids, or from 1 amino acid to about 14 amino acids, 1 amino acid to about 12 amino acids, 1 amino acid to about 10 amino acids, 1 amino acid to about 8 amino acids, 1 amino acid to about 6 amino acids, 1 amino acid to about 4 amino acids, about 2 amino acids to about 100 amino acids, about 2 amino acids to about 90 amino acids, about 2 amino acids to about 80 amino acids, about 2 amino acids to about 70 amino acids, about 2 amino acids to about 60 amino acids, about 2 amino acids to about 50 amino acids, about 2 amino acids to about 45 amino acids, about 2 amino acids to about 40 amino acids, about 2 amino acids to about 35 amino acids, about 2 amino acids to about 30 amino acids, about 2 amino acids to about 25 amino acids, about 2 amino acids to about 2 4 amino acids, about 2 amino acids to about 22 amino acids, about 2 amino acids to about 20 amino acids, about 2 amino acids to about 18 amino acids, about 2 amino acids to about 16 amino acids, about 2 amino acids to about 14 amino acids, about 2 amino acids to about 12 amino acids, about 2 amino acids to about 10 amino acids, about 2 amino acids to about 8 amino acids, about 2 amino acids to about 6 amino acids, about 2 amino acids to about 4 amino acids, about 4 amino acids to about 100 amino acids, about 4 amino acids to about 90 amino acids, about 4 amino acids to about 80 amino acids, about 4 amino acids to about 70 amino acids, about 4 amino acids to about 60 amino acids, about 4 amino acids to about 50 amino acids, about 4 amino groups Acid to about 45 amino acids, about 4 amino acids to about 40 amino acids, about 4 amino acids to about 35 amino acids, about 4 amino acids to about 30 amino acids, about 4 amino acids to about 25 amino acids, about 4 amino acids to about 24 amino acids, about 4 amino acids to about 22 amino acids, about 4 amino acids to about 20 amino acids, about 4 amino acids to about 18 amino acids, about 4 amino acids to about 16 amino acids, about 4 amino acids to about 14 amino acids, about 4 amino acids to about 12 amino acids, about 4 amino acids to about 10 amino acids, about 4 amino acids to about 8 amino acids, about 4 amino acids to about 6 amino acids, about 6 amino acids to about 100 amino acids, about 6 amino acids to about 90 amino acids,About 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 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. Approximately 8 amino acids to approximately 16 amino acids, approximately 8 amino acids to approximately 14 amino acids, approximately 8 amino acids to approximately 12 amino acids, approximately 8 amino acids to approximately 10 amino acids, approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 90 amino acids, approximately 10 amino acids to approximately 80 amino acids, approximately 10 amino acids to approximately 70 amino acids, approximately 10 amino acids to approximately 60 amino acids, approximately 10 amino acids to approximately 50 amino acids, approximately 10 amino acids to approximately 45 amino acids, approximately 10 amino acids to approximately 40 amino acids, approximately 10 amino acids to approximately 35 amino acids, approximately 10 amino acids to approximately 30 amino acids, approximately 10 amino acids to approximately 25 amino acids, approximately 10 amino acids to approximately... 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 Approximately 40 amino acids, approximately 14 amino acids to approximately 35 amino acids, approximately 14 amino acids to approximately 30 amino acids, approximately 14 amino acids to approximately 25 amino acids, approximately 14 amino acids to approximately 24 amino acids, approximately 14 amino acids to approximately 22 amino acids, approximately 14 amino acids to approximately 20 amino acids, approximately 14 amino acids to approximately 18 amino acids, approximately 14 amino acids to approximately 16 amino acids, approximately 16 amino acids to approximately 100 amino acids, approximately 16 amino acids to approximately 90 amino acids, approximately 16 amino acids to approximately 80 amino acids, approximately 16 amino acids to approximately 70 amino acids, approximately 16 amino acids to approximately 60 amino acids, approximately 16 amino acids to approximately 50 amino acids, approximately 16 amino acids to approximately 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 Approximately 40 amino acids, approximately 18 amino acids to approximately 35 amino acids, approximately 18 amino acids to approximately 30 amino acids, approximately 18 amino acids to approximately 25 amino acids, approximately 18 amino acids to approximately 24 amino acids, approximately 18 amino acids to approximately 22 amino acids, approximately 18 amino acids to approximately 20 amino acids, approximately 20 amino acids to approximately 100 amino acids, approximately 20 amino acids to approximately 90 amino acids, approximately 20 amino acids to approximately 80 amino acids, approximately 20 amino acids to approximately 70 amino acids, approximately 20 amino acids to approximately 60 amino acids, approximately 20 amino acids to approximately 50 amino acids, approximately 20 amino acids to approximately 45 amino acids, approximately 20 amino acids to approximately 40 amino acids, approximately 20 amino acids to approximately 35 amino acids,Approximately 20 amino acids to approximately 30 amino acids, approximately 20 amino acids to approximately 25 amino acids, approximately 20 amino acids to approximately 24 amino acids, approximately 20 amino acids to approximately 22 amino acids, approximately 22 amino acids to approximately 100 amino acids, approximately 22 amino acids to approximately 90 amino acids, approximately 22 amino acids to approximately 80 amino acids, approximately 22 amino acids to approximately 70 amino acids, approximately 22 amino acids to approximately 60 amino acids, approximately 22 amino acids to approximately 50 amino acids, approximately 22 amino acids to approximately 45 amino acids, approximately 22 amino acids to approximately 40 amino acids, approximately 22 amino acids to approximately 35 amino acids, approximately 22 amino acids to approximately 30 amino acids, approximately 22 amino acids to approximately 25 amino acids, approximately 22 amino acids to approximately 24 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids to about 30 amino acids, about 30 amino acids to about 100 amino acids, about 30 amino acids to about 90 amino acids, about 30 amino acids to about 80 amino acids, about 30 amino acids to about 70 amino acids, about 30 amino acids to about 60 amino acids, about 30 amino acids to about 50 amino acids, about 30 amino acids to about 45 amino acids, about 30 amino acids to about 40 amino acids, about 30 amino acids to about 35 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 50 amino acids, about 35 amino acids to about 45 amino acids, about 35 amino acids to about 40 amino acids, about 40 amino acids to about 100 amino acids, about 40 amino acids to about 90 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 100 amino acids, about 45 amino acids to about 90 amino acids, about 45 amino acids to about 80 amino acids, about 45 amino acids to about 70 amino acids, about 45 amino acids to about 60 amino acids, about 45 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, about 50 amino acids to about 90 amino acids, about 50 amino acids to about 80 amino acids, about 50 amino acids to about 70 amino acids, about 50 amino acids to about 60 amino acids, about 60 amino acids to about 100 amino acids.Approximately 60 amino acids to approximately 90 amino acids, approximately 60 amino acids to approximately 80 amino acids, approximately 60 amino acids to approximately 70 amino acids, approximately 70 amino acids to approximately 100 amino acids, approximately 70 amino acids to approximately 90 amino acids, approximately 70 amino acids to approximately 80 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 80 amino acids to approximately 90 amino acids, or approximately 90 amino acids to approximately 100 amino acids.

[0121] In some embodiments, the linker is enriched with glycine (Gly or G) residues. In some embodiments, the linker is enriched with serine (Ser or S) residues. In some embodiments, the linker is enriched with both glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences.

[0122] In some embodiments, the adapter sequence may comprise or consist of the following: GGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the adapter sequence may be encoded by a nucleic acid comprising or consisting of the following: GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 14). In some embodiments, the adapter sequence may comprise or consist of the following: GGGSGGGS (SEQ ID NO: 15).

[0123] Target-binding domain

[0124] In some embodiments of any multi-chain chimeric polypeptide described herein, the first target-binding domain, the second target-binding domain, and / or one or more additional target-binding domains may be antigen-binding domains that specifically bind to a ligand of TGF-βRII (e.g., any exemplary antigen-binding domain described herein or known in the art) or soluble interleukin or cytokine receptors that specifically bind to a ligand of TGF-βRII (e.g., any exemplary soluble interleukin receptor or soluble cytokine receptor described herein).

