Methods for treating age-related and inflammatory diseases

Abstract

The present invention provides a pharmaceutical composition for treating cancer in a given subject. [Solution] A pharmaceutical composition is provided comprising (i) a therapeutically effective amount of a polychain chimeric polypeptide; and (ii) a therapeutically effective amount of dexamethasone, wherein the polychain chimeric polypeptide comprises a first chimeric polypeptide comprising (a) a first target-binding domain consisting of a specific sequence; a soluble tissue factor domain consisting of a specific sequence; and a first domain of a pair of affinity domains consisting of a specific sequence; and (b) a second chimeric polypeptide comprising a second target-binding domain consisting of a specific sequence; and a second domain of a pair of affinity domains consisting of a specific sequence, wherein the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first and second domains of the pair of affinity domains.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 975,141, filed on 11 February 2020, which is incorporated in its entirety by reference herein.

[0002] Technical field This disclosure relates to the field of biotechnology, more specifically to methods for treating inflammatory and age-related diseases. [Background technology]

[0003] background Human aging is associated with increased systemic inflammation (Ferrucci et al., Blood 105(6):2294-2299, 2005; Dinarello, Am J Clin Nutr 83(2):447S-455S, 2006). The process linking inflammation and aging is called inflamm-aging (Franceschi et al., Ann NY Acad Sci 908:244-254, 2000). Inflamm-aging is characterized by a state of chronic, low-grade sterile inflammation, leading to the accumulation of senescent cells and persistent activation of inflammasomes. The aging process is associated with significant changes that affect the immune system and cause various age-related pathologies.

[0004] Senescent cells are associated with, for example, glaucoma (Liton et al., 2005), idiopathic pulmonary fibrosis (IPF) (Yanai et al., 2015, Schafer et al., 2017), atherosclerosis (Uryga and Bennett, 2016, Childs et al., 2016), and cirrhosis / NAFLD (Krizhanovsky et al., 2008, Kim et al. al., 2013, Ogrodnik et al., 2017, Wiemann et al., 2002), glomerulosclerosis (Melk et al., 2003, Melk et al., 2004, Maker et al., 2016), type 2 diabetes (Chen et al., 2009, Helman et al., 2016), cachexia (Berry et al. al., 2017, Xu et al. It is involved in the pathogenesis of several different diseases, including (al., 2015, Baker et al., 2016), sarcopenia (Sousa-Victor et al., 2014, Cosgrove et al., 2014, Chang et al., 2016), osteoarthritis (Price et al., 2002, Kuyinu et al., 2016, Jeon et al., 2017), cancer (Latz et al., Sem.Immunol. 40:61-73, 2018), arthritis (Latz et al., Sem.Immunol. 40:61-73, 2018), and neurodegenerative diseases (Latz et al., Sem.Immunol. 40:61-73, 2018).

[0005] The function of the immune system is to detect and respond to tissue damage or invasion by pathogenic microorganisms. Innate immune cells, the first line of defense against infection, express germline-encoded pattern recognition receptors (PRRs) that recognize conserved pathogen-associated molecular patterns (PAMPs) specific to microorganisms (Janeway, Cold Spring Harb Symp Quant Biol 54 Pt 1:1-13, 1989; Gong et al., Nat Rev Immunol 20(2):95-112, 2020). Danger signals released by infected or damaged cells are also recognized by damage-associated molecular pattern (DAMP) receptors (Gong et al., Nat Rev Immunol 20(2):95-112, 2020). Both PAMP and DAMP can initiate an innate immune response by activating classical PRRs such as Toll-like receptors (TLRs), as well as germline-encoded receptors such as NOD-like receptors (NLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), C-type lectin receptors (CLRs), and intracellular DNA sensors (Cao, Nat. Rev. Immunol. 16(1):35-50, 2016). DAMP can also be sensed by several other receptors. These include advanced glycation end product (RAGE) receptors (Hudson et al., Annu Rev Med 69:349-364, 2018; Teissier et al., Biogerontology 20(3):279-301, 2019), trigger receptors (TREMs) expressed in bone marrow cells (Ford et al., Curr Opin Immunol 21(1):38-46, 2009), several G protein-coupled receptors (GPCRs) (Heng et al., Annu Rev Pharmacol Toxicol 54:227-249, 2014; Weiss et al., Trends Immunol 39(10):815-829, 2018), and ion channels (Eisenhut et al., Pflugers Arch 461(4):401-421, 2011).

[0006] The inflammatory response initiated by DAMP is unrelated to pathogen infection and is therefore called aseptic inflammation (Chen et al., Nat. Rev. Immunol. 10(12):826-837, 2010). DAMP can activate both non-immune cells and innate immune cells (Chen et al., Nat. Rev. Immunol. 10(12):826-837, 2010). The activation of these cells leads to the production of cytokines and chemokines, which then recruit inflammatory cells and activate an adaptive immune response (Chen et al., Nat. Rev. Immunol. 10(12):826-837, 2010). Some DAMPs are also known to directly activate adaptive immune cells (Lau et al., J Exp Med 202(9):1171-1177, 2005; Qin et al., J Immunol 199(1):72-81, 2017). While aseptic inflammation plays a crucial role in tissue repair and regeneration to re-establish tissue hemostasis after harmful injury, unresolved chronic inflammation resulting from repeated tissue damage or in response to an excess of innate immune triggers present in the tissue is detrimental to the host and can lead to aseptic inflammatory diseases, including cancer, metabolic diseases (e.g., diabetes), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease), and autoimmune diseases (e.g., multiple sclerosis) (Roh et al., Immune Netw 18(4):e27, 2018).

[0007] Inflammasomes are large multimeric protein complexes containing cytoplasmic pattern recognition receptors, adapter proteins including caspase recruitment domains (ASCs), apoptosis-related Speck-like proteins, and caspase-1 (Lamkanfi et al., Cell 157(5):1013-1022, 2014). Their assembly in innate immune cells and other cells is triggered by various stimuli, leading to the activation of caspase-1, which subsequently cleaves pro-IL-1β into IL-1β (Latz et al., Nat Rev Immunol 13(6):397-411, 2013; Walsh et al., Nat Rev Neurosci 15(2):84-97, 2014). A diverse range of inflammasomes have been discovered to date. Among the various inflammasomes identified, the pyrin domain-containing 3 (NLRP3) inflammasome of the nucleotide-binding oligomerized domain leucine-rich repeat-containing receptor (NLR) family is the best characterized (Swanson et al., Nat Rev Immunol 19(8):477-489, 2019). NLRs are recognized as important sensors for pathogens and danger signals. The NLRP3 inflammasome has a two-step activation mechanism: "priming" involving the induction of pro-IL-1β and NLRP3, and "activation" in which a functional inflammasome complex is assembled after the uptake of PAMP or DAMP. The pathologies of various diseases, including Alzheimer's disease (Heneka et al., Nature 493(7434):674-678, 2013), Parkinson's disease (Heneka et al., Nat Rev Neurosci 19(10):610-621, 2018), and atherosclerosis (Jin et al., J Am Heart Assoc 8(12):e012219, 2019), are associated with hyperactivation of the NLRP3 inflammasome.