[0125] In some embodiments of any multi-chain chimeric polypeptide described herein, the first target-binding domain, the second target-binding domain, and / or one or more additional target-binding domains may each independently have the following total amino acid counts: about 5 amino acids to about 1000 amino acids, about 5 amino acids to about 950 amino acids, about 5 amino acids to about 900 amino acids, about 5 amino acids to about 850 amino acids, about 5 amino acids to about 800 amino acids, about 5 amino acids to about 750 amino acids, about 5 amino acids to about 700 amino acids, about 5 amino acids to about 650 amino acids, about 5 amino acids to about 600 amino acids, about 5 amino acids to about 550 amino acids, about 5 amino acids to about 5 00 amino acids, about 5 amino acids to about 450 amino acids, about 5 amino acids to about 400 amino acids, about 5 amino acids to about 350 amino acids, about 5 amino acids to about 300 amino acids, about 5 amino acids to about 280 amino acids, about 5 amino acids to about 260 amino acids, about 5 amino acids to about 240 amino acids, about 5 amino acids to about 220 amino acids, about 5 amino acids to about 200 amino acids, about 5 amino acids to about 195 amino acids, about 5 amino acids to about 190 amino acids, about 5 amino acids to about 185 amino acids, about 5 amino acids to about 180 amino acids, about 5 amino acids to about 175 amino acids, about 5 amino acids to about 170 amino acids. About 5 amino acids to about 165 amino acids, about 5 amino acids to about 160 amino acids, about 5 amino acids to about 155 amino acids, about 5 amino acids to about 150 amino acids, about 5 amino acids to about 145 amino acids, about 5 amino acids to about 140 amino acids, about 5 amino acids to about 135 amino acids, about 5 amino acids to about 130 amino acids, about 5 amino acids to about 125 amino acids, about 5 amino acids to about 120 amino acids, about 5 amino acids to about 115 amino acids, about 5 amino acids to about 110 amino acids, about 5 amino acids to about 105 amino acids, about 5 amino acids to about 100 amino acids, about 5 amino acids to about 95 amino acids, about 5 amino acids to about 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.Approximately 10 amino acids to approximately 1000 amino acids, approximately 10 amino acids to approximately 950 amino acids, approximately 10 amino acids to approximately 900 amino acids, approximately 10 amino acids to approximately 850 amino acids, approximately 10 amino acids to approximately 800 amino acids, approximately 10 amino acids to approximately 750 amino acids, approximately 10 amino acids to approximately 700 amino acids, approximately 10 amino acids to approximately 650 amino acids, approximately 10 amino acids to approximately 600 amino acids, approximately 10 amino acids to approximately 550 amino acids, approximately 10 amino acids to approximately 500 amino acids, approximately 10 amino acids to approximately 450 amino acids, approximately 10 amino acids to approximately 400 amino acids, approximately 10 amino acids to approximately 350 amino acids, approximately 10 amino acids to Approximately 300 amino acids, approximately 10 amino acids to approximately 280 amino acids, approximately 10 amino acids to approximately 260 amino acids, approximately 10 amino acids to approximately 240 amino acids, approximately 10 amino acids to approximately 220 amino acids, approximately 10 amino acids to approximately 200 amino acids, approximately 10 amino acids to approximately 195 amino acids, approximately 10 amino acids to approximately 190 amino acids, approximately 10 amino acids to approximately 185 amino acids, approximately 10 amino acids to approximately 180 amino acids, approximately 10 amino acids to approximately 175 amino acids, approximately 10 amino acids to approximately 170 amino acids, approximately 10 amino acids to approximately 165 amino acids, approximately 10 amino acids to approximately 160 amino acids, approximately 10 amino acids to approximately 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. 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.Approximately 15 amino acids to approximately 900 amino acids, approximately 15 amino acids to approximately 850 amino acids, approximately 15 amino acids to approximately 800 amino acids, approximately 15 amino acids to approximately 750 amino acids, approximately 15 amino acids to approximately 700 amino acids, approximately 15 amino acids to approximately 650 amino acids, approximately 15 amino acids to approximately 600 amino acids, approximately 15 amino acids to approximately 550 amino acids, approximately 15 amino acids to approximately 500 amino acids, approximately 15 amino acids to approximately 450 amino acids, approximately 15 amino acids to approximately 400 amino acids, approximately 15 amino acids to approximately 350 amino acids, approximately 15 amino acids to approximately 300 amino acids, approximately 15 amino acids to approximately 280 amino acids, approximately 15 amino acids to approximately 260 amino acids, about 15 amino acids to about 240 amino acids, about 15 amino acids to about 220 amino acids, about 15 amino acids to about 200 amino acids, about 15 amino acids to about 195 amino acids, about 15 amino acids to about 190 amino acids, about 15 amino acids to about 185 amino acids, about 15 amino acids to about 180 amino acids, about 15 amino acids to about 175 amino acids, about 15 amino acids to about 170 amino acids, about 15 amino acids to about 165 amino acids, about 15 amino acids to about 160 amino acids, about 15 amino acids to about 155 amino acids, about 15 amino acids to about 150 amino acids, about 15 amino acids to about 145 amino acids, about 15 amino acids to about 140 amino acids, about 15 amino acids to about 135 amino acids, about 15 amino acids to about 130 amino acids, about 15 amino acids to about 125 amino acids, about 15 amino acids to about 120 amino acids, about 15 amino acids to about 115 amino acids, about 15 amino acids to about 110 amino acids, about 15 amino acids to about 105 amino acids, about 15 amino acids to about 100 amino acids, about 15 amino acids to about 95 amino acids, about 15 amino acids to about 90 amino acids, about 15 amino acids to about 85 amino acids, about 15 amino acids to about 80 amino acids, about 15 amino acids to about 75 amino acids, about 15 amino acids to about 70 amino acids. Approximately 15 amino acids to approximately 65 amino acids, approximately 15 amino acids to approximately 60 amino acids, approximately 15 amino acids to approximately 55 amino acids, approximately 15 amino acids to approximately 50 amino acids, approximately 15 amino acids to approximately 45 amino acids, approximately 15 amino acids to approximately 40 amino acids, approximately 15 amino acids to approximately 35 amino acids, approximately 15 amino acids to approximately 30 amino acids, approximately 15 amino acids to approximately 25 amino acids, approximately 15 amino acids to approximately 20 amino acids, approximately 20 amino acids to approximately 1000 amino acids, approximately 20 amino acids to approximately 950 amino acids, approximately 20 amino acids to approximately 900 amino acids, approximately 20 amino acids to approximately 850 amino acids, approximately 20 amino acids to approximately 800 amino acids.Approximately 20 amino acids to approximately 750 amino acids, approximately 20 amino acids to approximately 700 amino acids, approximately 20 amino acids to approximately 650 amino acids, approximately 20 amino acids to approximately 600 amino acids, approximately 20 amino acids to approximately 550 amino acids, approximately 20 amino acids to approximately 500 amino acids, approximately 20 amino acids to approximately 450 amino acids, approximately 20 amino acids to approximately 400 amino acids, approximately 20 amino acids to approximately 350 amino acids, approximately 20 amino acids to approximately 300 amino acids, approximately 20 amino acids to approximately 280 amino acids, approximately 20 amino acids to approximately 260 amino acids, approximately 20 amino acids to approximately 240 amino acids, approximately 20 amino acids to approximately 220 amino acids, approximately 20 amino acids to approximately 200 amino acids, about 20 amino acids to about 195 amino acids, about 20 amino acids to about 190 amino acids, about 20 amino acids to about 185 amino acids, about 20 amino acids to about 180 amino acids, about 20 amino acids to about 175 amino acids, about 20 amino acids to about 170 amino acids, about 20 amino acids to about 165 amino acids, about 20 amino acids to about 160 amino acids, about 20 amino acids to about 155 amino acids, about 20 amino acids to about 150 amino acids, about 20 amino acids to about 145 amino acids, about 20 amino acids to about 140 amino acids, about 20 amino acids to about 135 amino acids, about 20 amino acids to about 130 amino acids, about 20 amino acids to about 125 amino acids, about 20 amino acids to about 120 amino acids, about 20 amino acids to about 115 amino acids, about 20 amino acids to about 110 amino acids, about 20 amino acids to about 105 amino acids, about 20 amino acids to about 100 amino acids, about 20 amino acids to about 95 amino acids, about 20 amino acids to about 90 amino acids, about 20 amino acids to about 85 amino acids, about 20 amino acids to about 80 amino acids, about 20 amino acids to about 75 amino acids, about 20 amino acids to about 70 amino acids, about 20 amino acids to about 65 amino acids, about 20 amino acids to about 60 amino acids, about 20 amino acids to about 55 amino acids, about 2 0 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.Approximately 25 amino acids to approximately 550 amino acids, approximately 25 amino acids to approximately 500 amino acids, approximately 25 amino acids to approximately 450 amino acids, approximately 25 amino acids to approximately 400 amino acids, approximately 25 amino acids to approximately 350 amino acids, approximately 25 amino acids to approximately 300 amino acids, approximately 25 amino acids to approximately 280 amino acids, approximately 25 amino acids to approximately 260 amino acids, approximately 25 amino acids to approximately 240 amino acids, approximately 25 amino acids to approximately 220 amino acids, approximately 25 amino acids to approximately 200 amino acids, approximately 25 amino acids to approximately 195 amino acids, approximately 25 amino acids to approximately 190 amino acids, approximately 25 amino acids to approximately 185 amino acids, approximately 25 amino acids to approximately 180 amino acids, about 25 amino acids to about 175 amino acids, about 25 amino acids to about 170 amino acids, about 25 amino acids to about 165 amino acids, about 25 amino acids to about 160 amino acids, about 25 amino acids to about 155 amino acids, about 25 amino acids to about 150 amino acids, about 25 amino acids to about 145 amino acids, about 25 amino acids to about 140 amino acids, about 25 amino acids to about 135 amino acids, about 25 amino acids to about 130 amino acids, about 25 amino acids to about 125 amino acids, about 25 amino acids to about 120 amino acids, about 25 amino acids to about 115 amino acids, about 25 amino acids to about 110 amino acids, about 2 5 amino acids to about 105 amino acids, about 25 amino acids to about 100 amino acids, about 25 amino acids to about 95 amino acids, about 25 amino acids to about 90 amino acids, about 25 amino acids to about 85 amino acids, about 25 amino acids to about 80 amino acids, about 25 amino acids to about 75 amino acids, about 25 amino acids to about 70 amino acids, about 25 amino acids to about 65 amino acids, about 25 amino acids to about 60 amino acids, about 25 amino acids to about 55 amino acids, about 25 amino acids to about 50 amino acids, about 25 amino acids to about 45 amino acids, about 25 amino acids to about 40 amino acids, about 25 amino acids to about 35 amino acids, about 25 amino acids About 30 amino acids, 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.Approximately 30 amino acids to approximately 300 amino acids, approximately 30 amino acids to approximately 280 amino acids, approximately 30 amino acids to approximately 260 amino acids, approximately 30 amino acids to approximately 240 amino acids, approximately 30 amino acids to approximately 220 amino acids, approximately 30 amino acids to approximately 200 amino acids, approximately 30 amino acids to approximately 195 amino acids, approximately 30 amino acids to approximately 190 amino acids, approximately 30 amino acids to approximately 185 amino acids, approximately 30 amino acids to approximately 180 amino acids, approximately 30 amino acids to approximately 175 amino acids, approximately 30 amino acids to approximately 170 amino acids, approximately 30 amino acids to approximately 165 amino acids, approximately 30 amino acids to approximately 160 amino acids, approximately 30 amino acids to approximately 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 The sequence of amino acids is approximately 80 amino acids, approximately 30 amino acids, approximately 75 amino acids, approximately 30 amino acids, approximately 65 amino acids, approximately 30 amino acids, approximately 55 amino acids, approximately 45 amino acids, approximately 30 amino acids, approximately 35 amino acids, approximately 1000 amino acids, approximately 950 amino acids, approximately 900 amino acids, approximately 850 amino acids, approximately 800 amino acids, and approximately 35 amino acids. Approximately 750 amino acids, approximately 35 amino acids to approximately 700 amino acids, approximately 35 amino acids to approximately 650 amino acids, approximately 35 amino acids to approximately 600 amino acids, approximately 35 amino acids to approximately 550 amino acids, approximately 35 amino acids to approximately 500 amino acids, approximately 35 amino acids to approximately 450 amino acids, approximately 35 amino acids to approximately 400 amino acids, approximately 35 amino acids to approximately 350 amino acids, approximately 35 amino acids to approximately 300 amino acids, approximately 35 amino acids to approximately 280 amino acids, approximately 35 amino acids to approximately 260 amino acids, approximately 35 amino acids to approximately 240 amino acids, approximately 35 amino acids to approximately 220 amino acids, approximately 35 amino acids to approximately 200 amino acids.Approximately 35 amino acids to approximately 195 amino acids, approximately 35 amino acids to approximately 190 amino acids, approximately 35 amino acids to approximately 185 amino acids, approximately 35 amino acids to approximately 180 amino acids, approximately 35 amino acids to approximately 175 amino acids, approximately 35 amino acids to approximately 170 amino acids, approximately 35 amino acids to approximately 165 amino acids, approximately 35 amino acids to approximately 160 amino acids, approximately 35 amino acids to approximately 155 amino acids, approximately 35 amino acids to approximately 150 amino acids, approximately 35 amino acids to approximately 145 amino acids, approximately 35 amino acids to approximately 140 amino acids, approximately 35 amino acids to approximately 135 amino acids, approximately 35 amino acids to approximately 130 amino acids, approximately 35 amino acids to approximately 1... 25 amino acids, about 35 amino acids to about 120 amino acids, about 35 amino acids to about 115 amino acids, about 35 amino acids to about 110 amino acids, about 35 amino acids to about 105 amino acids, about 35 amino acids to about 100 amino acids, about 35 amino acids to about 95 amino acids, about 35 amino acids to about 90 amino acids, about 35 amino acids to about 85 amino acids, about 35 amino acids to about 80 amino acids, about 35 amino acids to about 75 amino acids, about 35 amino acids to about 70 amino acids, about 35 amino acids to about 65 amino acids, about 35 amino acids to about 60 amino acids, about 35 amino acids to about 55 amino acids, about 35 amino acids to about 50 amino acids. Amino acids, approximately 35 amino acids to approximately 45 amino acids, approximately 35 amino acids to approximately 40 amino acids, approximately 40 amino acids to approximately 1000 amino acids, approximately 40 amino acids to approximately 950 amino acids, approximately 40 amino acids to approximately 900 amino acids, approximately 40 amino acids to approximately 850 amino acids, approximately 40 amino acids to approximately 800 amino acids, approximately 40 amino acids to approximately 750 amino acids, approximately 40 amino acids to approximately 700 amino acids, approximately 40 amino acids to approximately 650 amino acids, approximately 40 amino acids to approximately 600 amino acids, approximately 40 amino acids to approximately 550 amino acids, approximately 40 amino acids to approximately 500 amino acids, approximately 40 amino acids to approximately 450 amino acids, approximately 40 amino acids Approximately 400 amino acids, approximately 40 amino acids to approximately 350 amino acids, approximately 40 amino acids to approximately 300 amino acids, approximately 40 amino acids to approximately 280 amino acids, approximately 40 amino acids to approximately 260 amino acids, approximately 40 amino acids to approximately 240 amino acids, approximately 40 amino acids to approximately 220 amino acids, approximately 40 amino acids to approximately 200 amino acids, approximately 40 amino acids to approximately 195 amino acids, approximately 40 amino acids to approximately 190 amino acids, approximately 40 amino acids to approximately 185 amino acids, approximately 40 amino acids to approximately 180 amino acids, approximately 40 amino acids to approximately 175 amino acids, approximately 40 amino acids to approximately 170 amino acids, approximately 40 amino acids to approximately 165 amino acids.Approximately 40 amino acids to approximately 160 amino acids, approximately 40 amino acids to approximately 155 amino acids, approximately 40 amino acids to approximately 150 amino acids, approximately 40 amino acids to approximately 145 amino acids, approximately 40 amino acids to approximately 140 amino acids, approximately 40 amino acids to approximately 135 amino acids, approximately 40 amino acids to approximately 130 amino acids, approximately 40 amino acids to approximately 125 amino acids, approximately 40 amino acids to approximately 120 amino acids, approximately 40 amino acids to approximately 115 amino acids, approximately 40 amino acids to approximately 110 amino acids, approximately 40 amino acids to approximately 105 amino acids, approximately 40 amino acids to approximately 100 amino acids, approximately 40 amino acids to approximately 95 amino acids, approximately 40 amino acids to approximately 90 1 amino acid, about 40 amino acids to about 85 amino acids, about 40 amino acids to about 80 amino acids, about 40 amino acids to about 75 amino acids, about 40 amino acids to about 70 amino acids, about 40 amino acids to about 65 amino acids, about 40 amino acids to about 60 amino acids, about 40 amino acids to about 55 amino acids, about 40 amino acids to about 50 amino acids, about 40 amino acids to about 45 amino acids, about 45 amino acids to about 1000 amino acids, about 45 amino acids to about 950 amino acids, about 45 amino acids to about 900 amino acids, about 45 amino acids to about 850 amino acids, about 45 amino acids to about 800 amino acids, about 45 amino acids to about 750 amino acids Amino acids, approximately 45 amino acids to approximately 700 amino acids, approximately 45 amino acids to approximately 650 amino acids, approximately 45 amino acids to approximately 600 amino acids, approximately 45 amino acids to approximately 550 amino acids, approximately 45 amino acids to approximately 500 amino acids, approximately 45 amino acids to approximately 450 amino acids, approximately 45 amino acids to approximately 400 amino acids, approximately 45 amino acids to approximately 350 amino acids, approximately 45 amino acids to approximately 300 amino acids, approximately 45 amino acids to approximately 280 amino acids, approximately 45 amino acids to approximately 260 amino acids, approximately 45 amino acids to approximately 240 amino acids, approximately 45 amino acids to approximately 220 amino acids, approximately 45 amino acids to approximately 200 amino acids, approximately 45 amino groups Acid to about 195 amino acids, about 45 amino acids to about 190 amino acids, about 45 amino acids to about 185 amino acids, about 45 amino acids to about 180 amino acids, about 45 amino acids to about 175 amino acids, about 45 amino acids to about 170 amino acids, about 45 amino acids to about 165 amino acids, about 45 amino acids to about 160 amino acids, about 45 amino acids to about 155 amino acids, about 45 amino acids to about 150 amino acids, about 45 amino acids to about 145 amino acids, about 45 amino acids to about 140 amino acids, about 45 amino acids to about 135 amino acids, about 45 amino acids to about 130 amino acids, about 45 amino acids to about 125 amino acids.Approximately 45 amino acids to approximately 120 amino acids, approximately 45 amino acids to approximately 115 amino acids, approximately 45 amino acids to approximately 110 amino acids, approximately 45 amino acids to approximately 105 amino acids, approximately 45 amino acids to approximately 100 amino acids, approximately 45 amino acids to approximately 95 amino acids, approximately 45 amino acids to approximately 90 amino acids, approximately 45 amino acids to approximately 85 amino acids, approximately 45 amino acids to approximately 80 amino acids, approximately 45 amino acids to approximately 75 amino acids, approximately 45 amino acids to approximately 70 amino acids, approximately 45 amino acids to approximately 65 amino acids, approximately 45 amino acids to approximately 60 amino acids, approximately 45 amino acids to approximately 55 amino acids, approximately 45 amino acids to approximately 50 amino acids, approximately 5 0 amino acids to about 1000 amino acids, about 50 amino acids to about 950 amino acids, about 50 amino acids to about 900 amino acids, about 50 amino acids to about 850 amino acids, about 50 amino acids to about 800 amino acids, about 50 amino acids to about 750 amino acids, about 50 amino acids to about 700 amino acids, about 50 amino acids to about 650 amino acids, about 50 amino acids to about 600 amino acids, about 50 amino acids to about 550 amino acids, about 50 amino acids to about 500 amino acids, about 50 amino acids to about 450 amino acids, about 50 amino acids to about 400 amino acids, about 50 amino acids to about 350 amino acids, about 50 amino acids to about 30 0 amino acids, about 50 amino acids to about 280 amino acids, about 50 amino acids to about 260 amino acids, about 50 amino acids to about 240 amino acids, about 50 amino acids to about 220 amino acids, about 50 amino acids to about 200 amino acids, about 50 amino acids to about 195 amino acids, about 50 amino acids to about 190 amino acids, about 50 amino acids to about 185 amino acids, about 50 amino acids to about 180 amino acids, about 50 amino acids to about 175 amino acids, about 50 amino acids to about 170 amino acids, about 50 amino acids to about 165 amino acids, about 50 amino acids to about 160 amino acids, about 50 amino acids to about 155 amino acids, about 50 The sequence of amino acids is: approximately 150 amino acids, approximately 50 amino acids, approximately 145 amino acids, approximately 50 amino acids, approximately 135 amino acids, approximately 130 amino acids, approximately 125 amino acids, approximately 120 amino acids, approximately 115 amino acids, approximately 110 amino acids, approximately 105 amino acids, approximately 100 amino acids, approximately 95 amino acids, approximately 85 amino acids, approximately 80 amino acids.Approximately 50 amino acids to approximately 75 amino acids, approximately 50 amino acids to approximately 70 amino acids, approximately 50 amino acids to approximately 65 amino acids, approximately 50 amino acids to approximately 60 amino acids, approximately 50 amino acids to approximately 55 amino acids, approximately 55 amino acids to approximately 1000 amino acids, approximately 55 amino acids to approximately 950 amino acids, approximately 55 amino acids to approximately 900 amino acids, approximately 55 amino acids to approximately 850 amino acids, approximately 55 amino acids to approximately 800 amino acids, approximately 55 amino acids to approximately 750 amino acids, approximately 55 amino acids to approximately 700 amino acids, approximately 55 amino acids to approximately 650 amino acids, approximately 55 amino acids to approximately 600 amino acids, approximately 55 amino acids to approximately 550 amino acids. Amino acids, approximately 55 amino acids to approximately 500 amino acids, approximately 55 amino acids to approximately 450 amino acids, approximately 55 amino acids to approximately 400 amino acids, approximately 55 amino acids to approximately 350 amino acids, approximately 55 amino acids to approximately 300 amino acids, approximately 55 amino acids to approximately 280 amino acids, approximately 55 amino acids to approximately 260 amino acids, approximately 55 amino acids to approximately 240 amino acids, approximately 55 amino acids to approximately 220 amino acids, approximately 55 amino acids to approximately 200 amino acids, approximately 55 amino acids to approximately 195 amino acids, approximately 55 amino acids to approximately 190 amino acids, approximately 55 amino acids to approximately 185 amino acids, approximately 55 amino acids to approximately 180 amino acids, approximately 55 amino acids About 175 amino acids, about 55 amino acids to about 170 amino acids, about 55 amino acids to about 165 amino acids, about 55 amino acids to about 160 amino acids, about 55 amino acids to about 155 amino acids, about 55 amino acids to about 150 amino acids, about 55 amino acids to about 145 amino acids, about 55 amino acids to about 140 amino acids, about 55 amino acids to about 135 amino acids, about 55 amino acids to about 130 amino acids, about 55 amino acids to about 125 amino acids, about 55 amino acids to about 120 amino acids, about 55 amino acids to about 115 amino acids, about 55 amino acids to about 110 amino acids, about 55 amino acids to about 105 amino acids. Approximately 55 amino acids to approximately 100 amino acids, approximately 55 amino acids to approximately 95 amino acids, approximately 55 amino acids to approximately 90 amino acids, approximately 55 amino acids to approximately 85 amino acids, approximately 55 amino acids to approximately 80 amino acids, approximately 55 amino acids to approximately 75 amino acids, approximately 55 amino acids to approximately 70 amino acids, approximately 55 amino acids to approximately 65 amino acids, approximately 55 amino acids to approximately 60 amino acids, approximately 60 amino acids to approximately 1000 amino acids, approximately 60 amino acids to approximately 950 amino acids, approximately 60 amino acids to approximately 900 amino acids, approximately 60 amino acids to approximately 850 amino acids, approximately 60 amino acids to approximately 800 amino acids, approximately 60 amino acids to approximately 750 amino acids.Approximately 60 amino acids to approximately 700 amino acids, approximately 60 amino acids to approximately 650 amino acids, approximately 60 amino acids to approximately 600 amino acids, approximately 60 amino acids to approximately 550 amino acids, approximately 60 amino acids to approximately 500 amino acids, approximately 60 amino acids to approximately 450 amino acids, approximately 60 amino acids to approximately 400 amino acids, approximately 60 amino acids to approximately 350 amino acids, approximately 60 amino acids to approximately 300 amino acids, approximately 60 amino acids to approximately 280 amino acids, approximately 60 amino acids to approximately 260 amino acids, approximately 60 amino acids to approximately 240 amino acids, approximately 60 amino acids to approximately 220 amino acids, approximately 60 amino acids to approximately 200 amino acids, approximately 60 amino acids to approximately 19 5 amino acids, about 60 amino acids to about 190 amino acids, about 60 amino acids to about 185 amino acids, about 60 amino acids to about 180 amino acids, about 60 amino acids to about 175 amino acids, about 60 amino acids to about 170 amino acids, about 60 amino acids to about 165 amino acids, about 60 amino acids to about 160 amino acids, about 60 amino acids to about 155 amino acids, about 60 amino acids to about 150 amino acids, about 60 amino acids to about 145 amino acids, about 60 amino acids to about 140 amino acids, about 60 amino acids to about 135 amino acids, about 60 amino acids to about 130 amino acids, about 60 amino acids to about 125 amino acids, about 60 amino acids 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 The sequence of amino acids is approximately 850 amino acids, approximately 65 amino acids, approximately 750 amino acids, approximately 700 amino acids, approximately 65 amino acids, approximately 65 amino acids, approximately 600 amino acids, approximately 550 amino acids, approximately 450 amino acids, approximately 400 amino acids, approximately 350 amino acids, approximately 300 amino acids, approximately 280 amino acids, approximately 260 amino acids, and approximately 240 amino acids.Approximately 65 amino acids to approximately 220 amino acids, approximately 65 amino acids to approximately 200 amino acids, approximately 65 amino acids to approximately 195 amino acids, approximately 65 amino acids to approximately 190 amino acids, approximately 65 amino acids to approximately 185 amino acids, approximately 65 amino acids to approximately 180 amino acids, approximately 65 amino acids to approximately 175 amino acids, approximately 65 amino acids to approximately 170 amino acids, approximately 65 amino acids to approximately 165 amino acids, approximately 65 amino acids to approximately 160 amino acids, approximately 65 amino acids to approximately 155 amino acids, approximately 65 amino acids to approximately 150 amino acids, approximately 65 amino acids to approximately 145 amino acids, approximately 65 amino acids to approximately 140 amino acids, approximately 65 amino acids to approximately 13... 5 amino acids, about 65 amino acids to about 130 amino acids, about 65 amino acids to about 125 amino acids, about 65 amino acids to about 120 amino acids, about 65 amino acids to about 115 amino acids, about 65 amino acids to about 110 amino acids, about 65 amino acids to about 105 amino acids, about 65 amino acids to about 100 amino acids, about 65 amino acids to about 95 amino acids, about 65 amino acids to about 90 amino acids, about 65 amino acids to about 85 amino acids, about 65 amino acids to about 80 amino acids, about 65 amino acids to about 75 amino acids, about 65 amino acids to about 70 amino acids, about 70 amino acids to about 1000 amino acids, about 70 amino acids to about 95 amino acids. 0 amino acids, approximately 70 amino acids to approximately 900 amino acids, approximately 70 amino acids to approximately 850 amino acids, approximately 70 amino acids to approximately 800 amino acids, approximately 70 amino acids to approximately 750 amino acids, approximately 70 amino acids to approximately 700 amino acids, approximately 70 amino acids to approximately 650 amino acids, approximately 70 amino acids to approximately 600 amino acids, approximately 70 amino acids to approximately 550 amino acids, approximately 70 amino acids to approximately 500 amino acids, approximately 70 amino acids to approximately 450 amino acids, approximately 70 amino acids to approximately 400 amino acids, approximately 70 amino acids to approximately 350 amino acids, approximately 70 amino acids to approximately 300 amino acids, approximately 70 amino acids to approximately 280 amino acids, approximately 70 amino acids The amino acids are approximately 260 amino acids, approximately 70 amino acids are approximately 240 amino acids, approximately 70 amino acids are approximately 220 amino acids, approximately 70 amino acids are approximately 200 amino acids, approximately 70 amino acids are approximately 195 amino acids, approximately 70 amino acids are approximately 190 amino acids, approximately 70 amino acids are approximately 185 amino acids, approximately 70 amino acids are approximately 180 amino acids, approximately 70 amino acids are approximately 175 amino acids, approximately 70 amino acids are approximately 170 amino acids, approximately 70 amino acids are approximately 165 amino acids, approximately 70 amino acids are approximately 160 amino acids, approximately 70 amino acids are approximately 155 amino acids, approximately 70 amino acids are approximately 150 amino acids, and approximately 70 amino acids are approximately 145 amino acids.Approximately 70 amino acids to approximately 140 amino acids, approximately 70 amino acids to approximately 135 amino acids, approximately 70 amino acids to approximately 130 amino acids, approximately 70 amino acids to approximately 125 amino acids, approximately 70 amino acids to approximately 120 amino acids, approximately 70 amino acids to approximately 115 amino acids, approximately 70 amino acids to approximately 110 amino acids, approximately 70 amino acids to approximately 105 amino acids, approximately 70 amino acids to approximately 100 amino acids, approximately 70 amino acids to approximately 95 amino acids, approximately 70 amino acids to approximately 90 amino acids, approximately 70 amino acids to approximately 85 amino acids, approximately 70 amino acids to approximately 80 amino acids, approximately 70 amino acids to approximately 75 amino acids, approximately 75 amino acids to approximately 1000 amino acids. Acid, approximately 75 amino acids to approximately 950 amino acids, approximately 75 amino acids to approximately 900 amino acids, approximately 75 amino acids to approximately 850 amino acids, approximately 75 amino acids to approximately 800 amino acids, approximately 75 amino acids to approximately 750 amino acids, approximately 75 amino acids to approximately 700 amino acids, approximately 75 amino acids to approximately 650 amino acids, approximately 75 amino acids to approximately 600 amino acids, approximately 75 amino acids to approximately 550 amino acids, approximately 75 amino acids to approximately 500 amino acids, approximately 75 amino acids to approximately 450 amino acids, approximately 75 amino acids to approximately 400 amino acids, approximately 75 amino acids to approximately 350 amino acids, approximately 75 amino acids to approximately 300 amino acids, approximately 75 amino acids to approximately 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 Approximately 75 amino acids to about 145 amino acids, approximately 75 amino acids to about 140 amino acids, approximately 75 amino acids to about 135 amino acids, approximately 75 amino acids to about 130 amino acids, approximately 75 amino acids to about 125 amino acids, approximately 75 amino acids to about 120 amino acids, approximately 75 amino acids to about 115 amino acids, approximately 75 amino acids to about 110 amino acids, approximately 75 amino acids to about 105 amino acids, approximately 75 amino acids to about 100 amino acids, approximately 75 amino acids to about 95 amino acids, approximately 75 amino acids to about 90 amino acids, approximately 75 amino acids to about 85 amino acids, approximately 75 amino acids to about 80 amino acids, approximately 80 amino acids to about 1000 amino acids.Approximately 80 amino acids to approximately 950 amino acids, approximately 80 amino acids to approximately 900 amino acids, approximately 80 amino acids to approximately 850 amino acids, approximately 80 amino acids to approximately 800 amino acids, approximately 80 amino acids to approximately 750 amino acids, approximately 80 amino acids to approximately 700 amino acids, approximately 80 amino acids to approximately 650 amino acids, approximately 80 amino acids to approximately 600 amino acids, approximately 80 amino acids to approximately 550 amino acids, approximately 80 amino acids to approximately 500 amino acids, approximately 80 amino acids to approximately 450 amino acids, approximately 80 amino acids to approximately 400 amino acids, approximately 80 amino acids to approximately 350 amino acids, approximately 80 amino acids to approximately 300 amino acids, approximately 80 amino acids to approximately 280 amino acids. 1 amino acid, 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 About 145 amino acids, about 80 amino acids to about 140 amino acids, about 80 amino acids to about 135 amino acids, about 80 amino acids to about 130 amino acids, about 80 amino acids to about 125 amino acids, about 80 amino acids to about 120 amino acids, about 80 amino acids to about 115 amino acids, about 80 amino acids to about 110 amino acids, about 80 amino acids to about 105 amino acids, about 80 amino acids to about 100 amino acids, about 80 amino acids to about 95 amino acids, about 80 amino acids to about 90 amino acids, about 80 amino acids to about 85 amino acids, about 85 amino acids to about 1000 amino acids, about 85 amino acids to about 950 amino acids, about 85 Amino acids to about 900 amino acids, about 85 amino acids to about 850 amino acids, about 85 amino acids to about 800 amino acids, about 85 amino acids to about 750 amino acids, about 85 amino acids to about 700 amino acids, about 85 amino acids to about 650 amino acids, about 85 amino acids to about 600 amino acids, about 85 amino acids to about 550 amino acids, about 85 amino acids to about 500 amino acids, about 85 amino acids to about 450 amino acids, about 85 amino acids to about 400 amino acids, about 85 amino acids to about 350 amino acids, about 85 amino acids to about 300 amino acids, about 85 amino acids to about 280 amino acids, about 85 amino acids to about 260 amino acids.Approximately 85 amino acids to approximately 240 amino acids, approximately 85 amino acids to approximately 220 amino acids, approximately 85 amino acids to approximately 200 amino acids, approximately 85 amino acids to approximately 195 amino acids, approximately 85 amino acids to approximately 190 amino acids, approximately 85 amino acids to approximately 185 amino acids, approximately 85 amino acids to approximately 180 amino acids, approximately 85 amino acids to approximately 175 amino acids, approximately 85 amino acids to approximately 170 amino acids, approximately 85 amino acids to approximately 165 amino acids, approximately 85 amino acids to approximately 160 amino acids, approximately 85 amino acids to approximately 155 amino acids, approximately 85 amino acids to approximately 150 amino acids, approximately 85 amino acids to approximately 145 amino acids, approximately 85 amino acids to Approximately 140 amino acids, approximately 85 amino acids to approximately 135 amino acids, approximately 85 amino acids to approximately 130 amino acids, approximately 85 amino acids to approximately 125 amino acids, approximately 85 amino acids to approximately 120 amino acids, approximately 85 amino acids to approximately 115 amino acids, approximately 85 amino acids to approximately 110 amino acids, approximately 85 amino acids to approximately 105 amino acids, approximately 85 amino acids to approximately 100 amino acids, approximately 85 amino acids to approximately 95 amino acids, approximately 85 amino acids to approximately 90 amino acids, approximately 90 amino acids to approximately 1000 amino acids, approximately 90 amino acids to approximately 950 amino acids, approximately 90 amino acids to approximately 900 amino acids, approximately 90 amino acids to approximately 850 amino acids. Approximately 90 amino acids to approximately 800 amino acids, approximately 90 amino acids to approximately 750 amino acids, approximately 90 amino acids