[0008] Aseptic inflammation can also arise from the accumulation of senescent cells. Cellular senescence is defined as irreversible cell cycle arrest that occurs in response to cellular stress and prevents the transmission of defects to the next generation (Collado et al., Nat. Rev. Cancer 10(1):51-57, 2010; McHugh et al., J Cell Biol 217(1):65-77, 2018). Cellular senescence plays an important protective role in development, tissue hemostasis, and wound healing (Munoz-Espin et al., Cell 155(5):1104-1118, 2013; Storer et al., Cell 155(5):1119-1130, 2013; Demaria et al., Dev Cell 31(6):722-733, 2014; Yun et al., Elife 4, 2015). Cellular senescence is accompanied by a pro-inflammatory phenotype, known as the senescence-associated secretory phenotype (SASP) (McHugh et al., J Cell Biol 217(1):65-77, 2018). SASP is characterized by the release of inflammatory cytokines, chemokines, growth factors, and proteases. This enhances cellular senescence through autocrine and paracrine signaling, instructing immune cells to mobilize and eliminate senescent cells. Therefore, cellular senescence and SASP are crucial physiological responses that maintain homeostasis at the cellular, tissue, and organ levels. However, when damage persists or senescence progresses, the elimination of senescent cells is impaired, and dysfunctional cells accumulate. These uneliminated, accumulated SASPs derived from senescent cells are a persistent, long-term source of inflammatory factors that create an inflammatory microenvironment, resulting in a variety of pathological manifestations (Munoz-Espin et al., Nat Rev Mol Cell Biol 15(7):482-496, 2014; van Deursen, Nature 509(7501):439-446, 2014; McHugh et al., J Cell Biol 217(1):65-77, 2018). In addition, some SASP factors stimulate inflammasome-containing cells, thereby increasing the risk of inducing aseptic inflammation by stimulating long-term inflammasome activation.

[0009] Atherosclerosis is a chronic inflammatory disease resulting from an imbalance in lipid metabolism and an maladaptive immune response caused by the accumulation of cholesterol-containing macrophages in the arterial walls. Dyslipidemia increases the number of circulating monocytes recruited to mouse atherosclerotic plaques in a multi-step process involving chemokine-chemokine receptor pairs and endothelial adhesion molecules, such as selectins and adhesion molecules. The recruited monocytes differentiate into macrophages or dendritic cells in the intima, where they take up atherogenic lipoproteins via macropinocytosis or scavenger receptor-mediated pathways. The resulting foam cells secrete pro-inflammatory cytokines and chemokines, amplifying the inflammatory response, and also secrete retention factors that promote macrophage chemotaxis. These accumulating macrophages experience endoplasmic reticulum stress, which, if prolonged, triggers apoptosis. This cell death, coupled with defective efferocytosis, leads to the formation of a necrotic core, a characteristic of progressive plaques (Moore et al., Nat Rev Immunol 13:709-721, 2013).

[0010] In humans, low circulating Treg levels are associated with an increased risk of acute coronary insufficiency syndrome, and stable plaques contain more Tregs than unstable plaques (Dietel et al., Atherosclerosis 230:92-99, 2013). These findings are supported by studies in a hypercholesterolemia mouse model that showed a decrease in the number of Tregs in circulating blood and plaque, as well as a reduced inhibitory function during the progression of atherosclerosis (Maganto-Garcia et al., Circulation 124:185-195, 2011). Notably, in a mouse model of atherosclerosis progression using anti-CD25 antibodies, Treg depletion or diphtheria toxin target depletion in FoxP3+ cells exacerbates the disease (Klingenberg et al., J Clin Invest. 123:1323-1334, 2013), while adoptive Treg transplantation halts disease progression (Ait-Oufella el al., Nat Med. 12:178-180, 2006). Recent studies suggest that Tregs enable atherosclerosis regression by suppressing the pro-inflammatory response of macrophages and T cells in progress, and by retraining macrophages into a pro-degradation state that promotes tissue repair and plaque contraction. Tregs are essential for the enrichment of M2-like macrophages in degenerative plaques and for licensing key pro-degradation macrophage functions, such as the elimination of apoptotic cells, the production of specific pro-degradation lipid mediators, and the upregulation of receptors that sense these mediators. Treg-derived cytokines such as IL-10 and TGF-β can suppress the inflammatory response of macrophages, promote selective activation, and increase efferocytosis. M2 macrophages can also secrete IL-10 and TGF-β, which may then maintain iTregs, and this interaction may be synergistic in promoting tissue repair in plaques.The Treg-dependent increase in efferocytosis and decrease in necrotic core area in degenerative plaques, corresponding to an increase in smooth muscle cells in the fibrous capsule, suggests that Tregs are a key contributing factor in enabling tissue repair functions that promote plaque stability (Sharma et al., Circ Res. 127:335-353, 2020).

[0011] Regulatory T (Treg) cells are essential mediators of peripheral tolerance to self and non-self antigens (Sakaguchi et al., Cell 133(5):775-787, 2008; Sakaguchi et al, Annu Rev Immunol. 38:541-566, 2020). Treg cells are cells with innate immunity, antigen-presenting cell (APC) function, and adaptive B, CD4 + , or CD8 +This immunomodulatory control is achieved through multiple inhibitory mechanisms that inhibit the effector T (Teff) cell response (Sakaguchi et al., Int Immunol 21(10):1105-1111, 2009). Treg cells play a central role in overall immunomodulation in the host. Changes in the development, homeostasis, or function of Treg cells can make these cells susceptible to a variety of conditions, including allergies, autoimmunity, graft rejection, cancer, and responses to immunotherapy (Sakaguchi et al., Annu Rev Immunol. 38:541-566, 2020). Current research focuses on the development of novel therapies that enhance Treg cell function in vivo using cytokines and small molecular weight drugs to support endogenous Treg cell proliferation or activation, ex vivo engineered Treg cells in autologous adoptive cell therapy (ACT) that promotes immunomodulation under autoimmune conditions, or antigen-specific Treg cells such as chimeric antigen receptor Treg (CAR-Treg) cells that enhance resistance to allergic inflammation (Ferreira et al., Nat Rev Drug Discov 18(10):749-769, 2019). Excellent safety profiles have been demonstrated in patients receiving Treg cells (Esensten et al., J Allergy Clin Immunol 142(6):1710-1718, 2018).Early clinical studies have used Treg cells to treat acute and chronic graft-versus-host diseases (Brunstein et al., Blood 117(3):1061-1070, 2011; Di Ianni et al., Blood 117(14):3921-3928, 2011; Martelli et al., Blood 124(4):638-644, 2014; Theil et al., Cytotherapy 17(4):473-486, 2015; Brunstein et al., Blood 127(8):1044-1051, 2016), autoimmune diseases, and neurodegenerative diseases (Thonhoff et al., Neurol Neuroimmunol Neuroinflamm 5(4):e465, 2018; Dall'Era et al., Arthritis Rheumatol Promising results were shown in the prevention and treatment of (71(3):431-440, 2019). [Overview of the project]

[0012] overview Regulatory T (Treg) cells are essential mediators of peripheral tolerance to self and non-self antigens and overall immunomodulation in the host (Sakaguchi et al., Cell 133(5):775-787, 2008; Sakaguchi et al, Annu Rev Immunol. 38:541-566, 2020). Treg cells achieve this immunomodulatory control through multiple inhibitory mechanisms. These include IL-2 deprivation, secretion of inhibitory cytokines (i.e., IL-10 and TGF-β), and acquisition of costimulatory molecules from antigen-presenting cells through high-affinity binding to CTLA-4 (Oberle et al., J Immunol 179(6):3578-3587, 2007; Tang et al., Nat Immunol 9(3):239-244, 2008; Zheng et al., J Immunol 181(3):1683-1691, 2008). The adenosine triphosphate (ATP)-adenosine pathway is also utilized by regulatory Tregs as an important modulator of innate and adaptive immunity.

[0013] CD39 is a major ectonucleotidase widely expressed in immune cells (e.g., Tregs), endothelial cells, and tumor cells, and hydrolyzes ATP and adenosine diphosphate (ADP) to adenosine monophosphate (AMP) (Moesta et al., Nat Rev Immunol 20(12):739-755, 2020). Subsequently, AMP is hydrolyzed to adenosine by CD73. Adenosine binds to its receptors A1, A2A, A2B, and A3, which are presented to immune cells. The A2A and A2B receptors (A2AR and A2BR) are Gs-coupled receptors that increase intracellular cAMP and PKA levels and play a major role in adenosine-induced immunosuppression in a cAMP-dependent manner. A1 and A3 receptors (A1R and A3R) are Gi / o-coupled receptors that reduce intracellular cAMP, which is favorable for cell activation, and are therefore generally considered to be immunosuppressive adenosine receptors. In humans, A1R, A2AR, and A3R exhibit high affinity for adenosine, while A2BR has significantly lower affinity. A2A and A2BR are expressed in immune cells (Feng et al., Cancer Cell Int 20:110, 2020). Recently, human CD39 hiRegulatory T cells have been shown to exhibit greater stability, higher Foxp3 expression, and suppressive capacity under inflammatory conditions (Gu et al., Cell Mol Immunol 14(6):521-528, 2017). Changes in the development, homeostasis, or function of Treg cells may make these cells more susceptible to various pathological conditions, including allergies, autoimmunity, graft rejection, cancer, and responses to immunotherapy (Sakaguchi et al, Annu Rev Immunol. 38:541-566, 2020). Current research focuses on the development of novel therapies that enhance Treg cell function in vivo using cytokines and low molecular weight drugs to support endogenous Treg cell proliferation or activation, ex vivo engineered Treg cells in autologous adoptive cell therapy (ACT) that promotes immunomodulation under autoimmune conditions, or antigen-specific Treg cells such as chimeric antigen receptor Treg (CAR-Treg) cells that enhance resistance to allergic inflammation (Ferreira et al., Nat Rev Drug Discov 18(10):749-769, 2019). The present invention is a method for treating inflammatory senescence and / or any aging-related conditions by using Treg cells to inactivate inflammasomes and then using immune cells activated by one or more immunotherapies to reduce senescent cells accumulated in the individual. A method is provided that suppresses inflammasome-related diseases and senescence-related diseases. The Treg cells may be in vivo enhanced endogenous Treg cells or ex vivo engineered Treg cells used under ACT conditions. Immune cells for eliminating senescent cells can be activated in vivo by immunotherapy or generated by ex vivo stimulation and proliferation methods to support ACT administration.

[0014] The present invention suppresses inflammasome activity (e.g., using 2t2, anti-tissue factor antibodies, RAGE (Advanced Glycation End Product Trap), anti-CD36 antibodies, or adoptive cell therapy (e.g., using immune cells treated with 2t2 and 3t28, anti-tissue factor CAR-Treg cells, or anti-CD36 CAR Treg cells) and reduces the accumulation of senescent cells (e.g., using anti-tissue factor antibodies, anti-CD26 antibodies, or anti-CD36 antibodies, optionally using TGFRt15-TGFRs, TGFRt15-TGFRs, adoptive cell therapy (e.g., immune cells treated with 18t15-12s, immune cells treated with 18t15-12s and 7t15-21s, anti-tissue factor CAR NK cells, or anti-CD26 CAR) This method utilizes approaches that reduce inflammation by further using NK cells. This method includes immunotherapy, mAbs targeting inflammation and senescence, and various combinations of activated and / or manipulated NK and T cells, attacking inflammatory senescence through multiple channels to eliminate the direct causes of inflammation and the underlying development and persistence of these causes. For example, an anti-inflammatory approach against the inflammasome is facilitated by regulatory T cells. In addition, a senescent cell approach may be NK cell-mediated. This method addresses both chronic inflammatory activity, a major factor in inflammatory senescence, and the underlying contribution of the accumulation of senescent cells that produce senescence-associated secretory phenotype (SASP) factors that maintain inflammasome activity.

[0015] Methods for treating inflammasome-associated disorders and senescent cell-associated disorders are provided. Treg cells may be in vivo enhanced endogenous Treg cells or ex vivo engineered Treg cells used under ACT conditions. Immune cells for senescent cell elimination may be activated in vivo by immunotherapy or generated by ex vivo stimulation and proliferation methods to support ACT administration.

[0016] A method for treating age-related diseases or inflammatory diseases in a subject is provided herein, comprising administering to the subject (i) a therapeutically effective amount of an NK cell activator and / or NK cells and / or monoclonal antibody, and (ii) a therapeutically effective amount of a Treg cell activator and / or Treg cells and / or monoclonal antibody and / or advanced glycation end product (AGE) inhibitor.

[0017] In some embodiments of the methods described herein, the age-related disease is inflammatory aging-related. In some embodiments of the methods described herein, (i) is administered to the subject substantially simultaneously with (ii). In some embodiments of the methods described herein, (i) is administered to the subject before (ii) is administered to the subject. In some embodiments of the methods described herein, (ii) is administered to the subject before (i) is administered to the subject.

[0018] In some embodiments of the methods described herein, the method comprises administering a therapeutically effective dose of NK cells to a subject. In some embodiments of the methods described herein, the NK cells are autologous, haplotype-matched, or allogeneic NK cells isolated from peripheral blood, isolated from umbilical cord blood, or isolated and differentiated from iPSCs. In some embodiments of the methods described herein, the methods further comprise isolating NK cells from a subject and culturing the isolated NK cells in a liquid culture medium under conditions sufficient to induce or increase NK cell proliferation, wherein the NK cells are administered to the subject after the isolation and culture steps. In some embodiments of the methods described herein, the liquid culture medium comprises a multi-chain chimeric polypeptide. In some embodiments of the methods described herein, the NK cells comprise a chimeric antigen receptor. In some embodiments of the methods described herein, the chimeric antigen receptor comprises an extracellular domain that specifically binds to tissue factor or CD26.

[0019] In some embodiments of the methods described herein, the method comprises administering a therapeutically effective amount of an NK cell activator and / or a monoclonal antibody to a subject. In some embodiments of the methods described herein, the NK cell activator is one or more polychain chimeric polypeptides. In some embodiments of the methods described herein, the monoclonal antibody is one or more anti-tissue factor antibodies and / or anti-CD26 antibodies. In some embodiments of the methods described herein, the NK cell activator comprises one or more polychain chimeric polypeptides, and the monoclonal antibody comprises one or more anti-tissue factor antibodies and / or anti-CD26 antibodies.

[0020] In some embodiments of the methods described herein, the method comprises administering a therapeutically effective dose of Treg cells to a subject. In some embodiments of the methods described herein, the Treg cells are autologous Treg cells, haplotype-matched Treg cells, or allogeneic Treg cells isolated from peripheral blood or umbilical cord blood. In some embodiments of the methods described herein, the method further comprises culturing the isolated Treg cells in a liquid culture medium under conditions sufficient to induce or increase Treg cell proliferation, wherein the Treg cells are administered to a subject after the isolation and culture steps.

[0021] In some embodiments of any of the methods described herein, the step of isolating Treg cells from a subject comprises obtaining a sample comprising Treg cells from the subject and isolating the Treg cells from the sample using an antibody or ligand capable of binding to CD39. In some embodiments of any of the methods described herein, the step of isolating Treg cells from a sample comprises mixing the sample with an antibody or ligand capable of binding to CD39 under conditions that allow binding of the antibody of the ligand to Treg cells expressing CD39 and separating the Treg cells bound to the antibody or ligand from other components in the sample, thereby isolating the Treg cells. In some embodiments of any of the methods described herein, the antibody is a mouse, humanized, or human antibody or an antigen-binding fragment thereof, and / or the antibody or ligand is labeled with at least one of biotin, avidin, streptavidin, or a fluorescent dye or is bound to a particle, bead, resin, or solid support. In some embodiments of any of the methods described herein, the separation comprises the use of flow cytometry, fluorescence-activated cell sorting (FACS), centrifugation, or a column, plate, particle, or bead-based method.

[0022] In some embodiments of any of the methods described herein, the autologous Treg cells, haplotype-matched Treg cells, or allogeneic Treg cells are isolated from a sample comprising fresh or frozen peripheral blood, umbilical cord blood, peripheral blood mononuclear cells, lymphocytes, CD4 + T cells, or Treg cells. In some embodiments of any of the methods described herein, the Treg cells are CD4 + CD25 + Foxp3 + cells. In some embodiments of any of the methods described herein, the Treg cells are CD4 + CD25 + CD127dim -These are cells. In some embodiments of the methods described herein, Treg cells are immunosuppressive in vitro and in vivo.