to approximately 700 amino acids, approximately 90 amino acids to approximately 650 amino acids, approximately 90 amino acids to approximately 600 amino acids, approximately 90 amino acids to approximately 550 amino acids, approximately 90 amino acids to approximately 500 amino acids, approximately 90 amino acids to approximately 450 amino acids, approximately 90 amino acids to approximately 400 amino acids, approximately 90 amino acids to approximately 350 amino acids, approximately 90 amino acids to approximately 300 amino acids, approximately 90 amino acids to approximately 280 amino acids, approximately 90 amino acids to approximately 260 amino acids, approximately 90 amino acids to approximately 240 amino acids, approximately 90 amino acids to Approximately 220 amino acids, approximately 90 amino acids to approximately 200 amino acids, approximately 90 amino acids to approximately 195 amino acids, approximately 90 amino acids to approximately 190 amino acids, approximately 90 amino acids to approximately 185 amino acids, approximately 90 amino acids to approximately 180 amino acids, approximately 90 amino acids to approximately 175 amino acids, approximately 90 amino acids to approximately 170 amino acids, approximately 90 amino acids to approximately 165 amino acids, approximately 90 amino acids to approximately 160 amino acids, approximately 90 amino acids to approximately 155 amino acids, approximately 90 amino acids to approximately 150 amino acids, approximately 90 amino acids to approximately 145 amino acids, approximately 90 amino acids to approximately 140 amino acids, approximately 90 amino acids to approximately 135 amino acids.Approximately 90 amino acids to approximately 130 amino acids, approximately 90 amino acids to approximately 125 amino acids, approximately 90 amino acids to approximately 120 amino acids, approximately 90 amino acids to approximately 115 amino acids, approximately 90 amino acids to approximately 110 amino acids, approximately 90 amino acids to approximately 105 amino acids, approximately 90 amino acids to approximately 100 amino acids, approximately 90 amino acids to approximately 95 amino acids, approximately 95 amino acids to approximately 1000 amino acids, approximately 95 amino acids to approximately 950 amino acids, approximately 95 amino acids to approximately 900 amino acids, approximately 95 amino acids to approximately 850 amino acids, approximately 95 amino acids to approximately 800 amino acids, approximately 95 amino acids to approximately 750 amino acids, approximately 95 amino acids to approximately 70 0 amino acids, approximately 95 amino acids to approximately 650 amino acids, approximately 95 amino acids to approximately 600 amino acids, approximately 95 amino acids to approximately 550 amino acids, approximately 95 amino acids to approximately 500 amino acids, approximately 95 amino acids to approximately 450 amino acids, approximately 95 amino acids to approximately 400 amino acids, approximately 95 amino acids to approximately 350 amino acids, approximately 95 amino acids to approximately 300 amino acids, approximately 95 amino acids to approximately 280 amino acids, approximately 95 amino acids to approximately 260 amino acids, approximately 95 amino acids to approximately 240 amino acids, approximately 95 amino acids to approximately 220 amino acids, approximately 95 amino acids to approximately 200 amino acids, approximately 95 amino acids to approximately 195 amino acids, approximately 95 amino acids The amino acids are approximately 190 amino acids, approximately 95 amino acids are approximately 185 amino acids, approximately 95 amino acids are approximately 180 amino acids, approximately 95 amino acids are approximately 175 amino acids, approximately 95 amino acids are approximately 170 amino acids, approximately 95 amino acids are approximately 165 amino acids, approximately 95 amino acids are approximately 160 amino acids, approximately 95 amino acids are approximately 155 amino acids, approximately 95 amino acids are approximately 150 amino acids, approximately 95 amino acids are approximately 145 amino acids, approximately 95 amino acids are approximately 140 amino acids, approximately 95 amino acids are approximately 135 amino acids, approximately 95 amino acids are approximately 130 amino acids, approximately 95 amino acids are approximately 125 amino acids, approximately 95 amino acids are approximately 120 amino acids. Approximately 95 amino acids to approximately 115 amino acids, approximately 95 amino acids to approximately 110 amino acids, approximately 95 amino acids to approximately 105 amino acids, approximately 95 amino acids to approximately 100 amino acids, approximately 100 amino acids to approximately 1000 amino acids, approximately 100 amino acids to approximately 950 amino acids, approximately 100 amino acids to approximately 900 amino acids, approximately 100 amino acids to approximately 850 amino acids, approximately 100 amino acids to approximately 800 amino acids, approximately 100 amino acids to approximately 750 amino acids, approximately 100 amino acids to approximately 700 amino acids, approximately 100 amino acids to approximately 650 amino acids, approximately 100 amino acids to approximately 600 amino acids, approximately 100 amino acids to approximately 550 amino acids.Approximately 100 amino acids to approximately 500 amino acids, approximately 100 amino acids to approximately 450 amino acids, approximately 100 amino acids to approximately 400 amino acids, approximately 100 amino acids to approximately 350 amino acids, approximately 100 amino acids to approximately 300 amino acids, approximately 100 amino acids to approximately 280 amino acids, approximately 100 amino acids to approximately 260 amino acids, approximately 100 amino acids to approximately 240 amino acids, approximately 100 amino acids to approximately 220 amino acids, approximately 100 amino acids to approximately 200 amino acids, approximately 100 amino acids to approximately 195 amino acids, approximately 100 amino acids to approximately 190 amino acids, approximately 100 amino acids to approximately 185 amino acids, approximately 100 amino acids to approximately 180 100 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 Approximately 110 amino acids, approximately 100 amino acids to approximately 105 amino acids, approximately 105 amino acids to approximately 1000 amino acids, approximately 105 amino acids to approximately 950 amino acids, approximately 105 amino acids to approximately 900 amino acids, approximately 105 amino acids to approximately 850 amino acids, approximately 105 amino acids to approximately 800 amino acids, approximately 105 amino acids to approximately 750 amino acids, approximately 105 amino acids to approximately 700 amino acids, approximately 105 amino acids to approximately 650 amino acids, approximately 105 amino acids to approximately 600 amino acids, approximately 105 amino acids to approximately 550 amino acids, approximately 105 amino acids to approximately 500 amino acids, approximately 105 amino acids to approximately 450 amino acids, approximately 105 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their components. A direct translation wouldn't be meaningful without further context.Approximately 105 amino acids to approximately 165 amino acids, approximately 105 amino acids to approximately 160 amino acids, approximately 105 amino acids to approximately 155 amino acids, approximately 105 amino acids to approximately 150 amino acids, approximately 105 amino acids to approximately 145 amino acids, approximately 105 amino acids to approximately 140 amino acids, approximately 105 amino acids to approximately 135 amino acids, approximately 105 amino acids to approximately 130 amino acids, approximately 105 amino acids to approximately 125 amino acids, approximately 105 amino acids to approximately 120 amino acids, approximately 105 amino acids to approximately 115 amino acids, approximately 105 amino acids to approximately 110 amino acids, approximately 110 amino acids to approximately 1000 amino acids, approximately 110 amino acids to approximately 950 amino acids. 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 Approximately 280 amino acids, approximately 110 amino acids to approximately 260 amino acids, approximately 110 amino acids to approximately 240 amino acids, approximately 110 amino acids to approximately 220 amino acids, approximately 110 amino acids to approximately 200 amino acids, approximately 110 amino acids to approximately 195 amino acids, approximately 110 amino acids to approximately 190 amino acids, approximately 110 amino acids to approximately 185 amino acids, approximately 110 amino acids to approximately 180 amino acids, approximately 110 amino acids to approximately 175 amino acids, approximately 110 amino acids to approximately 170 amino acids, approximately 110 amino acids to approximately 165 amino acids, approximately 110 amino acids to approximately 160 amino acids, approximately 110 amino acids to approximately 155 amino acids, approximately 110 Amino acids to about 150 amino acids, about 110 amino acids to about 145 amino acids, about 110 amino acids to about 140 amino acids, about 110 amino acids to about 135 amino acids, about 110 amino acids to about 130 amino acids, about 110 amino acids to about 125 amino acids, about 110 amino acids to about 120 amino acids, about 110 amino acids to about 115 amino acids, about 115 amino acids to about 1000 amino acids, about 115 amino acids to about 950 amino acids, about 115 amino acids to about 900 amino acids, about 115 amino acids to about 850 amino acids, about 115 amino acids to about 800 amino acids, about 115 amino acids to about 750 amino acids.Approximately 115 amino acids to approximately 700 amino acids, approximately 115 amino acids to approximately 650 amino acids, approximately 115 amino acids to approximately 600 amino acids, approximately 115 amino acids to approximately 550 amino acids, approximately 115 amino acids to approximately 500 amino acids, approximately 115 amino acids to approximately 450 amino acids, approximately 115 amino acids to approximately 400 amino acids, approximately 115 amino acids to approximately 350 amino acids, approximately 115 amino acids to approximately 300 amino acids, approximately 115 amino acids to approximately 280 amino acids, approximately 115 amino acids to approximately 260 amino acids, approximately 115 amino acids to approximately 240 amino acids, approximately 115 amino acids to approximately 220 amino acids, approximately 115 amino acids to approximately 200 amino acids. 115 amino acids to about 195 amino acids, about 115 amino acids to about 190 amino acids, about 115 amino acids to about 185 amino acids, about 115 amino acids to about 180 amino acids, about 115 amino acids to about 175 amino acids, about 115 amino acids to about 170 amino acids, about 115 amino acids to about 165 amino acids, about 115 amino acids to about 160 amino acids, about 115 amino acids to about 155 amino acids, about 115 amino acids to about 150 amino acids, about 115 amino acids to about 145 amino acids, about 115 amino acids to about 140 amino acids, about 115 amino acids to about 135 amino acids, about 115 amino acids to Approximately 130 amino acids, approximately 115 amino acids to approximately 125 amino acids, approximately 115 amino acids to approximately 120 amino acids, approximately 120 amino acids to approximately 1000 amino acids, approximately 120 amino acids to approximately 950 amino acids, approximately 120 amino acids to approximately 900 amino acids, approximately 120 amino acids to approximately 850 amino acids, approximately 120 amino acids to approximately 800 amino acids, approximately 120 amino acids to approximately 750 amino acids, approximately 120 amino acids to approximately 700 amino acids, approximately 120 amino acids to approximately 650 amino acids, approximately 120 amino acids to approximately 600 amino acids, approximately 120 amino acids to approximately 550 amino acids, approximately 120 amino acids to approximately 500 amino acids, approximately 120 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their composition. A direct translation wouldn't be meaningful without further context.Approximately 120 amino acids to approximately 170 amino acids, approximately 120 amino acids to approximately 165 amino acids, approximately 120 amino acids to approximately 160 amino acids, approximately 120 amino acids to approximately 155 amino acids, approximately 120 amino acids to approximately 150 amino acids, approximately 120 amino acids to approximately 145 amino acids, approximately 120 amino acids to approximately 140 amino acids, approximately 120 amino acids to approximately 135 amino acids, approximately 120 amino acids to approximately 130 amino acids, approximately 120 amino acids to approximately 125 amino acids, approximately 125 amino acids to approximately 1000 amino acids, approximately 125 amino acids to approximately 950 amino acids, approximately 125 amino acids to approximately 900 amino acids, approximately 125 amino acids to approximately 850 amino acids. 125 amino acids to about 800 amino acids, about 125 amino acids to about 750 amino acids, about 125 amino acids to about 700 amino acids, about 125 amino acids to about 650 amino acids, about 125 amino acids to about 600 amino acids, about 125 amino acids to about 550 amino acids, about 125 amino acids to about 500 amino acids, about 125 amino acids to about 450 amino acids, about 125 amino acids to about 400 amino acids, about 125 amino acids to about 350 amino acids, about 125 amino acids to about 300 amino acids, about 125 amino acids to about 280 amino acids, about 125 amino acids to about 260 amino acids, about 125 amino acids to Approximately 240 amino acids, approximately 125 amino acids to approximately 220 amino acids, approximately 125 amino acids to approximately 200 amino acids, approximately 125 amino acids to approximately 195 amino acids, approximately 125 amino acids to approximately 190 amino acids, approximately 125 amino acids to approximately 185 amino acids, approximately 125 amino acids to approximately 180 amino acids, approximately 125 amino acids to approximately 175 amino acids, approximately 125 amino acids to approximately 170 amino acids, approximately 125 amino acids to approximately 165 amino acids, approximately 125 amino acids to approximately 160 amino acids, approximately 125 amino acids to approximately 155 amino acids, approximately 125 amino acids to approximately 150 amino acids, approximately 125 amino acids to approximately 145 amino acids, approximately 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.Approximately 130 amino acids to approximately 450 amino acids, approximately 130 amino acids to approximately 400 amino acids, approximately 130 amino acids to approximately 350 amino acids, approximately 130 amino acids to approximately 300 amino acids, approximately 130 amino acids to approximately 280 amino acids, approximately 130 amino acids to approximately 260 amino acids, approximately 130 amino acids to approximately 240 amino acids, approximately 130 amino acids to approximately 220 amino acids, approximately 130 amino acids to approximately 200 amino acids, approximately 130 amino acids to approximately 195 amino acids, approximately 130 amino acids to approximately 190 amino acids, approximately 130 amino acids to approximately 185 amino acids, approximately 130 amino acids to approximately 180 amino acids, approximately 130 amino acids to approximately 175 amino acids. Amino acids, approximately 130 amino acids to approximately 170 amino acids, approximately 130 amino acids to approximately 165 amino acids, approximately 130 amino acids to approximately 160 amino acids, approximately 130 amino acids to approximately 155 amino acids, approximately 130 amino acids to approximately 150 amino acids, approximately 130 amino acids to approximately 145 amino acids, approximately 130 amino acids to approximately 140 amino acids, approximately 130 amino acids to approximately 135 amino acids, approximately 135 amino acids to approximately 1000 amino acids, approximately 135 amino acids to approximately 950 amino acids, approximately 135 amino acids to approximately 900 amino acids, approximately 135 amino acids to approximately 850 amino acids, approximately 135 amino acids to approximately 800 amino acids, approximately 135 amino acids to Approximately 750 amino acids, approximately 135 amino acids to approximately 700 amino acids, approximately 135 amino acids to approximately 650 amino acids, approximately 135 amino acids to approximately 600 amino acids, approximately 135 amino acids to approximately 550 amino acids, approximately 135 amino acids to approximately 500 amino acids, approximately 135 amino acids to approximately 450 amino acids, approximately 135 amino acids to approximately 400 amino acids, approximately 135 amino acids to approximately 350 amino acids, approximately 135 amino acids to approximately 300 amino acids, approximately 135 amino acids to approximately 280 amino acids, approximately 135 amino acids to approximately 260 amino acids, approximately 135 amino acids to approximately 240 amino acids, approximately 135 amino acids to approximately 220 amino acids, approximately 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.Approximately 140 amino acids to approximately 950 amino acids, approximately 140 amino acids to approximately 900 amino acids, approximately 140 amino acids to approximately 850 amino acids, approximately 140 amino acids to approximately 800 amino acids, approximately 140 amino acids to approximately 750 amino acids, approximately 140 amino acids to approximately 700 amino acids, approximately 140 amino acids to approximately 650 amino acids, approximately 140 amino acids to approximately 600 amino acids, approximately 140 amino acids to approximately 550 amino acids, approximately 140 amino acids to approximately 500 amino acids, approximately 140 amino acids to approximately 450 amino acids, approximately 140 amino acids to approximately 400 amino acids, approximately 140 amino acids to approximately 350 amino acids, approximately 140 amino acids to approximately 300 amino acids. 140 amino acids to about 280 amino acids, about 140 amino acids to about 260 amino acids, about 140 amino acids to about 240 amino acids, about 140 amino acids to about 220 amino acids, about 140 amino acids to about 200 amino acids, about 140 amino acids to about 195 amino acids, about 140 amino acids to about 190 amino acids, about 140 amino acids to about 185 amino acids, about 140 amino acids to about 180 amino acids, about 140 amino acids to about 175 amino acids, about 140 amino acids to about 170 amino acids, about 140 amino acids to about 165 amino acids, about 140 amino acids to about 160 amino acids, about 140 amino acids to Approximately 155 amino acids, approximately 140 amino acids to approximately 150 amino acids, approximately 140 amino acids to approximately 145 amino acids, approximately 145 amino acids to approximately 1000 amino acids, approximately 145 amino acids to approximately 950 amino acids, approximately 145 amino acids to approximately 900 amino acids, approximately 145 amino acids to approximately 850 amino acids, approximately 145 amino acids to approximately 800 amino acids, approximately 145 amino acids to approximately 750 amino acids, approximately 145 amino acids to approximately 700 amino acids, approximately 145 amino acids to approximately 650 amino acids, approximately 145 amino acids to approximately 600 amino acids, approximately 145 amino acids to approximately 550 amino acids, approximately 145 amino acids to approximately 500 amino acids, approximately 145 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their composition. A direct translation wouldn't be meaningful without further context.Approximately 145 amino acids to approximately 170 amino acids, approximately 145 amino acids to approximately 165 amino acids, approximately 145 amino acids to approximately 160 amino acids, approximately 145 amino acids to approximately 155 amino acids, approximately 145 amino acids to approximately 150 amino acids, approximately 150 amino acids to approximately 1000 amino acids, approximately 150 amino acids to approximately 950 amino acids, approximately 150 amino acids to approximately 900 amino acids, approximately 150 amino acids to approximately 850 amino acids, approximately 150 amino acids to approximately 800 amino acids, approximately 150 amino acids to approximately 750 amino acids, approximately 150 amino acids to approximately 700 amino acids, approximately 150 amino acids to approximately 650 amino acids, approximately 150 amino acids to approximately 600 amino acids. 