[0023] In some embodiments of the methods described herein, the liquid culture medium comprises one or more single-chain chimeric polypeptides. In some embodiments of the methods described herein, the Treg cells comprises a chimeric antigen receptor. In some embodiments of the methods described herein, the chimeric antigen receptor comprises an extracellular domain that specifically binds to tissue factor or CD36.

[0024] In some embodiments of the methods described herein, the method comprises administering a therapeutically effective amount of a Treg cell activator and / or a monoclonal antibody and / or an AGE inhibitor to a subject. In some embodiments of the methods described herein, the Treg cell activator is one or more single-chain chimeric polypeptides. In some embodiments of the methods described herein, the monoclonal antibody is one or both of an anti-tissue factor antibody and / or an anti-CD36 antibody. In some embodiments of the methods described herein, the AGE inhibitor is a soluble RAGE trap. In some embodiments of the methods described herein, the Treg cell activator comprises one or more single-chain chimeric polypeptides, the monoclonal antibody comprises one or more of an anti-tissue factor antibody and / or an anti-CD36 antibody, and the AGE inhibitor comprises one or more soluble RAGE traps.

[0025] In some embodiments of the methods described herein, the polychain chimeric polypeptide comprises (a) a first chimeric polypeptide comprising (i) a first target-binding domain, (ii) a soluble tissue factor domain, and (iii) a first domain of a pair of affinity domains, and (b) a second chimeric polypeptide comprising (i) a second domain of a pair of affinity domains and (ii) a second target-binding domain, wherein the first chimeric polypeptide and the second chimeric polypeptide associate via binding of the first domain and the second domain of the pair of affinity domains.

[0026] In some embodiments of the methods described herein, the single-chain chimeric polypeptide comprises (i) a first target-binding domain, (ii) a soluble tissue factor domain, and (iii) a second target-binding domain.

[0027] In some embodiments of the methods described herein, age-related disorders are selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, hepatic fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

[0028] In some embodiments of the methods described herein, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Crohn's disease, multiple sclerosis, Guillain-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, and non-alcoholic steatohepatitis.

[0029] 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, either from the same or different species). For example, a chimeric polypeptide may comprise domains derived from at least two different naturally occurring human proteins. In some examples, a chimeric polypeptide may comprise a synthetic sequence domain (e.g., scFv) and a domain derived from a naturally occurring protein (e.g., a naturally occurring human protein). In some embodiments, a chimeric polypeptide may comprise at least two different domains (e.g., two different scFv) that are synthetic sequences.

[0030] An "antigen-binding domain" is one or more protein domains (e.g., formed from amino acids derived from a single polypeptide, or from amino acids derived from two or more polypeptides (e.g., the same or different polypeptides)) that can specifically bind to one or more different antigens. In some examples, an antigen-binding domain can bind to an antigen or epitope with similar specificity and affinity to 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. Additional examples of antigen-binding domains are known in the art.

[0031] A “soluble tissue factor domain” refers to a polypeptide having at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity) with a segment of wild-type mammalian tissue factor protein (e.g., wild-type human tissue factor protein) that lacks a transmembrane domain and an intracellular domain. Non-limiting examples of soluble tissue factor domains are described herein.

[0032] The term “soluble interleukin protein” is used herein to refer to mature secretory interleukin proteins or their biologically active fragments. In some examples, soluble interleukin proteins may contain sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to wild-type mature secretory mammalian interleukin proteins (e.g., wild-type human interleukin proteins) and that retain their biological activity. Non-limiting examples of soluble interleukin proteins are described herein.

[0033] The term “soluble cytokine protein” is used herein to refer to mature secretory cytokine proteins or their biologically active fragments. In some examples, soluble cytokine proteins may contain sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to wild-type mature secretory mammalian interleukin proteins (e.g., wild-type human interleukin proteins) and that retain their biological activity. Non-limiting examples of soluble cytokine proteins are described herein.

[0034] The term “soluble interleukin receptor” is used herein in its broadest sense and refers to polypeptides that lack a transmembrane domain (and optionally an intracellular domain) capable of binding to one or more of their native ligands under physiological conditions, e.g., in phosphate-buffered saline at room temperature. For example, a soluble interleukin receptor may include a sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) to the extracellular domain of a wild-type interleukin receptor and retains the ability to specifically bind to one or more of its native ligands, but lacks its transmembrane domain (and optionally further lacks an intracellular domain). Non-limiting examples of soluble interleukin receptors are described herein.

[0035] The term “soluble cytokine receptor” is used herein in its broadest sense and refers to polypeptides lacking a transmembrane domain (and optionally an intracellular domain) that can bind to one or more of its native ligands under physiological conditions, e.g., in phosphate-buffered saline at room temperature. For example, a soluble cytokine receptor may include a sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) to the extracellular domain of a wild-type cytokine receptor and retains the ability to specifically bind to one or more of its native ligands, but lacks its transmembrane domain (and optionally further lacks an intracellular domain). Non-limiting examples of soluble cytokine receptors are described herein.

[0036] The term “antibody” is used herein in its broadest sense and includes certain types of immunoglobulin molecules that contain one or more antigen-binding domains that specifically bind to an antigen or epitope. Antibodies specifically include, for example, intact antibodies (e.g., intact immunoglobulins), antibody fragments, and multispecific antibodies. An example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer. Examples of antibody additions are described herein. Examples of antibody additions are known in the art.

[0037] "Affinity" refers to the total strength of non-covalent interactions between an antigen-binding site and its binding partner (e.g., antigen or epitope). Unless otherwise indicated, as used herein, "affinity" refers to endogenous binding affinity, which reflects the 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 is given by the dissociation equilibrium constant (K). D ) can be expressed as follows. The dynamical components contributing to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including the methods described herein. Affinity can be determined, for example, using surface plasmon resonance (SPR) techniques (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®). Additional methods for determining the affinity of an antigen-binding domain to its corresponding antigen or epitope are known in the art.

[0038] As used in this specification, "single-chain polypeptide" refers to a single protein chain.

[0039] As used herein, "polychain polypeptide" refers to a polypeptide comprising two or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) protein chains (e.g., at least a first chimeric polypeptide and a second polypeptide) linked together via non-covalent bonds to form a quaternary structure.

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

[0041] The term "epitope" refers to a portion of an antigen that specifically binds to an antigen-binding domain. Epitopes may consist, for example, surface-contactable amino acid residues and / or sugar side chains, and may possess specific three-dimensional structural properties as well as specific charge properties. Conformational epitopes and non-conformational epitopes are distinguished in that binding to the former may be lost in the presence of a denaturing solvent, while binding to the latter may not be lost. Epitopes may include amino acid residues directly involved in binding and other amino acid residues not directly involved in binding. Methods for identifying epitopes to which an antigen-binding domain binds are known in the art.

[0042] The term “treatment” means improving at least one symptom of a disorder. In some examples, the disorder being treated is cancer, and improving at least one symptom of cancer includes reducing abnormal growth, gene expression, signaling, translation, and / or secretion of factors. Generally, a treatment method involves administering to a subject who requires such treatment, or who has been determined to require such treatment, a therapeutically effective amount of a composition that alleviates at least one symptom of the disorder.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in this invention, and other suitable methods and materials well known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit the scope. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated in their entirety by reference. In the event of any inconsistency, this specification, including its definitions, shall prevail.