150 amino acids to about 550 amino acids, about 150 amino acids to about 500 amino acids, about 150 amino acids to about 450 amino acids, about 150 amino acids to about 400 amino acids, about 150 amino acids to about 350 amino acids, about 150 amino acids to about 300 amino acids, about 150 amino acids to about 280 amino acids, about 150 amino acids to about 260 amino acids, about 150 amino acids to about 240 amino acids, about 150 amino acids to about 220 amino acids, about 150 amino acids to about 200 amino acids, about 150 amino acids to about 195 amino acids, about 150 amino acids to about 190 amino acids, about 150 amino acids to Approximately 185 amino acids, approximately 150 amino acids to approximately 180 amino acids, approximately 150 amino acids to approximately 175 amino acids, approximately 150 amino acids to approximately 170 amino acids, approximately 150 amino acids to approximately 165 amino acids, approximately 150 amino acids to approximately 160 amino acids, approximately 150 amino acids to approximately 155 amino acids, approximately 155 amino acids to approximately 1000 amino acids, approximately 155 amino acids to approximately 950 amino acids, approximately 155 amino acids to approximately 900 amino acids, approximately 155 amino acids to approximately 850 amino acids, approximately 155 amino acids to approximately 800 amino acids, approximately 155 amino acids to approximately 750 amino acids, approximately 155 amino acids to approximately 700 amino acids, approximately 155 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their composition. A direct translation wouldn't be meaningful without further context.Approximately 155 amino acids to approximately 190 amino acids, approximately 155 amino acids to approximately 185 amino acids, approximately 155 amino acids to approximately 180 amino acids, approximately 155 amino acids to approximately 175 amino acids, approximately 155 amino acids to approximately 170 amino acids, approximately 155 amino acids to approximately 165 amino acids, approximately 155 amino acids to approximately 160 amino acids, approximately 160 amino acids to approximately 1000 amino acids, approximately 160 amino acids to approximately 950 amino acids, approximately 160 amino acids to approximately 900 amino acids, approximately 160 amino acids to approximately 850 amino acids, approximately 160 amino acids to approximately 800 amino acids, approximately 160 amino acids to approximately 750 amino acids, approximately 160 amino acids to approximately 700 amino acids. 160 amino acids to about 650 amino acids, about 160 amino acids to about 600 amino acids, about 160 amino acids to about 550 amino acids, about 160 amino acids to about 500 amino acids, about 160 amino acids to about 450 amino acids, about 160 amino acids to about 400 amino acids, about 160 amino acids to about 350 amino acids, about 160 amino acids to about 300 amino acids, about 160 amino acids to about 280 amino acids, about 160 amino acids to about 260 amino acids, about 160 amino acids to about 240 amino acids, about 160 amino acids to about 220 amino acids, about 160 amino acids to about 200 amino acids, about 160 amino acids to Approximately 195 amino acids, approximately 160 amino acids to approximately 190 amino acids, approximately 160 amino acids to approximately 185 amino acids, approximately 160 amino acids to approximately 180 amino acids, approximately 160 amino acids to approximately 175 amino acids, approximately 160 amino acids to approximately 170 amino acids, approximately 160 amino acids to approximately 165 amino acids, approximately 165 amino acids to approximately 1000 amino acids, approximately 165 amino acids to approximately 950 amino acids, approximately 165 amino acids to approximately 900 amino acids, approximately 165 amino acids to approximately 850 amino acids, approximately 165 amino acids to approximately 800 amino acids, approximately 165 amino acids to approximately 750 amino acids, approximately 165 amino acids to approximately 700 amino acids, approximately 165 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their composition. A direct translation wouldn't be meaningful without further context.Approximately 165 amino acids to approximately 190 amino acids, approximately 165 amino acids to approximately 185 amino acids, approximately 165 amino acids to approximately 180 amino acids, approximately 165 amino acids to approximately 175 amino acids, approximately 165 amino acids to approximately 170 amino acids, approximately 170 amino acids to approximately 1000 amino acids, approximately 170 amino acids to approximately 950 amino acids, approximately 170 amino acids to approximately 900 amino acids, approximately 170 amino acids to approximately 850 amino acids, approximately 170 amino acids to approximately 800 amino acids, approximately 170 amino acids to approximately 750 amino acids, approximately 170 amino acids to approximately 700 amino acids, approximately 170 amino acids to approximately 650 amino acids, approximately 170 amino acids to approximately 600 amino acids. 170 amino acids to about 550 amino acids, about 170 amino acids to about 500 amino acids, about 170 amino acids to about 450 amino acids, about 170 amino acids to about 400 amino acids, about 170 amino acids to about 350 amino acids, about 170 amino acids to about 300 amino acids, about 170 amino acids to about 280 amino acids, about 170 amino acids to about 260 amino acids, about 170 amino acids to about 240 amino acids, about 170 amino acids to about 220 amino acids, about 170 amino acids to about 200 amino acids, about 170 amino acids to about 195 amino acids, about 170 amino acids to about 190 amino acids, about 170 amino acids to Approximately 185 amino acids, approximately 170 amino acids to approximately 180 amino acids, approximately 170 amino acids to approximately 175 amino acids, approximately 175 amino acids to approximately 1000 amino acids, approximately 175 amino acids to approximately 950 amino acids, approximately 175 amino acids to approximately 900 amino acids, approximately 175 amino acids to approximately 850 amino acids, approximately 175 amino acids to approximately 800 amino acids, approximately 175 amino acids to approximately 750 amino acids, approximately 175 amino acids to approximately 700 amino acids, approximately 175 amino acids to approximately 650 amino acids, approximately 175 amino acids to approximately 600 amino acids, approximately 175 amino acids to approximately 550 amino acids, approximately 175 amino acids to approximately 500 amino acids, approximately 175 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their composition. A direct translation wouldn't be meaningful without further context.Approximately 180 amino acids to approximately 950 amino acids, approximately 180 amino acids to approximately 900 amino acids, approximately 180 amino acids to approximately 850 amino acids, approximately 180 amino acids to approximately 800 amino acids, approximately 180 amino acids to approximately 750 amino acids, approximately 180 amino acids to approximately 700 amino acids, approximately 180 amino acids to approximately 650 amino acids, approximately 180 amino acids to approximately 600 amino acids, approximately 180 amino acids to approximately 550 amino acids, approximately 180 amino acids to approximately 500 amino acids, approximately 180 amino acids to approximately 450 amino acids, approximately 180 amino acids to approximately 400 amino acids, approximately 180 amino acids to approximately 350 amino acids, approximately 180 amino acids to approximately 300 amino acids. Amino acids, approximately 180 amino acids to approximately 280 amino acids, approximately 180 amino acids to approximately 260 amino acids, approximately 180 amino acids to approximately 240 amino acids, approximately 180 amino acids to approximately 220 amino acids, approximately 180 amino acids to approximately 200 amino acids, approximately 180 amino acids to approximately 195 amino acids, approximately 180 amino acids to approximately 190 amino acids, approximately 180 amino acids to approximately 185 amino acids, approximately 185 amino acids to approximately 1000 amino acids, approximately 185 amino acids to approximately 950 amino acids, approximately 185 amino acids to approximately 900 amino acids, approximately 185 amino acids to approximately 850 amino acids, approximately 185 amino acids to approximately 800 amino acids, approximately 185 amino acids to Approximately 750 amino acids, approximately 185 amino acids to approximately 700 amino acids, approximately 185 amino acids to approximately 650 amino acids, approximately 185 amino acids to approximately 600 amino acids, approximately 185 amino acids to approximately 550 amino acids, approximately 185 amino acids to approximately 500 amino acids, approximately 185 amino acids to approximately 450 amino acids, approximately 185 amino acids to approximately 400 amino acids, approximately 185 amino acids to approximately 350 amino acids, approximately 185 amino acids to approximately 300 amino acids, approximately 185 amino acids to approximately 280 amino acids, approximately 185 amino acids to approximately 260 amino acids, approximately 185 amino acids to approximately 240 amino acids, approximately 185 amino acids to approximately 220 amino acids, approximately 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.Approximately 190 amino acids to approximately 450 amino acids, approximately 190 amino acids to approximately 400 amino acids, approximately 190 amino acids to approximately 350 amino acids, approximately 190 amino acids to approximately 300 amino acids, approximately 190 amino acids to approximately 280 amino acids, approximately 190 amino acids to approximately 260 amino acids, approximately 190 amino acids to approximately 240 amino acids, approximately 190 amino acids to approximately 220 amino acids, approximately 190 amino acids to approximately 200 amino acids, approximately 190 amino acids to approximately 195 amino acids, approximately 195 amino acids to approximately 1000 amino acids, approximately 195 amino acids to approximately 950 amino acids, approximately 195 amino acids to approximately 900 amino acids, approximately 195 amino acids to approximately 850 amino acids. 195 amino acids to about 800 amino acids, about 195 amino acids to about 750 amino acids, about 195 amino acids to about 700 amino acids, about 195 amino acids to about 650 amino acids, about 195 amino acids to about 600 amino acids, about 195 amino acids to about 550 amino acids, about 195 amino acids to about 500 amino acids, about 195 amino acids to about 450 amino acids, about 195 amino acids to about 400 amino acids, about 195 amino acids to about 350 amino acids, about 195 amino acids to about 300 amino acids, about 195 amino acids to about 280 amino acids, about 195 amino acids to about 260 amino acids, about 195 amino acids to Approximately 240 amino acids, approximately 195 amino acids to approximately 220 amino acids, approximately 195 amino acids to approximately 200 amino acids, approximately 200 amino acids to approximately 1000 amino acids, approximately 200 amino acids to approximately 950 amino acids, approximately 200 amino acids to approximately 900 amino acids, approximately 200 amino acids to approximately 850 amino acids, approximately 200 amino acids to approximately 800 amino acids, approximately 200 amino acids to approximately 750 amino acids, approximately 200 amino acids to approximately 700 amino acids, approximately 200 amino acids to approximately 650 amino acids, approximately 200 amino acids to approximately 600 amino acids, approximately 200 amino acids to approximately 550 amino acids, approximately 200 amino acids to approximately 500 amino acids, approximately 200 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their composition. A direct translation wouldn't be meaningful without further context.Approximately 220 amino acids to approximately 700 amino acids, approximately 220 amino acids to approximately 650 amino acids, approximately 220 amino acids to approximately 600 amino acids, approximately 220 amino acids to approximately 550 amino acids, approximately 220 amino acids to approximately 500 amino acids, approximately 220 amino acids to approximately 450 amino acids, approximately 220 amino acids to approximately 400 amino acids, approximately 220 amino acids to approximately 350 amino acids, approximately 220 amino acids to approximately 300 amino acids, approximately 220 amino acids to approximately 280 amino acids, approximately 220 amino acids to approximately 260 amino acids, approximately 220 amino acids to approximately 240 amino acids, approximately 240 amino acids to approximately 1000 amino acids, approximately 240 amino acids to approximately 950 amino acids. 1 amino acid, about 240 amino acids to about 900 amino acids, about 240 amino acids to about 850 amino acids, about 240 amino acids to about 800 amino acids, about 240 amino acids to about 750 amino acids, about 240 amino acids to about 700 amino acids, about 240 amino acids to about 650 amino acids, about 240 amino acids to about 600 amino acids, about 240 amino acids to about 550 amino acids, about 240 amino acids to about 500 amino acids, about 240 amino acids to about 450 amino acids, about 240 amino acids to about 400 amino acids, about 240 amino acids to about 350 amino acids, about 240 amino acids to about 300 amino acids, about 240 amino acids to Approximately 280 amino acids, approximately 240 amino acids to approximately 260 amino acids, approximately 260 amino acids to approximately 1000 amino acids, approximately 260 amino acids to approximately 950 amino acids, approximately 260 amino acids to approximately 900 amino acids, approximately 260 amino acids to approximately 850 amino acids, approximately 260 amino acids to approximately 800 amino acids, approximately 260 amino acids to approximately 750 amino acids, approximately 260 amino acids to approximately 700 amino acids, approximately 260 amino acids to approximately 650 amino acids, approximately 260 amino acids to approximately 600 amino acids, approximately 260 amino acids to approximately 550 amino acids, approximately 260 amino acids to approximately 500 amino acids, approximately 260 amino acids to approximately 450 amino acids, approximately 260 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their components. A direct translation wouldn't be meaningful without further context.Approximately 280 amino acids to approximately 500 amino acids, approximately 280 amino acids to approximately 450 amino acids, approximately 280 amino acids to approximately 400 amino acids, approximately 280 amino acids to approximately 350 amino acids, approximately 280 amino acids to approximately 300 amino acids, approximately 300 amino acids to approximately 1000 amino acids, approximately 300 amino acids to approximately 950 amino acids, approximately 300 amino acids to approximately 900 amino acids, approximately 300 amino acids to approximately 850 amino acids, approximately 300 amino acids to approximately 800 amino acids, approximately 300 amino acids to approximately 750 amino acids, approximately 300 amino acids to approximately 700 amino acids, approximately 300 amino acids to approximately 650 amino acids, approximately 300 amino acids to approximately 600 amino acids. 1 amino acid, 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 Approximately 600 amino acids, approximately 350 amino acids to approximately 550 amino acids, approximately 350 amino acids to approximately 500 amino acids, approximately 350 amino acids to approximately 450 amino acids, approximately 350 amino acids to approximately 400 amino acids, approximately 400 amino acids to approximately 1000 amino acids, approximately 400 amino acids to approximately 950 amino acids, approximately 400 amino acids to approximately 900 amino acids, approximately 400 amino acids to approximately 850 amino acids, approximately 400 amino acids to approximately 800 amino acids, approximately 400 amino acids to approximately 750 amino acids, approximately 400 amino acids to approximately 700 amino acids, approximately 400 amino acids to approximately 650 amino acids, approximately 400 amino acids to approximately 600 amino acids, approximately 400 The numbers appear to be a series of amino acids and their components, possibly related to a list of amino acids and their composition. A direct translation wouldn't be meaningful without further context.Approximately 500 amino acids to approximately 1000 amino acids, approximately 500 amino acids to approximately 950 amino acids, approximately 500 amino acids to approximately 900 amino acids, approximately 500 amino acids to approximately 850 amino acids, approximately 500 amino acids to approximately 800 amino acids, approximately 500 amino acids to approximately 750 amino acids, approximately 500 amino acids to approximately 700 amino acids, approximately 500 amino acids to approximately 650 amino acids, approximately 500 amino acids to approximately 600 amino acids, approximately 500 amino acids to approximately 550 amino acids, approximately 550 amino acids to approximately 1000 amino acids, approximately 550 amino acids to approximately 950 amino acids, approximately 550 amino acids to approximately 900 amino acids, approximately 550 amino acids to Approximately 850 amino acids, approximately 550 amino acids to approximately 800 amino acids, approximately 550 amino acids to approximately 750 amino acids, approximately 550 amino acids to approximately 700 amino acids, approximately 550 amino acids to approximately 650 amino acids, approximately 550 amino acids to approximately 600 amino acids, approximately 600 amino acids to approximately 1000 amino acids, approximately 600 amino acids to approximately 950 amino acids, approximately 600 amino acids to approximately 900 amino acids, approximately 600 amino acids to approximately 850 amino acids, approximately 600 amino acids to approximately 800 amino acids, approximately 600 amino acids to approximately 750 amino acids, approximately 600 amino acids to approximately 700 amino acids, approximately 600 amino acids to approximately 650 amino acids, approximately 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, approximately 750 amino acids to approximately 950 amino acids, approximately 750 amino acids to approximately 900 amino acids, approximately 750 amino acids to approximately 850 amino acids, approximately 750 amino acids to approximately 800 amino acids, approximately 800 amino acids to approximately 1000 amino acids, approximately 800 amino acids to approximately 950 amino acids, approximately 800 amino acids to approximately 900 amino acids, approximately 800 amino acids to approximately 850 amino acids, approximately 850 amino acids to approximately 1000 amino acids, approximately 850 amino acids to approximately 950 amino acids, approximately 850 amino acids to approximately 900 amino acids, approximately 900 amino acids to approximately 1000 amino acids, approximately 900 amino acids to approximately 950 amino acids.It may contain approximately 950 amino acids to approximately 1000 amino acids.