[0044] Other features and advantages of the present invention will become apparent from the embodiments and drawings for carrying out the invention, as well as from the claims. [Brief explanation of the drawing]

[0045] [Figure 1] Polychain chimeric polypeptides: Exemplary figures of the first chimeric polypeptide (i) comprising a first target-binding domain (A), a soluble tissue factor domain, a first domain of a pair of affinity domains (soluble interleukin IL-15), and an additional target-binding domain (B), and (ii) a second chimeric polypeptide comprising a second domain of a pair of affinity domains (IL-15 receptor alphasci domain), a second target-binding domain (C), and an additional antigen-binding domain (D) are shown. The schematic diagram above shows the association of the first and second chimeric polypeptides via a pair of affinity domains. The schematic diagram below shows the order of domains within the first and second chimeric polypeptides. [Figure 2]Polychain chimeric polypeptides: Exemplary figures of the first chimeric polypeptide are shown, comprising (i) a first chimeric polypeptide comprising a first target-binding domain (A), a soluble tissue factor domain containing five amino acid substitutions to remove binding of the soluble tissue factor domain to FVIIa, a first domain of a pair of affinity domains (soluble interleukin IL-15 containing D8N or D8A amino acid substitutions), and an additional target-binding domain (B); and (ii) a second chimeric polypeptide comprising a second domain of a pair of affinity domains (IL-15 receptor alphasusci domain), a second target-binding domain (C), and an additional antigen-binding domain (D). The schematic diagram above shows the association of the first and second chimeric polypeptides via a pair of affinity domains. The schematic diagram below shows the order of domains within the first and second chimeric polypeptides. In any other embodiment of the polychain chimeric polypeptides described herein, the soluble tissue factor domain may include or consist of a soluble wild-type human tissue factor domain (containing or consisting of a continuous sequence within wild-type human tissue factor). [Figure 3] A schematic diagram of an exemplary IL-12 / IL-15RαSu DNA construct is shown. [Figure 4] A schematic diagram of an exemplary IL-18 / TF / IL-15 DNA construct is shown. [Figure 5] A schematic diagram illustrating the interaction between exemplary IL-12 / IL-15RαSu DNA constructs and IL-18 / TF / IL-15 DNA constructs is shown. [Figure 6] A schematic diagram of the interaction between an exemplary IL-12 / IL-15RαSu fusion protein and an IL-18 / TF / IL-15 fusion protein, resulting in the IL-18 / TF / IL-15:IL-12 / IL-15RαSu complex (18t15-12s), is shown. [Figure 7] This shows a chromatogram of purified elution from an anti-TF antibody affinity column at 18T15-12S. [Figure 8]Exemplary chromatographic profiles of anti-TF antibody / SEC-purified 18t15-12s protein after elution on an analytical size exclusion column are shown, demonstrating the separation of monomeric multiprotein 18t15-12s complexes from protein aggregates. [Figure 9] Examples of 4-12% SDS-PAGE of the 18t15-12s complex after disulfide bond reduction are shown. Lane 1: SeeBlue Plus2 marker, Lane 2: 18t15-12s purified by anti-tissue factor antibody affinity column (0.5 μg), Lane 3: 18t15-12s purified by anti-tissue factor antibody affinity column (1 μg). [Figure 10] SDS-PAGE analysis of deglycosylated and non-deglycosylated 18t15-12s is shown. Lane 1: Anti-tissue factor antibody affinity column purified 18t15-12s (0.5 μg) (non-deglycosylated), Lane 2: Anti-TF antibody purified 18t15-12s (1 μg) (non-deglycosylated), Lane 3: 18t15-12s (1 μg) (deglycosylated), Lane 4: Mark12 non-stained marker. [Figure 11] This shows a sandwich ELISA of an 18t15-12s complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-12 detection antibody (BAF 219). [Figure 12] This shows a sandwich ELISA of an 18t15-12s complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-15 detection antibody (BAM 247). [Figure 13] This shows a sandwich ELISA of an 18t15-12s complex containing an anti-human tissue factor capture antibody and a biotinylated anti-human IL-18 detection antibody (D045-6). [Figure 14] This shows a sandwich ELISA of an 18t15-12s complex containing an anti-human tissue factor (I43) capture antibody and an anti-human tissue factor detection antibody. [Figure 15] This shows the proliferation of IL-15-dependent 32Dβ cells mediated by the 18T15-12S complex (white square) and recombinant IL-15 (black square). [Figure 16]The 18T15-12S complex (white square) shows the biological activity of IL-18, while recombinant IL-18 (black square) and recombinant IL-12 (black circle) function as positive and negative controls, respectively. [Figure 17] The 18T15-12S complex (white square) shows the biological activity of IL-12, with recombinant IL-12 (black circle) and recombinant IL-18 (black square) functioning as positive and negative controls, respectively. [Figure 18A] Figures 18A and 18B show the cell surface CD25 expression and cell surface CD69 expression of NK cells induced by the 18t15-12s complex. [Figure 18B] See the explanation in Figure 18A. [Figure 19] This shows a flow cytometry graph of intracellular interferon-gamma expression in NK cells induced by the 18T15-12S complex. [Figure 20] This study demonstrates the cytotoxicity of 18t15-12s-induced human NK cells against K562 cells. [Figure 21] A schematic diagram of an exemplary IL-7 / IL-15RαSu DNA construct is shown. [Figure 22] A schematic diagram of an exemplary IL-21 / TF / IL-15 DNA construct is shown. [Figure 23] A schematic diagram illustrating the interaction between exemplary IL-7 / IL-15RαSu DNA constructs and IL-21 / TF / IL-15 DNA constructs is shown. [Figure 24] A schematic diagram of the interaction between an exemplary IL-7 / IL-15RαSu fusion protein and an IL-21 / TF / IL-15 fusion protein, resulting in the IL-21 / TF / IL-15:IL-7 / IL-15RαSu complex (21t15-7s), is shown. [Figure 25]The study demonstrated the cytotoxic activity of proliferating NK cells against K562 human tumor cells, showing that NK cells stimulated with 21t15-7s + anti-TF IgG1 antibody exhibited higher specific lysis of K562 cells than NK cells not stimulated with 21t15-7s + anti-TF IgG1 antibody. [Figure 26] A schematic diagram of an exemplary IL-21 / IL-15RαSu DNA construct is shown. [Figure 27] A schematic diagram of an exemplary IL-7 / TF / IL-15 DNA construct is shown. [Figure 28] A schematic diagram of the interaction between exemplary IL-21 / IL-15RαSu DNA constructs and exemplary IL-7 / TF / IL-15 DNA constructs is shown. [Figure 29] A schematic diagram of the interaction between an exemplary IL-21 / IL-15RαSu fusion protein and an exemplary IL-7 / TF / IL-15 fusion protein, which result in the IL-7 / TF / IL-15:IL-21 / IL-15RαSU complex (7t15-21s), is shown. [Figure 30] The size exclusion chromatography (SEC) profiles of anti-TF IgG1 antibody, 7t15-21s, and a complex containing an equal amount of anti-TF IgG1 antibody and 7t15-21s are shown. [Figure 31] This shows the oxygen consumption rate (OCR) in picomoles / min for human NK cells (2 × 10⁶ cells / mL) isolated from the blood of two different donors. [Figure 32] This shows the extracellular acidification rate (ECAR) in mpH / min for human NK cells (2 × 10⁶ cells / mL) isolated from the blood of two different donors. [Figure 33] A schematic diagram of the TGFRt15-TGFRs structure is shown. [Figure 34] A schematic diagram of the additional TGFRt15-TGFRs structure is shown. [Figure 35] The results of TGFβ1 inhibition by TGFRt15-TGFRs and TGFR-Fc are shown. [Figure 36]Figures 36A and 36B show the results of detecting IL-15 and TGFβRII in TGFRt15-TGFRs using the corresponding antibodies with ELISA. [Figure 37] This line graph shows the chromatographic profile of the cell culture supernatant containing TGFRt15-TGFRs protein after binding to and elution from an anti-TF antibody resin. [Figure 38] The analytical SEC profiles of TGFRt15-TGFRs are shown. [Figure 39] The TGFRt15-TGFRs before and after deglycosylation, as analyzed by reduced SDS-PAGE, are shown. [Figure 40] Figures 40A and 40B show the spleen weight and percentage of immune cell types in TGFRt15-TGFRs-treated mice and control-treated mice. Figure 40A shows the spleen weight in TGFRt15-TGFRs-treated mice compared to PBS controls. Figure 40B shows the percentages of CD4+ T cells, CD8+ T cells, and NK cells in TGFRt15-TGFRs-treated mice compared to PBS controls. [Figure 41] Figures 41A and 41B show spleen weight and immune stimulation over 92 hours in mice treated with TGFRt15-TGFRs. Figure 41A shows spleen weight in mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours post-treatment. Figure 41B shows the percentage of immune cells in mice treated with TGFRt15-TGFRs at 16, 24, 48, 72, and 92 hours post-treatment. [Figure 42] Figures 42A and 42B show the time course of Ki67 and granzyme B expression in mice treated with TGFRt15-TGFRs. [Figure 43] This demonstrates the enhancement of splenic cell cytotoxicity by TGFRt15-TGFRs in C57BL / 6 mice. [Figure 44]This shows changes in tumor size in a mouse model of pancreatic cancer in response to PBS treatment, chemotherapy alone, TGFRt15-TGFRs alone, or a combination of chemotherapy and TGFRt15-TGFRs. [Figure 45] This shows the cytotoxicity of NK cells isolated from mice treated with TGFRt15-TGFRs. [Figure 46] This shows the changes in the surface phenotype of lymphocyte populations after stimulation at 18T15-12S, 18T15-12S16, and 7T15-21S. [Figure 47] This shows increased phospho-STAT4 and phospho-STAT5 levels in NK cells after stimulation at 18t15-12s. [Figure 48A] Figures 48A to 48C show in vivo stimulation of Treg cells, NK cells, and CD8+ T cells in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 48B] See the explanation in Figure 48A. [Figure 48C] See the explanation in Figure 48A. [Figure 49A] Figures 49A to 49C show immunostimulation in C57BL / 6 mice after treatment with TGFRt15-TGFRs. [Figure 49B] See the explanation in Figure 49A. [Figure 49C] See the explanation in Figure 49A. [Figure 50] Figures 50A and 50B show in vivo induction of NK cell and CD8+ T cell proliferation in ApoE- / - mice fed a Western diet and treated with TGFRt15-TGFRs. [Figure 51] Figures 51A and 51B show the enhanced cytotoxicity of NK cells after treatment with TGFRt15-TGFRs. [Figure 52] Figures 52A and 52B show the enhancement of ADCC activity in NK cells after treatment with TGFRt15-TGFRs. [Figure 53A]Figures 53A to 53H show the antitumor activity of TGFRt15-TGFRs + anti-TRP1 antibody (TA99) in combination with chemotherapy in a melanoma mouse model. [Figure 53B] See the explanation in Figure 53A. [Figure 53C] See the explanation in Figure 53A. [Figure 53D] See the explanation in Figure 53A. [Figure 53E] See the explanation in Figure 53A. [Figure 53F] See the explanation in Figure 53A. [Figure 53G] See the explanation in Figure 53A. [Figure 53H] See the explanation in Figure 53A. [Figure 54] Figures 54A to 54C show the improvement of Western diet-induced hyperglycemia in ApoE- / - mice by TGFRt15-TGFRs. [Figure 55] This shows cell surface staining that summarizes the differentiation of NK cells into cytokine-induced memory-like NK cells (CIML-NK cells) after stimulation with 18T15-12S and culture in rhIL15. [Figure 56] This shows the upregulation of CD44hi memory T cells upon treatment with TGFRt15-TGFRs. [Figure 57] This is a schematic diagram of an example αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide. [Figure 58] This chromatograph shows the elution of exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptides from an anti-tissue factor antibody affinity column. [Figure 59] This chromatograph shows the elution of an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide from a Superdex 200 Increase 10 / 300 GL gel filtration column. [Figure 60]This is an exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide on a sodium dodecyl sulfate polyacrylamide gel (4-12% NuPage bis-Tris gel) purified using an anti-tissue factor antibody affinity column. [Figure 61] This graph shows the ELISA quantification of exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptides performed using the method described in Example 38. Purified tissue factor was used as a control. [Figure 62] This graph shows the ability of the exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide to stimulate CD25 expression in CD4+ T cells isolated from the blood of two donors. These experiments were performed as described in Example 39. [Figure 63] This graph shows the ability of the exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide to stimulate CD25 expression in CD8+ T cells isolated from the blood of two donors. These experiments were performed as described in Example 39. [Figure 64] This graph shows the ability of the exemplary αCD3scFv / TF / αCD28scFv single-chain chimeric polypeptide to stimulate CD69 expression in CD4+ T cells isolated from the blood of two donors. These experiments were performed as described in Example 39. [Figure 65] This is a schematic diagram of an exemplary IL-2 / TF / IL-2 single-chain chimeric polypeptide. [Figure 66] This shows the IL-2 activity of IL-2 / TF / IL-2 compared to recombinant IL-2 using a 32Dβ cell proliferation assay. [Figure 67] This shows the IL-2 activity of IL-2 / TF / IL-2 compared to recombinant IL-2 using a CTLL-2 cell proliferation assay. [Figure 68] This shows fasting blood glucose levels in ApoE- / - mice fed a standard solid diet or a high-fat diet and treated with PBS control (untreated) or IL-2 / TF / IL-2. [Figure 69]This shows the ratio of CD4+CD25+FoxP3+T regulatory cells in blood lymphocytes from ApoE- / - mice fed a standard solid diet or a high-fat diet and treated with PBS control (untreated) or IL-2 / TF / IL-2. [Figure 70] This line graph shows the chromatographic profile of the IL-2 / TF / IL-2 protein-containing cell culture supernatant after binding to and elution of the anti-TF antibody resin. [Figure 71] The analytical SEC profile of IL-2 / TF / IL-2 is shown. [Figure 72] Figures 72A and 72B show the reduced SDS-PAGE analysis of IL-2 / TF / IL-2 before and after deglycosylation. Figure 16A shows the reduced SDS-PAGE analysis of IL-2 / TF / IL-2 before deglycosylation. Figure 16B shows the reduced SDS-PAGE analysis of IL-2 / TF / IL-2 after deglycosylation. [Figure 73] Figures 73A and 73B show the results of immunostimulation in C57BL / 6 mice using IL-2 / TF / IL-2. Figure 73A shows the spleen weight after treatment with IL-2 / TF / IL-2. Figure 73B shows the percentage of immune cell types after treatment with IL-2 / TF / IL-2. [Figure 74] This shows the upregulation of CD25 expression in CD4+ T cells in mice treated with IL-2 / TF / IL-2. [Figure 75] This shows the pharmacokinetics of IL-2 / TF / IL-2 in C57BL / 6 mice. [Figure 76] Figures 76A and 76B show the effect of IL-2 / TF / IL-2 on attenuating high-fat diet-induced atherosclerotic plaque formation in ApoE- / - mice. Figure 20A shows representative images of atherosclerotic plaques from ApoE- / - mice fed a standard solid diet or a high-fat diet and treated with either PBS control or IL-2 / TF / IL-2. Figure 76B shows the results of quantitative analysis of atherosclerotic plaques in each group. [Figure 77]This shows the fasting blood glucose levels of IL-2 / TF / IL-2 treated mice compared to control mice. [Figure 78] This shows the percentage of CD4+CD25+FoxP3+Treg in blood lymphocytes from mice treated with IL-2 / TF / IL-2 and control mice. [Figure 79] Figures 79A to 79C are a series of graphs showing the immunostimulation in C57BL / 6 mice after treatment with 2t2. [Figure 80] Figures 80A–80C are a series of graphs showing in vivo stimulation of Treg cells, NK cells, and CD8+ T cells in ApoE- / - mice fed a Western diet and treated with 2t2. [Figure 81] Figures 81A to 81C are a series of graphs showing the induction of splenocyte proliferation by 2t2 in C57BL / 6 mice. [Figure 82] Figures 82A and 82B are a series of graphs showing the in vivo induction of NK cell and CD8+ T cell proliferation in ApoE- / - mice fed a Western diet and treated with 2t2. [Figure 83] Figures 83A to 83C are a series of graphs showing the improvement of Western diet-induced hyperglycemia in ApoE- / - mice by 2t2. [Figure 84] This shows the upregulation of CD44 memory T cells during 2t2 treatment. [Figure 85] Figures 85A to 85C show the pStat5a response of human hematopoietic lymphocytes in CD4+CD25hiTreg cells, CD4+CD25-Tcon cells, or CD8+Tcon cells in response to 2t2 or IL2 treatment. Figure 85A shows the pSTAT5 response in CD4+CD25hiTreg cells. Figure 34B shows the pSTAT5 response in CD4+CD25-Tcon cells. Figure 85C shows the pSTAT5 response in CD8+Tcon cells. [Figure 86] This graph shows plasma hemoglobin A1C levels in