[0126] Any target-binding domain described in this article can dissociate with the following equilibrium constant (K). D Ligands binding to TGF-βRII: less than 1x10 -7 M, less than 1x10 -8 M, less than 1x10 -9 M, less than 1x10 -10 M, less than 1x10 -11 M, less than 1x10 -12 M, or less than 1x10 -13 M. In some embodiments, the antigen-binding protein constructs provided herein can be used as follows K D Binding to the identification antigen: approximately 1 x 10 -3 M to approximately 1x10 -5 M, approximately 1x10 -4 M to approximately 1x10 -6 M, approximately 1x10 -5 M to approximately 1x10 -7 M, approximately 1x10 -6 M to approximately 1x10 - 8 M, approximately 1x10 -7 M to approximately 1x10 -9 M, approximately 1x10 -8 M to approximately 1x10 -10 M, or approximately 1x10 -9 M to approximately 1x10 -11 M (inclusive).

[0127] Any target-binding domain described herein can bind to a ligand of TGF-βRII (e.g., TGF-β) in the following K... DCombination: about 1 pM to about 30 nM (e.g., about 1 pM to about 25 nM, about 1 pM to about 20 nM, about 1 pM to about 15 nM, about 1 pM to about 10 nM, about 1 pM to about 5 nM, about 1 pM to about 2 nM, about 1 pM to about 1 nM, about 1 pM to about 950 pM, about 1 pM to about 900 pM, about 1 pM to about 850 pM, about 1 pM to about 800 pM, about 1 pM to about 750 pM, about 1 pM to about 700 pM, about 1 pM to about 650 pM, about 1 pM to about 600 pM, about 1 pM to about 550 pM, about 1 pM to about 500 pM, about 1 pM to about 450 pM, about 1 pM to about 400 pM, about 1 pM to about 350 pM, about 1 pM to about 300 pM, about 1 pM to about 250 pM, about 1 pM to about 200 pM, about 1 pM to about 150 pM, about 1 pM to about 100 pM, about 1 pM to about 90 pM, about 1 pM to about 80 pM, about 1 pM to about 70 pM, about 1 pM to about 60 pM, about 1 pM to about 50 pM, about 1 pM to about 40 pM, about 1 pM to about 30 pM, about 1 pM to about 20 pM, about 1 pM to about 10 pM, about 1 pM to about 5 pM, about 1 pM to about 4 pM, about 1 pM to about 3 pM, about 1 pM to about 2 pM, about 2 pM to about 30 nM, about 2 pM to about 25 nM, about 2 pM to about 20 nM, about 2 pM to about 15 nM, about 2 pM to about 10 nM, approximately 2pM to approximately 5nM, approximately 2pM to approximately 2nM, approximately 2pM to approximately 1nM, approximately 2pM to approximately 950pM, approximately 2pM to approximately 900pM, approximately 2pM to approximately 850pM, approximately 2pM to approximately 800pM, approximately 2pM to approximately 750pM, approximately 2pM to approximately 700pM, approximately 2pM to approximately 650pM, approximately 2pM to approximately 600pM, approximately 2pM to approximately 550pM, approximately 2pM to approximately 500pM, approximately 2pM to approximately 450pM, approximately 2pM to approximately 400pM, approximately 2pM to approximately 350pM, approximately 2pM to approximately 300pM, approximately 2pM to approximately 250pM, approximately 2pM to approximately 200pM, approximately 2pM to approximately 150pM, approximately 2pM to approximately 100pM, Approximately 2 pM to approximately 90 pM, approximately 2 pM to approximately 80 pM, approximately 2 pM to approximately 70 pM, approximately 2 pM to approximately 60 pM, approximately 2 pM to approximately 50 pM, approximately 2 pM to approximately 40 pM, approximately 2 pM to approximately 30 pM, approximately 2 pM to approximately 20 pM, approximately 2 pM to approximately 10 pM, approximately 2 pM to approximately 5 pM, approximately 2 pM to approximately 4 pM, approximately 2 pM to approximately 3 pM, approximately 5 pM to approximately 30 nM, approximately 5 pM to approximately 25 nM, approximately 5 pM to approximately 20 nM, approximately 5 pM to approximately 15 nM, approximately 5 pM to approximately 10 nM, approximately 5 pM to approximately 5 nM, approximately 5 pM to approximately 2 nM, approximately 5 pM to approximately 1 nM, approximately 5 pM to approximately 950 pM, approximately 5 pM to approximately 900 pM, approximately 5 pM to approximately 850 pM,Approximately 5 pM to approximately 800 pM, approximately 5 pM to approximately 750 pM, approximately 5 pM to approximately 700 pM, approximately 5 pM to approximately 650 pM, approximately 5 pM to approximately 600 pM, approximately 5 pM to approximately 550 pM, approximately 5 pM to approximately 500 pM, approximately 5 pM to approximately 450 pM, approximately 5 pM to approximately 400 pM, approximately 5 pM to approximately 350 pM, approximately 5 pM to approximately 300 pM, approximately 5 pM to approximately 250 pM, approximately 5 pM to approximately 200 pM, approximately 5 pM to approximately 150 pM, approximately 5 pM to approximately 100 pM, approximately 5 pM to approximately 90 pM, approximately 5 pM to approximately 80 pM, approximately 5 pM to approximately 70 pM, approximately 5 pM to approximately 60 pM, approximately 5 pM to approximately 50 pM, approximately 5 pM to approximately 40 pM, approximately 5pM to about 30pM, about 5pM to about 20pM, about 5pM to about 10pM, about 10pM to about 30nM, about 10pM to about 25nM, about 10pM to about 20nM, about 10pM to about 15nM, about 10pM to about 10nM, about 10pM to about 5nM, about 10pM to about 2nM, about 10pM to about 1nM, about 10pM to about 950pM, about 10pM to about 900pM, about 10pM to about 850pM, about 10pM to about 800pM, about 10pM to about 750pM, about 10pM to about 700pM, about 10pM to about 650pM, about 10pM to about 600pM, about 10pM to about 550pM, about 10pM to Approximately 500 pM, approximately 10 pM to approximately 450 pM, approximately 10 pM to approximately 400 pM, approximately 10 pM to approximately 350 pM, approximately 10 pM to approximately 300 pM, approximately 10 pM to approximately 250 pM, approximately 10 pM to approximately 200 pM, approximately 10 pM to approximately 150 pM, approximately 10 pM to approximately 100 pM, approximately 10 pM to approximately 90 pM, approximately 10 pM to approximately 80 pM, approximately 10 pM to approximately 70 pM, approximately 10 pM to approximately 60 pM, approximately 10 pM to approximately 50 pM, approximately 10 pM to approximately 40 pM, approximately 10 pM to approximately 30 pM, approximately 10 pM to approximately 20 pM, approximately 15 pM to approximately 30 nM, approximately 15 pM to approximately 25 nM, approximately 15 pM to approximately 20 nM, approximately 15 p M to approximately 15 nmM, approximately 15 pM to approximately 10 nmM, approximately 15 pM to approximately 5 nmM, approximately 15 pM to approximately 2 nmM, approximately 15 pM to approximately 1 nmM, approximately 15 pM to approximately 950 pM, approximately 15 pM to approximately 900 pM, approximately 15 pM to approximately 850 pM, approximately 15 pM to approximately 800 pM, approximately 15 pM to approximately 750 pM, approximately 15 pM to approximately 700 pM, approximately 15 pM to approximately 650 pM, approximately 15 pM to approximately 600 pM, approximately 15 pM to approximately 550 pM, approximately 15 pM to approximately 500 pM, approximately 15 pM to approximately 450 pM, approximately 15 pM to approximately 400 pM, approximately 15 pM to approximately 350 pM, approximately 15 pM to approximately 300 pM, approximately 15 pM to approximately 250 pM,Approximately 15 pM to approximately 200 pM, approximately 15 pM to approximately 150 pM, approximately 15 pM to approximately 100 pM, approximately 15 pM to approximately 90 pM, approximately 15 pM to approximately 80 pM, approximately 15 pM to approximately 70 pM, approximately 15 pM to approximately 60 pM, approximately 15 pM to approximately 50 pM, approximately 15 pM to approximately 40 pM, approximately 15 pM to approximately 30 pM, approximately 15 pM to approximately 20 pM, approximately 20 pM to approximately 30 nM, approximately 20 pM to approximately 25 nM, approximately 20 pM to approximately 20 nM, approximately 20 pM to approximately 15 nM, approximately 20 pM to approximately 10 nM, approximately 20 pM to approximately 5 nM, approximately 20 pM to approximately 2 nM, approximately 20 pM to approximately 1 nM, approximately 20 pM to approximately 950 pM, approximately 20 pM to approximately 900pM, approximately 20pM to approximately 850pM, approximately 20pM to approximately 800pM, approximately 20pM to approximately 750pM, approximately 20pM to approximately 700pM, approximately 20pM to approximately 650pM, approximately 20pM to approximately 600pM, approximately 20pM to approximately 550pM, approximately 20pM to approximately 500pM, approximately 20pM to approximately 450pM, approximately 20pM to approximately 400pM, approximately 20pM to approximately 350pM, approximately 20pM to approximately 300pM, approximately 20pM to approximately 250pM, approximately 20pM to approximately 20pM, approximately 200pM to approximately 150pM, approximately 20pM to approximately 100pM, approximately 20pM to approximately 90pM, approximately 20pM to approximately 80pM, approximately 20pM to approximately 70pM M, approximately 20 pM to approximately 60 pM, approximately 20 pM to approximately 50 pM, approximately 20 pM to approximately 40 pM, approximately 20 pM to approximately 30 pM, approximately 30 pM to approximately 30 nM, approximately 30 pM to approximately 25 nM, approximately 30 pM to approximately 30 nM, approximately 30 pM to approximately 15 nM, approximately 30 pM to approximately 10 nM, approximately 30 pM to approximately 5 nM, approximately 30 pM to approximately 2 nM, approximately 30 pM to approximately 1 nM, approximately 30 pM to approximately 950 pM, approximately 30 pM to approximately 900 pM, approximately 30 pM to approximately 850 pM, approximately 30 pM to approximately 800 pM, approximately 30 pM to approximately 750 pM, approximately 30 pM to approximately 700 pM, approximately 30 pM to approximately 650 pM, approximately 30 pM to approximately 600 pM, approximately 30pM to approximately 550pM, approximately 30pM to approximately 500pM, approximately 30pM to approximately 450pM, approximately 30pM to approximately 400pM, approximately 30pM to approximately 350pM, approximately 30pM to approximately 300pM, approximately 30pM to approximately 250pM, approximately 30pM to approximately 200pM, approximately 30pM to approximately 150pM, approximately 30pM to approximately 100pM, approximately 30pM to approximately 90pM, approximately 30pM to approximately 80pM, approximately 30pM to approximately 70pM, approximately 30pM to approximately 60pM, approximately 30pM to approximately 50pM, approximately 30pM to approximately 40pM, approximately 40pM to approximately 30nM, approximately 40pM to approximately 25nM, approximately 40pM to approximately 30nM, approximately 40pM to approximately 15nM,Approximately 40 pM to approximately 10 nM, approximately 40 pM to approximately 5 nM, approximately 40 pM to approximately 2 nM, approximately 40 pM to approximately 1 nM, approximately 40 pM to approximately 950 pM, approximately 40 pM to approximately 900 pM, approximately 40 pM to approximately 850 pM, approximately 40 pM to approximately 800 pM, approximately 40 pM to approximately 750 pM, approximately 40 pM to approximately 700 pM, approximately 40 pM to approximately 650 pM, approximately 40 pM to approximately 600 pM, approximately 40 pM to approximately 550 pM, approximately 40 pM to approximately 500 pM, approximately 40 pM to approximately 450 pM, approximately 40 pM to approximately 400 pM, approximately 40 pM to approximately 350 pM, approximately 40 pM to approximately 300 pM, approximately 40 pM to approximately 250 pM, approximately 40 pM to approximately 200pM, approximately 40pM to approximately 150pM, approximately 40pM to approximately 100pM, approximately 40pM to approximately 90pM, approximately 40pM to approximately 80pM, approximately 40pM to approximately 70pM, approximately 40pM to approximately 60pM, approximately 40pM to approximately 50pM, approximately 50pM to approximately 30nM, approximately 50pM to approximately 25nM, approximately 50pM to approximately 30nM, approximately 50pM to approximately 15nM, approximately 50pM to approximately 10nM, approximately 50pM to approximately 5nM, approximately 50pM to approximately 2nM, approximately 50pM to approximately 1nM, approximately 50pM to approximately 950pM, approximately 50pM to approximately 900pM, approximately 50pM to approximately 850pM, approximately 50pM to approximately 800pM, approximately 50pM to approximately 750pM pM, approximately 50 pM to approximately 700 pM, approximately 50 pM to approximately 650 pM, approximately 50 pM to approximately 600 pM, approximately 50 pM to approximately 550 pM, approximately 50 pM to approximately 500 pM, approximately 50 pM to approximately 450 pM, approximately 50 pM to approximately 400 pM, approximately 50 pM to approximately 350 pM, approximately 50 pM to approximately 300 pM, approximately 50 pM to approximately 250 pM, approximately 50 pM to approximately 200 pM, approximately 50 pM to approximately 150 pM, approximately 50 pM to approximately 100 pM, approximately 50 pM to approximately 90 pM, approximately 50 pM to approximately 80 pM, approximately 50 pM to approximately 70 pM, approximately 50 pM to approximately 60 pM, approximately 60 pM to approximately 30 nM, approximately 60 pM to approximately 25 nM, approximately 60 pM to about 30 nM, about 60 pM to about 15 nM, about 60 pM to about 10 nM, about 60 pM to about 5 nM, about 60 pM to about 2 nM, about 60 pM to about 1 nM, about 60 pM to about 950 pM, about 60 pM to about 900 pM, about 60 pM to about 850 pM, about 60 pM to about 800 pM, about 60 pM to about 750 pM, about 60 pM to about 700 pM, about 60 pM to about 650 pM, about 60 pM to about 600 pM, about 60 pM to about 550 pM, about 60 pM to about 500 pM, about 60 pM to about 450 pM, about 60 pM to about 400 pM, about 60 pM to about 350 pM, about 60 pM to about 300 pMApproximately 60 pM to approximately 250 pM, approximately 60 pM to approximately 200 pM, approximately 60 pM to approximately 150 pM, approximately 60 pM to approximately 100 pM, approximately 60 pM to approximately 90 pM, approximately 60 pM to approximately 80 pM, approximately 60 pM to approximately 70 pM, approximately 70 pM to approximately 30 nM, approximately 70 pM to approximately 25 nM, approximately 70 pM to approximately 30 nM, approximately 70 pM to approximately 15 nM, approximately 70 pM to approximately 10 nM, approximately 70 pM to approximately 5 nM, approximately 70 pM to approximately 2 nM, approximately 70 pM to approximately 1 nM, approximately 70 pM to approximately 950 pM, approximately 70 pM to approximately 900 pM, approximately 70 pM to approximately 850 pM, approximately 70 pM to approximately 800 pM, approximately 70 pM to approximately 750 pM, approximately 70 pM M to approximately 700 pM, approximately 70 pM to approximately 650 pM, approximately 70 pM to approximately 600 pM, approximately 70 pM to approximately 550 pM, approximately 70 pM to approximately 500 pM, approximately 70 pM to approximately 450 pM, approximately 70 pM to approximately 400 pM, approximately 70 pM to approximately 350 pM, approximately 70 pM to approximately 300 pM, approximately 70 pM to approximately 250 pM, approximately 70 pM to approximately 200 pM, approximately 70 pM to approximately 150 pM, approximately 70 pM to approximately 100 pM, approximately 70 pM to approximately 90 pM, approximately 70 pM to approximately 80 pM, approximately 80 pM to approximately 30 nM, approximately 80 pM to approximately 25 nM, approximately 80 pM to approximately 30 nM, approximately 80 pM to approximately 15 nM, approximately 80 pM to approximately 10 nM, approximately 80pM to about 5nM, about 80pM to about 2nM, about 80pM to about 1nM, about 80pM to about 950pM, about 80pM to about 900pM, about 80pM to about 850pM, about 80pM to about 800pM, about 80pM to about 750pM, about 80pM to about 700pM, about 80pM to about 650pM, about 80pM to about 600pM, about 80pM to about 550pM, about 80pM to about 500pM, about 80pM to about 450pM, about 80pM to about 400pM, about 80pM to about 350pM, about 80pM to about 300pM, about 80pM to about 250pM, about 80pM to about 200pM, about 80pM to about 150pM pM, approximately 80 pM to approximately 100 pM, approximately 80 pM to approximately 90 pM, approximately 90 pM to approximately 30 nM, approximately 90 pM to approximately 25 nM, approximately 90 pM to approximately 30 nM, approximately 90 pM to approximately 15 nM, approximately 90 pM to approximately 10 nM, approximately 90 pM to approximately 5 nM, approximately 90 pM to approximately 2 nM, approximately 90 pM to approximately 1 nM, approximately 90 pM to approximately 950 pM, approximately 90 pM to approximately 900 pM, approximately 90 pM to approximately 850 pM, approximately 90 pM to approximately 800 pM, approximately 90 pM to approximately 750 pM, approximately 90 pM to approximately 700 pM, approximately 90 pM to approximately 650 pM, approximately 90 pM to approximately 600 pM, approximately 90 pM to approximately 550 pM, approximately 90 pM to approximately 500 pM,Approximately 90 pM to approximately 450 pM, approximately 90 pM to approximately 400 pM, approximately 90 pM to approximately 350 pM, approximately 90 pM to approximately 300 pM, approximately 90 pM to approximately 250 pM, approximately 90 pM to approximately 200 pM, approximately 90 pM to approximately 150 pM, approximately 90 pM to approximately 100 pM, approximately 100 pM to approximately 30 nM, approximately 100 pM to approximately 25 nM, approximately 100 pM to approximately 30 nM, approximately 100 pM to approximately 15 nM, approximately 100 pM to approximately 10 nM, approximately 100 pM to approximately 5 nM, approximately 100 pM to approximately 2 nM, approximately 100 pM to approximately 1 nM, approximately 100 pM to approximately 950 pM, approximately 100 pM to approximately 900 pM, approximately 100 pM to approximately 850 pM, approximately 100pM to about 800pM, about 100pM to about 750pM, about 100pM to about 700pM, about 100pM to about 650pM, about 100pM to about 600pM, about 100pM to about 550pM, about 100pM to about 500pM, about 100pM to about 450pM, about 100pM to about 400pM, about 100pM to about 350pM, about 100pM to about 300pM, about 100pM to about 250pM, about 100pM to about 200pM, about 100pM to about 150pM, about 150pM to about 30nM, about 150pM to about 25nM, about 150pM to about 30nM, about 150pM to about 15nM, about 1 50pM to about 10nM, about 150pM to about 5nM, about 150pM to about 2nM, about 150pM to about 1nM, about 150pM to about 950pM, about 150pM to about 900pM, about 150pM to about 850pM, about 150pM to about 800pM, about 150pM to about 750pM, about 150pM to about 700pM, about 150pM to about 650pM, about 150pM to about 600pM, about 150pM to about 550pM, about 150pM to about 500pM, about 150pM to about 450pM, about 150pM to about 400pM, about 150pM to about 350pM, about 150pM to about 300pM, about 150pM Approximately 250 pM, approximately 150 pM to approximately 200 pM, approximately 200 pM to approximately 30 nM, approximately 200 pM to approximately 25 nM, approximately 200 pM to approximately 30 nM, approximately 200 pM to approximately 15 nM, approximately 200 pM to approximately 10 nM, approximately 200 pM to approximately 5 nM, approximately 200 pM to approximately 2 nM, approximately 200 pM to approximately 1 nM, approximately 200 pM to approximately 950 pM, approximately 200 pM to approximately 900 pM, approximately 200 pM to approximately 850 pM, approximately 200 pM to approximately 800 pM, approximately 200 pM to approximately 750 pM, approximately 200 pM to approximately 700 pM, approximately 200 pM to approximately 650 pM, approximately 200 pM to approximately 600 pM, approximately 200 pM to approximately 550 pM,Approximately 200 pM to approximately 500 pM, approximately 200 pM to approximately 450 pM, approximately 200 pM to approximately 400 pM, approximately 200 pM to approximately 350 pM, approximately 200 pM to approximately 300 pM, approximately 200 pM to approximately 250 pM, approximately 300 pM to approximately 30 nM, approximately 300 pM to approximately 25 nM, approximately 300 pM to approximately 30 nM, approximately 300 pM to approximately 15 nM, approximately 300 pM to approximately 10 nM, approximately 300 pM to approximately 5 nM, approximately 300 pM to approximately 2 nM, approximately 300 pM to approximately 1 nM, approximately 300 pM to approximately 950 pM, approximately 300 pM to approximately 900 pM, approximately 300 pM to approximately 850 pM, approximately 300 pM to approximately 800 pM, approximately 300 pM to Approximately 750 pM, approximately 300 pM to approximately 700 pM, approximately 300 pM to approximately 650 pM, approximately 300 pM to approximately 600 pM, approximately 300 pM to approximately 550 pM, approximately 300 pM to approximately 500 pM, approximately 300 pM to approximately 450 pM, approximately 300 pM to approximately 400 pM, approximately 300 pM to approximately 350 pM, approximately 400 pM to approximately 30 nM, approximately 400 pM to approximately 25 nM, approximately 400 pM to approximately 30 nM, approximately 400 pM to approximately 15 nM, approximately 400 pM to approximately 10 nM, approximately 400 pM to approximately 5 nM, approximately 400 pM to approximately 2 nM, approximately 400 pM to approximately 1 nM, approximately 400 pM to approximately 950 pM, approximately 400 pM to approximately 900 pM, Approximately 400 pM to approximately 850 pM, approximately 400 pM to approximately 800 pM, approximately 400 pM to approximately 750 pM, approximately 400 pM to approximately 700 pM, approximately 400 pM to approximately 650 pM, approximately 400 pM to approximately 600 pM, approximately 400 pM to approximately 550 pM, approximately 400 pM to approximately 500 pM, approximately 500 pM to approximately 30 nM, approximately 500 pM to approximately 25 nM, approximately 500 pM to approximately 30 nM, approximately 500 pM to approximately 15 nM, approximately 500 pM to approximately 10 nM, approximately 500 pM to approximately 5 nM, approximately 500 pM to approximately 2 nM, approximately 500 pM to approximately 1 nM, approximately 500 pM to approximately 950 pM, approximately 500 pM to approximately 900 pM, approximately 500 pM to Approximately 850 pM, approximately 500 pM to approximately 800 pM, approximately 500 pM to approximately 750 pM, approximately 500 pM to approximately 700 pM, approximately 500 pM to approximately 650 pM, approximately 500 pM to approximately 600 pM, approximately 500 pM to approximately 550 pM, approximately 600 pM to approximately 30 nM, approximately 600 pM to approximately 25 nM, approximately 600 pM to approximately 30 nM, approximately 600 pM to approximately 15 nM, approximately 600 pM to approximately 10 nM, approximately 600 pM to approximately 5 nM, approximately 600 pM to approximately 2 nM, approximately 600 pM to approximately 1 nM, approximately 600 pM to approximately 950 pM, approximately 600 pM to approximately 900 pM, approximately 600 pM to approximately 850 pM, approximately 600 pM to approximately 800 pM,Approximately 600 pM to approximately 750 pM, approximately 600 pM to approximately 700 pM, approximately 600 pM to approximately 650 pM, approximately 700 pM to approximately 30 nM, approximately 700 pM to approximately 25 nM, approximately 700 pM to approximately 30 nM, approximately 700 pM to approximately 15 nM, approximately 700 pM to approximately 10 nM, approximately 700 pM to approximately 5 nM, approximately 700 pM to approximately 2 nM, approximately 700 pM to approximately 1 nM, approximately 700 pM to approximately 950 pM, approximately 700 pM to approximately 900 pM, approximately 700 pM to approximately 850 pM, approximately 700 pM to approximately 800 pM, approximately 700 pM to approximately 7 50pM, approximately 800pM to approximately 30nM, approximately 800pM to approximately 25nM, approximately 800pM to approximately 30nM, approximately 800pM to approximately 15nM, approximately 800pM to approximately 10nM, approximately 800pM to approximately 5nM, approximately 800pM to approximately 2nM, approximately 800pM to approximately 1nM, approximately 800pM to approximately 950pM, approximately 800pM to approximately 900pM, approximately 800pM to approximately 850pM, approximately 900pM to approximately 30nM, approximately 900pM to approximately 25nM, approximately 900pM to approximately 30nM, approximately 900pM to approximately 15nM, approximately 900pM to approximately 10 nM, about 900 pM to about 5 nM, about 900 pM to about 2 nM, about 900 pM to about 1 nM, about 900 pM to about 950 pM, about 1 nM to about 30 nM, about 1 nM to about 25 nM, about 1 nM to about 20 nM, about 1 nM to about 15 nM, about 1 nM to about 10 nM, about 1 nM to about 5 nM, about 2 nM to about 30 nM, about 2 nM to about 25 nM, about 2 nM to about 20 nM, about 2 nM to about 15 nM, about 2 nM to about 10 nM, about 2 nM to about 5 nM, about 4 nM to about 30 nM, about 4 nM to about 25 nM, about 4nM to about 20nM, about 4nM to about 15nM, about 4nM to about 10nM, about 4nM to about 5nM, about 5nM to about 30nM, about 5nM to about 25nM, about 5nM to about 20nM, about 5nM to about 15nM, about 5nM to about 10nM, about 10nM to about 30nM, about 10nM to about 25nM, about 10nM to about 20nM, about 10nM to about 15nM, about 15nM to about 30nM, about 15nM to about 25nM, about 15nM to about 20nM, about 20nM to about 30nM, and about 20nM to about 25nM.

[0128] Any target-binding domain described in this article can bind to the ligand of TGFβRII via the following K... DCombination: 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).

[0129] The Kk of any antigen-binding protein construct described herein can be determined using a variety of different methods known in the art (e.g., electrophoretic mobility variation assays, membrane filtration binding assays, surface plasmon resonance assays, and biomolecular binding kinetic assays). D value.

[0130] Antigen-binding domain

[0131] In some embodiments of any multi-chain chimeric peptides 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 peptides, 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 peptides, the first target-binding domain and the second target-binding domain comprise the same amino acid sequence.

[0132] In some embodiments of any multi-chain chimeric polypeptide described herein, the first target-binding domain and the second target-binding domain specifically bind to different antigens.

[0133] In some embodiments of any multi-chain chimeric peptide described herein, one or both of the first target-binding domain and the second target-binding domain are antigen-binding domains.

[0134] In some embodiments of any multi-chain chimeric polypeptide described herein, the antigen-binding domain includes or is an scFv or single-domain antibody (e.g., a VHH or VNAR domain).

[0135] In some instances, antigen-binding domains (e.g., any antigen-binding domains described herein) can specifically bind to ligands of TGF-βRIIs (see, for example, antigen-binding domains that can specifically bind to TGF-β as described in US2021 / 0061897, US2020 / 0399358, US2020 / 0392221, US2019 / 0315850, US2019 / 0092846, US2021 / 0403545, US2021 / 0355204 and US2019 / 0177406, each of which is incorporated herein by reference).

[0136] The antigen-binding domains present in any multi-chain chimeric polypeptide described herein are each independently selected from: VHH domains, VNAR domains, and scFv. In some embodiments, any antigen-binding domain described herein is BiTe, (scFv)2, nanobodies, nanobodies-HSA, DART, TandAb, sc biantibodies, sc biantibodies-CH3, scFv-CH-CL-scFv, HSA bodies, sc biantibodies-HAS, or tandem-scFv. Other examples of antigen-binding domains that can be used in any multi-chain chimeric polypeptide are known in the art.

[0137] The VHH domain is a single monomeric variable antibody domain found in camelids. The VNAR domain is a single monomeric variable antibody domain found in cartilaginous fish. The VHH domain and V... NARNon-restrictive aspects of the domain are described in, for example, Cromie et al., Curr. Top. Med. Chem. [Current Topics in Medicinal Chemistry] 15:2543-2557, 2016; DeGenst et al., Dev. Comp. Immunol. [Developmental and Comparative Immunology] 30:187-198, 2006; De Meyer et al., Trends Biotechnol. [Trends in Biotechnology] 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. [Trends in Biochemistry] 26:230-235, 2001; Muyldermans, Ann. Rev. Biochem. [Annual Review of Biochemistry] 82:775-797, 2013; Rahbarizadeh et al., Immunol. Invest. [Immunological Research] 40:299-338, 2011; Van Audenhove et al., E BioMedicine [E Biomedicine] 8:40-48, 2016; Van Bockstaele et al., Curr. Opin. Investig. Drugs [Current Views on Drug Research] 10:1212-1224, 2009; Vincke et al., Methods Mol. Biol. [Methods of Molecular Biology] 911:15-26, 2012; and Wesolowski et al., Med. Microbiol. Immunol. [Medical Microbiology and Immunology] 198:157-174, 2009.

[0138] In some embodiments, the antigen-binding domains of the multi-chain chimeric peptides described herein are all VHH domains, or at least one antigen-binding domain is a VHH domain. In some embodiments, the antigen-binding domains of the multi-chain chimeric peptides described herein are all VNAR domains, or at least one antigen-binding domain is a VNAR domain. In some embodiments, the antigen-binding domains of the multi-chain chimeric peptides described herein are all scFv domains, or at least one antigen-binding domain is an scFv domain.

[0139] In some embodiments, two or more polypeptides present in a multi-chain chimeric polypeptide can be assembled (e.g., non-covalently assembled) to form any antigen-binding domain described herein, such as antigen-binding fragments of antibodies (e.g., any antigen-binding fragment of antibodies described herein), VHH-scAb, VHH-Fab, dual scFab, F(ab')2, dual antibody, cross-Mab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common light chain, knobs-in-holes assembly, charge pair, Fab arm exchange, SEED body, LUZ-Y, Fcab, κλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, biantibody-CH3, triplet, microantibody, small body, TriBi small body, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc biantibody-Fc, biantibody-Fc, tandem scFv-Fc, intracellular antibody, dock and lock, lmmTAC, IgG-IgG conjugate, Cov-X-body, and scFv1-PEG-scFv2. See, for example, Spiess et al., Mol. Immunol. [Molecular Immunology] 67:95-106, 2015, the entire contents of which are incorporated herein by reference to describe these elements. Non-limiting examples of antigen-binding fragments of antibodies include Fv fragments, Fab fragments, F(ab')2 fragments, and Fab' fragments. Further examples of antigen-binding fragments of antibodies are antigen-binding fragments of IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., antigen-binding fragments of human or humanized IgG, such as 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, such as human or humanized IgA1 or IgA2); antigen-binding fragments of IgD (e.g., antigen-binding fragments of human or humanized IgD); antigen-binding fragments of IgE (e.g., antigen-binding fragments of human or humanized IgE); or antigen-binding fragments of IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0140] The “Fv” fragment is a non-covalently linked dimer consisting of a heavy chain variable domain and a light chain variable domain.

[0141] In addition to the variable structural domains of the heavy and light chains of the Fv segment, the “Fab” segment also includes the constant structural domain of the light chain and the first constant structural domain (C) of the heavy chain. H1 ).

[0142] The “F(ab')2” segment comprises two Fab segments connected by disulfide bonds near the hinge region.

[0143] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to multivalent and multispecific binding proteins, as described in, for example, DiGiammarino et al., Methods Mol. Biol. [Molecular Biology Methods] 899:145-156, 2012; Jakob et al., MABs [Antibodies] 5:358-363, 2013; and U.S. Patent Nos. 7,612,181; 8,258,268; 8,586,714; 8,716,450; 8,722,855; 8,735,546; and 8,822,645, the entire contents of which are incorporated herein by reference.

[0144] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014. In some embodiments, any antigen-binding domain described herein may bind to antigens selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

[0145] Other examples and aspects of antigen-binding domains are known in the art.

[0146] soluble receptors

[0147] In some embodiments of any multi-chain chimeric polypeptide described herein, one or both of the first and second target-binding domains are soluble interleukin receptors, soluble cytokine receptors, or ligand receptors. In some embodiments, the soluble receptor is a soluble TGF-β receptor II (TGF-βRII) (see, for example, those described in Yung et al., Am. J. Resp. Crit. CareMed. [American Journal of Respiratory and Critical Care Medicine] 194(9):1140-1151, 2016) or a soluble TGF-βRIII (see, for example, those described in Heng et al., Placenta [Placenta] 57:320, 2017). In some embodiments, one or both of the first and second target-binding domains are soluble TGF-β receptor II (TGF-βRII). In some embodiments, the first and second target-binding domains are soluble TGF-βRII.

[0148] In some embodiments, the first target-binding domain comprises a first sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:2, and a second sequence that is at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99% identical to SEQ ID NO:2, wherein the first and second sequences are separated by a connector. In some embodiments, the first target-binding domain comprises the first sequence of SEQ ID NO:2 and the second sequence of SEQ ID NO:2.

[0149] In some embodiments, the first target-binding domain comprises 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% of the sequence identical to SEQ ID NO:4. In some embodiments, the first target-binding domain comprises the sequence of SEQ ID NO:4.

[0150] In some embodiments, the second target-binding domain comprises a first sequence having 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% identity with SEQ ID NO:2, and a second sequence having 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% identity with SEQ ID NO:2, wherein the first and second sequences are separated by a connector. In some embodiments, the second target-binding domain comprises the first sequence of SEQ ID NO:2 and the second sequence of SEQ ID NO:2.

[0151] In some embodiments, the second target-binding domain comprises a sequence having 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% identity with SEQ ID NO:4. In some embodiments, the second target-binding domain comprises the sequence of SEQ ID NO:4.

[0152] Other examples of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0153] Other target-binding domains

[0154] In some embodiments of any multi-chain chimeric polypeptide, the first chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art), wherein at least one of the one or more additional antigen-binding domains is located between a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide may further include a linker sequence (e.g., any exemplary soluble tissue factor domain described herein) between at least one of a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and one or more additional target-binding domains (e.g., any exemplary target-binding domain described herein or known in the art), and / or a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domain described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein).

[0155] In some embodiments of any multi-chain chimeric polypeptide described herein, the first chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains located at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to a first domain of an affinity domain pair in the first chimeric polypeptide (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of the one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art).

[0156] In some embodiments of any multi-chain chimeric peptides described herein, at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is disposed at the N-terminus and / or C-terminus of the first chimeric peptide, and at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is located between a soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein or known in the art) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) disposed at the N-terminus is directly adjacent to the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) or the first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein). In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any linker sequence described herein or known in the art) disposed between at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) and the first target-binding domain (e.g., any exemplary first domain of any exemplary affinity pair described herein). In some embodiments, at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) disposed at the C-terminus is directly adjacent to the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) or the first domain of the affinity pair (e.g., any exemplary first domain of any exemplary affinity pair described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) disposed between at least one additional target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) and the first target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) or the first domain of an affinity pair (e.g., any exemplary first domain described herein of any exemplary affinity pair).In some embodiments, at least one of one or more additional target-binding domains (e.g., any exemplary soluble tissue factor domain described herein) located between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and the first domain of the affinity domain pair (e.g., any first domain described herein or any exemplary affinity domain pair described herein) is directly adjacent to the first domain of the soluble tissue factor domain and / or the first domain of the affinity domain pair. In some embodiments, the first chimeric polypeptide further comprises a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) disposed between: (i) a soluble tissue factor domain (e.g., any exemplary soluble tissue factor described herein) and at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domain described herein or known in the art), the additional target-binding domain being located between the soluble tissue factor domain (e.g., any exemplary soluble tissue factor domain described herein) and a first domain of an affinity domain pair (e.g., any exemplary first domain of any exemplary affinity domain pair described herein), and / or (ii) a first domain of an affinity domain pair and at least one of one or more additional target-binding domains, the additional target-binding domain being located between the soluble tissue factor domain and the first domain of the affinity domain pair.