aged mice after treatment with PBS or TGFRt15-TGFRs and / or 2t2. [Figure 87A] Figures 87A to 87C are a series of graphs showing the gene expression levels of aging markers (IL-1α, IL-6, and PAI-1, respectively) in the tissues of aged mice after treatment with PBS, TGFRt15-TGFRs, and 2t2 (either TGFRt15-TGFRs administered first on day 0, followed by 2t2 on day 60, or 2t2 administered first on day 0, followed by TGFRt15-TGFRs on day 60). [Figure 87B] See the explanation in Figure 87A. [Figure 87C] See the explanation in Figure 87A. [Figure 88A] A schematic diagram of the experimental plan for inducing NASH in ApoE- / - mice by feeding them is shown. [Figure 88B] This graph shows the therapeutic effect on hydroxyproline content, which is associated with collagen accumulation and fibrosis in the liver of ApoE- / - mice. [Figure 89] Figure 89A is a schematic diagram of the experimental design for a high-fat diet-induced atherosclerosis animal model. Figure 89B is a table showing the effect of 2t2 treatment on IL-1β and MCP-1 plasma cytokine levels in ApoE- / - mice from which plasma samples were collected 3 days after the second injection. [Figure 90] This graph shows the effect of 2t2 treatment on triglyceride plasma levels in high-fat diet-induced ApoE- / - mice. [Figure 91] This graph shows the effect of 2t2 treatment on LDL plasma levels in high-fat diet-induced ApoE- / - mice. [Figure 92] This graph shows the effect of 2t2 administration on body weight in high-fat diet-induced ApoE- / - mice. [Figure 93] Figures 93A to 93E show exemplary physical appearances of mice that were fed either a control diet or a high-fat diet, and were either untreated or treated with TGFRt15-TGFRs, 2t2, or 21t15-TGFRs. [Figure 94]Figures 94A–94E are a series of images demonstrating that treatment with an IL-2-based molecule (2t2) can induce hair follicle formation after hair loss in a mouse model. Figure 94A is an image from a control mouse that underwent hair loss only after shaving; Figure 94B is an image from a mouse administered a low dose of IL-2 (1 mg / kg) after hair loss; and Figures 94C–94E are images from mice administered 0.3 mg / kg (Figure 94C), 1 mg / kg (Figure 94D), and 3 mg / kg (Figure 94E) of 2t2 after hair loss. Black arrows indicate anagen hair follicles that later extend into the dermis and promote hair growth. [Figure 95] This shows the total number of growing hair follicles counted per 10 fields of view for each treatment group. [Figure 96] This is an illustrative schematic diagram of an experimental design using a melanoma mouse model. [Figure 97-1] Figures 97A to 97H are graphs showing the effects of TGFRt15-TGFRs administration on the proliferation, enlargement, and activation of NK / T cells in the blood of a melanoma mouse model. [Figure 97-2] See the explanation in Figure 97-1. [Figure 98A] Figures 98A to 98C are graphs showing the effects of TGFRt15-TGFRs treatment on TGF-β1, TGF-β2, and TGF-β3 levels in the plasma of a melanoma mouse model. [Figure 98B] See the explanation in Figure 98A. [Figure 98C] See the explanation in Figure 98A. [Figure 99A] Figures 99A to 99E are graphs showing the effects of treatment with dexamethasone or a combination of TGFRt15-TGFRs and dexamethasone on plasma levels of IL-2, IL-1β, IL-6, and GM-CSF in a melanoma mouse model. [Figure 99B] See the explanation in Figure 99A. [Figure 99C] See the explanation in Figure 99A. [Figure 99D] See the explanation in Figure 99A. [Figure 99E]See the explanation in Figure 99A. [Figure 100] Figures 100A and 100B are graphs showing the effects of treatment with dexamethasone or a combination of TGFRt15-TGFRs and dexamethasone on NK cell levels and CD8+ T cell levels in the spleen of a melanoma mouse model. [Figure 101A] Figures 101A to 101C are a series of graphs showing the effects of treatment with physiological saline (black line), dexamethasone (dark gray line), or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 (light gray line) on the glycolytic activity of splenic cells. [Figure 101B] See the explanation in Figure 101A. [Figure 101C] See the explanation in Figure 101A. [Figure 102-1] Figures 102A to 102L are a series of graphs showing the effects of treatment with physiological saline, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on glycolytic activity (glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification) of splenocytes derived from a melanoma mouse model. [Figure 102-2] See the explanation in Figure 102-1. [Figure 102-3] See the explanation in Figure 102-1. [Figure 103A] Figures 103A to 103C are a series of graphs showing the effects of PBS, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on mitochondrial respiration in splenocytes derived from a melanoma mouse model. [Figure 103B] See the explanation in Figure 103A. [Figure 103C] See the explanation in Figure 103A. [Figure 104-1] Figures 104A to 104L are a series of graphs showing the effects of treatment with PBS, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on mitochondrial respiration (basal respiration, maximal respiration, respiratory reserve, and ATP production) in splenocytes derived from a melanoma mouse model. [Figure 104-2] See the explanation in Figure 104-1. [Figure 104-3] See the explanation in Figure 104-1. [Figure 105-1] Figures 105A to 105H are a series of graphs showing the effects of treatment with PBS, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on the infiltration of NK / Ki67 cells, CD8 / Ki67 cells, NK cells, CD8 cells, NK / CD25 cells, NK / granzyme B cells, CD8 / CD25 cells, and CD8 / granzyme B cells into melanoma tumors in a melanoma mouse model. [Figure 105-2] See the explanation in Figure 105-1. [Figure 106] Figure 106A is a schematic diagram of the experimental design for treatment-induced aging in B16F10 tumors in a mouse model of melanoma. Figures 106B–106E are a series of graphs showing the effects of DTX treatment on aging-related gene expression (DPP4, IL-6, p16, and p21, respectively) in mouse B16F10 tumor cells. [Figure 107A] This is a schematic diagram of the experimental design for treatment-induced aging in B16F10 tumors in a melanoma mouse model. [Figure 107B] Figures 107B to 107C are graphs showing the effects of treatment with saline, dexamethasone, or a combination of dexamethasone, TGFRt15-TGFRs, and TA99 on the expression of p21 and IL-6 in the B16F10 tumor of the melanoma tumor model, respectively. [Figure 107C] See the explanation in Figure 107B. [Figure 108A] This graph shows the effect of 2t2 or IL-2 on the proliferation of IL2Rαβγ-containing cells or IL-2Rβγ-containing cells. [Figure 108B] This graph shows the effect of IL-2 2t2 on the activation of human CD4+CD25+Treg pSTAT5 and human CD8+Tcon pSTAT5. [Figure 108C]This graph shows the effect of 2t2 or IL-2 on the activation of human CD4+CD25-Tcon pSTAT5 or human CD56bright NK pSTAT5. [Figure 108D] This graph shows the effect of 2t2 or IL-2 on the activation of CD56dim NK pSTAT5. [Figure 109A] This is a schematic diagram of an experiment studying the effects of 2t2 treatment in ApoE- / - mice fed a Western diet. [Figure 109B] This is a series of graphs showing the effects of 2t2 administration on CD25+Foxp3+Treg cell levels, CTLA4+Foxp3+Treg cell levels, and CD39+Foxp3+Treg cell levels in ApoE- / - mice fed a Western diet. [Figure 109C] This is a series of graphs showing the effects of 2t2 administration on CD4+ T cell levels, CD8+ T cell levels, and CD3-NK1.1+ NK cell levels in ApoE- / - mice fed a Western diet. [Figure 109D] This is a series of graphs showing the effects of 2t2 administration on plasma levels of IL-1β, MCP-1, and TNF-α in ApoE- / - mice fed a Western diet. [Figure 109E] This is a series of graphs showing the effects of 2t2 administration on plasma LDL cholesterol levels, fasting blood glucose levels, and HOMA-IR index in ApoE- / - mice fed a Western diet. [Figure 110A] Figures 110A to 110D are a series of graphs showing the levels of protein expression of aging markers (PAI1, IL-1α, CXCL1, and IL-2, respectively) in the plasma of aged mice after treatment with PBS, TGFRt15-TGFRs, and 2t2 (either TGFRt15-TGFRs administered first on day 0, followed by 2t2 on day 60, or 2t2 administered first on day 0, followed by TGFRt15-TGFRs on day 60). [Figure 110B] See the explanation in Figure 110A. [Figure 110C] See the explanation in Figure 110A. [Figure 110D] See the explanation in Figure 110A. [Modes for carrying out the invention]