[0157] In some embodiments of any multi-chain chimeric polypeptide described herein, the second chimeric polypeptide further includes one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) at the N-terminal and / or C-terminal ends of the second chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to a second domain of an affinity pair in the second chimeric polypeptide (e.g., any exemplary second domain of any exemplary affinity pair described herein).

[0158] In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) and a second domain of an affinity domain pair (e.g., any second domain of any exemplary affinity domain pair described herein). In some embodiments, at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) is directly adjacent to a second target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art) in the second chimeric polypeptide. In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., any exemplary linker sequence described herein or known in the art) between at least one of one or more additional target-binding domains (e.g., any exemplary target-binding domains described herein or known in the art) and a second target-binding domain (e.g., any exemplary target-binding domain described herein or known in the art).

[0159] In some embodiments of any multi-chain chimeric polypeptide described herein, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) first target-binding domains, second target-binding domains, and one or more additional target-binding domains specifically bind to the same antigen. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) first target-binding domains, second target-binding domains, and one or more additional target-binding domains specifically bind to the same epitope. In some embodiments, two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) first target-binding domains, second target-binding domains, and one or more additional target-binding domains comprise the same amino acid sequence. In some embodiments, the first target-binding domain, the second target-binding domain, and 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 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 one or more additional target-binding domains each contain the same amino acid sequence.

[0160] In some embodiments of any multi-chain chimeric peptide described herein, a first target-binding domain, a second target-binding domain, and one or more additional target-binding domains specifically bind to different antigens. In some embodiments of any multi-chain chimeric peptide described herein, one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the first target-binding domain, the second target-binding domain, and one or more target-binding domains are antigen-binding domains. In some embodiments, the first target-binding domain, the second target-binding domain, and one or more additional target-binding domains are each an antigen-binding domain (e.g., scFv or a single-domain antibody).

[0161] Affinity domain pairs

[0162] 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 and second chimeric polypeptides are associated through the binding of the first and second domains of the affinity domain pair. In some embodiments, the affinity domain pair is the sushi domain of the α chain of the human IL-15 receptor (IL15Rα) and soluble IL-15. The sushi domain, also known as a short concordant repeat or type 1 glycoprotein motif, is a common motif in protein-protein interactions. Sushi domains have been found on many protein-binding molecules, including complement components C1r, C1s, factor H, and C2m, as well as non-immune molecules factor XIII and β2-glycoprotein. A typical sushi domain has approximately 60 amino acid residues and contains four cysteine ​​residues (Ranganathan, Pac. Symp Biocomput. [Pacific Biocomputation Symposium] 2000: 155-67). The first cysteine ​​residue can form a disulfide bond with the third cysteine ​​residue, and the second cysteine ​​residue can form a disulfide bridge with the fourth cysteine ​​residue. In some embodiments where one member of the affinity domain pair is soluble IL-15, the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments where one member of the affinity domain pair is the α chain of the human IL-15 receptor (IL15Rα), the human IL15Rα is the mature, full-length IL15Rα. In some embodiments, the affinity domain pair is Bacillus RNase and Barnstar. In some embodiments, the affinity domain pair is PKA and AKAP. In some embodiments, the affinity domain pair is based on an adaptor / docking tag module of a mutated RNase I fragment (Rossi, ProcNatl Acad Sci USA. [Proceedings of the National Academy of Sciences] 103:6841-6846, 2006; Sharkey et al., Cancer Res. [Cancer Research] 68:5282-5290, 2008; Rossi et al., Trends Pharmacol Sci. [Trends in Pharmacology] 33:474-481, 2012) or on a SNARE module based on the interaction between synaptic fusion proteins, synaptic binding proteins, small synaptic vesicle proteins, and SNAP25 (Deyev et al., Nat Biotechnol. [Nature Biotechnology] 1486-1492, 2003).

[0163] In some embodiments, a first chimeric polypeptide of a multi-chain chimeric polypeptide includes a first domain of an affinity domain pair, and a second chimeric polypeptide of a multi-chain chimeric polypeptide includes a second domain of an affinity domain pair, wherein the first domain of the affinity domain pair and the second domain of the affinity domain pair are less than 1 x 10^646 ...-7 M, less than 1x10 -8 M, less than 1x10 -9 M, less than 1x10 -10 M, less than 1x10 -11 M, less than 1x10 -12 M, or less than 1x10 -13 The dissociation equilibrium constant (KD) of M is bound to each other. In some embodiments, the first domain of the affinity domain pair and the second domain of the affinity domain pair are bounded by the following K... D Combined: Approximately 1x10 -4 M to approximately 1x10 -6 M, approximately 1x10 -5 M to approximately 1x10 -7 M, approximately 1x10 -6 M to approximately 1x10 -8 M, approximately 1x10 -7 M to approximately 1x10 -9 M, approximately 1x10 -8 M to approximately 1x10 -10 M, approximately 1x10 -9 M to approximately 1x10 -11 M, approximately 1x10 -10 M to approximately 1x10 -12 M, approximately 1x10 - 11 M to approximately 1x10 -13 M, approximately 1x10 -4 M to approximately 1x10 -5 M, approximately 1x10 -5 M to approximately 1x10 -6 M, approximately 1x10 -6 M to approximately 1x10 -7 M, approximately 1x10 -7 M to approximately 1x10 -8 M, approximately 1x10 -8 M to approximately 1x10 -9 M, approximately 1x10 -9 M to approximately 1x10 -10 M, approximately 1x10 -10 M to approximately 1x10 - 11 M, approximately 1x10 -11 M to approximately 1x10 -12 M, or approximately 1x10 -12 M to approximately 1x10 -13M (inclusive). Any of the various methods known in the art (e.g., electrophoretic mobility variation measurement, membrane filtration binding measurement, surface plasmon resonance and biomolecular binding kinetics measurement, etc.) can be used to determine K, the binding of the first domain and the second domain of the affinity domain pair. D value.

[0164] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide includes a first domain of an affinity domain pair, and the second chimeric polypeptide of the multi-chain chimeric polypeptide includes a second domain of an affinity domain pair, wherein the length of the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both is about 10 to 100 amino acids. For example, the length of the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both can be about 10 to 100 amino acids 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, or about 55 to 100 amino acids long. 100 amino acids long, approximately 60 to 100 amino acids long, approximately 65 to 100 amino acids long, approximately 70 to 100 amino acids long, approximately 75 to 100 amino acids long, approximately 80 to 100 amino acids long, approximately 85 to 100 amino acids long, approximately 90 to 100 amino acids long, approximately 95 to 100 amino acids long, approximately 10 to 95 amino acids long, approximately 10 to 85 amino acids long, approximately 10 to 80 amino acids long. The amino acid length is approximately 10 to 75 amino acids, approximately 10 to 70 amino acids, approximately 10 to 65 amino acids, approximately 10 to 60 amino acids, approximately 10 to 55 amino acids, approximately 10 to 50 amino acids, approximately 10 to 45 amino acids, approximately 10 to 40 amino acids, approximately 10 to 35 amino acids, approximately 10 to 30 amino acids, approximately 10 to 25 amino acids, approximately 10 to 20 amino acids, approximately 10 to 15 amino acids. The length of the amino acid is approximately 20 to 30 amino acids, approximately 30 to 40 amino acids, approximately 40 to 50 amino acids, approximately 50 to 60 amino acids, approximately 60 to 70 amino acids, approximately 70 to 80 amino acids, approximately 80 to 90 amino acids, approximately 90 to 100 amino acids, approximately 20 to 90 amino acids, approximately 30 to 80 amino acids, approximately 40 to 70 amino acids, approximately 50 to 60 amino acids, or any range between these lengths. In some embodiments, the length of the first domain of the affinity domain pair, the second domain of the affinity domain pair, or both is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0165] In some embodiments, either the first and / or second domains of the affinity domain pair disclosed herein may include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more amino acids) at their N-terminus and / or C-terminus, provided that the function of the first and / or second domains of the affinity domain pair remains intact. For example, the sushi domain of the α-chain from the human IL-15 receptor (IL15Rα) may include one or more additional amino acids at its N-terminus and / or C-terminus while still retaining the ability to bind soluble IL-15. Alternatively or additionally, 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 of the α-chain from the human IL-15 receptor (IL15Rα).

[0166] Non-limiting examples of the sushi domain of the α chain from the IL-15 receptor α (IL15Rα) may include sequences having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity with ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO:16). In some embodiments, the sushi domain of the α chain from IL15Rα may be encoded by a nucleic acid comprising the following: ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO:17).

[0167] In some embodiments, soluble IL-15 may include a sequence having at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity with NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:18). In some embodiments, soluble IL-15 may be encoded by a nucleic acid comprising the following sequence: AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (SEQ ID NO:19).

[0168] In some embodiments, soluble IL-15 may include a D8N amino acid substitution. In some embodiments, soluble IL-15 (IL15D8N) having a D8N mutant may contain a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% identity with NWVNVISNLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO:62). In some embodiments, soluble IL-15 (IL15D8N) having a D8N mutant (e.g., SEQ ID NO:62) may be encoded by a nucleic acid containing the following sequence:

[0169] AACTGGGTGAATGTAATAAGTAATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAG TATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT(SEQ ID NO:63).

[0170] signal sequence

[0171] In some embodiments, the multi-chain chimeric polypeptide includes a first chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, the multi-chain chimeric polypeptide includes a second chimeric polypeptide comprising a signal sequence at its N-terminus. In some embodiments, both the first and second chimeric polypeptides of the multi-chain chimeric polypeptide comprise a signal sequence. Those skilled in the art will understand that a signal sequence is an amino acid sequence present at the N-terminus of many endogenously produced proteins that guides the protein into secretory pathways (e.g., guiding the protein to reside in certain intracellular organelles, reside in the cell membrane, or be secreted from the cell). Signal sequences are heterogeneous and their primary amino acid sequences vary considerably. However, signal sequences are typically 16 to 30 amino acids in length and include a hydrophilic, typically positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region containing a signal peptidase cleavage site.

[0172] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide include a signal sequence having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO:20). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide include a signal sequence encoded by the nucleic acid sequences ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO:21), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (SEQ ID NO:22), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (SEQ ID NO:23).

[0173] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide include a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 24). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide include a signal sequence having the amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO: 25). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide include a signal sequence having the amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 26). In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide include a signal sequence having the amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO:27). Those skilled in the art will recognize other suitable signal sequences that can be used in the first chimeric polypeptide and / or the second chimeric polypeptide of the multi-chain chimeric polypeptide described herein.

[0174] In some embodiments, the first chimeric polypeptide of the multi-chain chimeric polypeptide, the second chimeric polypeptide of the multi-chain chimeric polypeptide, or both contain a signal sequence of about 10 to 100 amino acids in length. For example, the length of the signal sequence can be approximately 10 to 100 amino acids, approximately 15 to 100 amino acids, approximately 20 to 100 amino acids, approximately 25 to 100 amino acids, approximately 30 to 100 amino acids, approximately 35 to 100 amino acids, approximately 40 to 100 amino acids, approximately 45 to 100 amino acids, approximately 50 to 100 amino acids, approximately 55 to 100 amino acids, approximately 60 to 100 amino acids, approximately 65 to 100 amino acids, approximately 70 to 100 amino acids, approximately 75 to 100 amino acids, approximately 80 to 100 amino acids, approximately 85 to 100 amino acids, approximately 90 to 100 amino acids, approximately 95 to 100 amino acids, approximately 10 to 95 amino acids, approximately 10 to 85 amino acids, approximately 10 to 80 amino acids, approximately 10 to 75 amino acids. 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 between these. In some embodiments, the signal sequence length is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0175] In some embodiments, any signal sequence disclosed herein may contain one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10 or more amino acids) at its N-terminus and / or C-terminus, provided that the function of the signal sequence remains intact. For example, a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO:28) may contain one or more additional amino acids at its N-terminus or C-terminus while still retaining the ability to guide the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide into the secretory pathway.

[0176] In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of the multi-chain chimeric polypeptide contain a signal sequence that directs the multi-chain chimeric polypeptide into the extracellular space. Such embodiments can be used to produce multi-chain chimeric polypeptides that are relatively easy to isolate and / or purify.

[0177] peptide tags

[0178] In some embodiments, the multi-chain chimeric polypeptide comprises a first chimeric polypeptide containing a peptide tag (e.g., at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, the multi-chain chimeric polypeptide comprises a second chimeric polypeptide containing a peptide tag (e.g., at the N-terminus or C-terminus of t...

Claims

1. A method for regulating the expression of one or more circadian clock genes in a tissue of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of the affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of the affinity domain pair; and (ii) a second target binding domain, in: The first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the affinity domain pair; and The first target binding domain and the second target binding domain each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

2. A method for treating a circadian clock gene disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide, the multi-chain chimeric polypeptide comprising: (a) 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, in: The first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the affinity domain pair; and The first target binding domain and the second target binding domain each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

3. A method for reducing the progression rate of a circadian clock gene disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a multi-chain chimeric polypeptide comprising: (a) a first chimeric polypeptide comprising: (i) a first target binding domain; (ii) a soluble tissue factor domain; and (iii) a first domain of the affinity domain pair; (b) a second chimeric polypeptide comprising: (i) a second domain of the affinity domain pair; and (ii) a second target binding domain, in: The first chimeric polypeptide and the second chimeric polypeptide are associated through binding of the first domain and the second domain of the affinity domain pair; and The first target binding domain and the second target binding domain each specifically bind to a ligand of TGF-β receptor II (TGF-βRII).

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

5. The method of any one of claims 1-3, wherein the first chimeric polypeptide further comprises a linker sequence between the first target binding domain and the soluble tissue factor domain in the first chimeric polypeptide.

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

7. The method of any one of claims 1-5, wherein the first chimeric polypeptide further comprises a linker sequence between the soluble tissue factor domain in the first chimeric polypeptide and the first domain of the affinity domain pair.

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

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

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

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

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

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

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

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

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

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

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

1.

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

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

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

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

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

2.

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

2.

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

3.

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

4.

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

4.

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

4.

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

6.

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

6.

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

6.

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

7.

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

68.

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

70.

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

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

2.

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

2.

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

3.

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

4.

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

4.

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

4.

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

5.

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

5.

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

5.

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

8.

46. ​​The method of any one of claims 1-45, wherein administration of the multi-chain chimeric polypeptide causes an increase in the expression level of one or more of Per, Cry, Nrld1, Nrld2, Tef, and Dbp compared to the one or more expression levels in a similar subject not administered the multi-chain chimeric polypeptide.

47. The method of any one of claims 1-46, wherein administration of the multi-chain chimeric polypeptide results in a decrease in the expression level of one or all of Arnt1, Npas2, and Ppard compared to one or more expression levels in a similar subject not administered the multi-chain chimeric polypeptide.

48. The method of any one of claims 1 and 4-47, wherein the tissue is cardiac tissue, kidney tissue, spleen tissue, pancreatic tissue, intestinal tissue, brain tissue and / or liver tissue.

49. The method of any one of claims 1 and 4-48, wherein administration of the multi-chain chimeric polypeptide results in increased expression of one or more of: Per, Cry, Nrld1, Nrld2, Tef, and Dbp.

50. The method of any one of claims 1 and 4-49, wherein administration of the multi-chain chimeric polypeptide results in decreased expression of one or more of: Arnt1, Npas2, and Ppard.

51. The method of any one of claims 1-50, wherein the subject has been diagnosed or identified as having a circadian clock gene disorder.

52. The method of any one of claims 1-50, wherein the subject has been identified as having an increased risk of developing a circadian clock gene disorder.

53. The method of claim 51 or 52, 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 disorders, cognitive disorders, schizophrenia, obesity, diabetes, Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), metabolic disorders, cancer, cardiovascular disease, and sleep disorders, physical impairment associated with aging and sarcopenia.

Images