[0046] Detailed explanation A method for treating an age-related disease or inflammatory disease in a subject is provided herein, comprising administering (i) a therapeutically effective amount of an NK cell activator and / or NK cells and / or monoclonal antibody, and (ii) a therapeutically effective amount of a Treg cell activator and / or Treg cells and / or monoclonal antibody and / or advanced glycation end product (AGE) inhibitor. In some embodiments, the age-related disease is inflammatory aging-related.

[0047] Methods for treating age-related and inflammatory diseases in the target population. In some embodiments of the methods described herein, (i) is administered to the subject substantially simultaneously with (ii). In some embodiments of the methods described herein, (i) is administered to the subject before (ii) is administered to the subject. In some embodiments of the methods described herein, (ii) is administered to the subject before (i) is administered to the subject.

[0048] In some embodiments of the methods described herein, the method comprises administering a therapeutically effective dose of NK cells to a subject. In some embodiments, the NK cells are autologous NK cells. In some embodiments, the method may further comprise isolating NK cells from a subject and culturing the isolated NK cells in a liquid culture medium under conditions sufficient to induce or increase NK cell proliferation, wherein the NK cells are administered to the subject after the isolation and culture steps. In some embodiments, the liquid culture medium comprises one or more polychain chimeric polypeptides (e.g., any of the exemplary polychain chimeric polypeptides described herein).

[0049] In some embodiments, NK cells include a chimeric antigen receptor (for example, the chimeric antigen receptor includes an extracellular domain that specifically binds to tissue factor or CD26).

[0050] In some embodiments, the method may involve administering a therapeutically effective amount of an NK cell activator to a subject. In some embodiments, the NK cell activator is one or more polychain chimeric polypeptides (e.g., one or more of the polychain chimeric polypeptides described herein). In some embodiments, the NK cell activator is one or more anti-tissue factor antibodies, anti-CD26 antibodies, and / or anti-CD36 antibodies. In some embodiments, the NK cell activator comprises one or more polychain chimeric polypeptides and one or more anti-tissue factor antibodies, anti-CD26 antibodies, and / or anti-CD36 antibodies.

[0051] In some embodiments, the method comprises administering a therapeutically effective dose of Treg cells to a subject. In some embodiments, the Treg cells are autologous Treg cells. In some embodiments, the method further comprises culturing isolated Treg cells in a liquid culture medium under conditions sufficient to induce or increase Treg cell proliferation, where the Treg cells are administered to the subject after the isolation and culture steps. In some embodiments, the liquid culture medium comprises one or more single-chain chimeric polypeptides.

[0052] In some embodiments, Treg cells include a chimeric antigen receptor (for example, a chimeric antigen receptor that includes an extracellular domain that specifically binds to tissue factor or CD36).

[0053] In some embodiments, the method involves administering a therapeutically effective amount of a Treg cell activator to a subject. In some embodiments, the Treg cell activator is one or more single-chain chimeric polypeptides (e.g., one or more of the single-chain chimeric polypeptides described herein). In some embodiments, the Treg cell activator is one or both of an anti-tissue factor antibody and an anti-CD36 antibody. In some embodiments, the Treg cell activator is a soluble RAGE trap.

[0054] In some embodiments, the Treg cell activator comprises one or more single-chain chimeric polypeptides and one or more of the following: an anti-tissue factor antibody, an anti-CD36 antibody, and a soluble RAGE trap.

[0055] In some embodiments, the method involves administering a therapeutically effective dose of a monoclonal antibody to a subject. In some embodiments, the monoclonal antibody includes one or more anti-tissue factor antibodies, anti-CD36 antibodies, and / or anti-CD36 antibodies that can directly or indirectly reduce inflammasome or senescent cell activity.

[0056] In some embodiments, the method involves administering a therapeutically effective dose of an advanced glycation end product (AGE) inhibitor to a subject. In some embodiments, the AGE inhibitor comprises one or more soluble RAGE traps that can directly or indirectly reduce the activity of inflammasomes or senescent cells.

[0057] In some embodiments of the methods described herein, the age-related disease is inflammatory aging-related. Non-limiting examples of age-related diseases are selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, lipodystrophy, atherosclerosis, cataracts, COPD, idiopathic pulmonary fibrosis, renal transplant failure, hepatic fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

[0058] Non-exclusive examples of inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, amyotrophic lateral sclerosis, diabetic nephropathy, CNS injury, Alzheimer's disease, Parkinson's disease, Crohn's disease, multiple sclerosis, Guillain-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, and non-alcoholic steatohepatitis.

[0059] In some cases, the subjects may be individuals identified or diagnosed with age-related diseases or chronic inflammation.

[0060] In some embodiments, these methods may result in a reduction in the number, severity, or frequency of one or more symptoms of age-related disease in a subject (for example, compared to the number, severity, or frequency of one or more symptoms of cancer in the subject before treatment).

[0061] In some cases, these methods can lead to a reduction in the number of senescent cells in a subject compared to the number of senescent cells in the subject before treatment (e.g., a reduction in the number of senescent cells in one or more specific tissues involved in and / or related to age-related diseases or disorders in the subject) (e.g., approximately 1% to 99% reduction, approximately 1% to 95% reduction, approximately 1% to 90% reduction, approximately 1% to 85% reduction, approximately 1% to 80% reduction, approximately 1% to 75% reduction, approximately 1% to 70% reduction, approximately 1% to 65% reduction, approximately 1% to 60% reduction, approximately 1% reduction). Small to approx. 55% decrease, approx. 1% decrease to approx. 50% decrease, approx. 1% decrease to approx. 45% decrease, approx. 1% decrease to approx. 40% decrease, approx. 1% decrease to approx. 35% decrease, approx. Small, approximately 1% decrease to approximately 5% decrease, approximately 5% decrease to approximately 99% decrease, approximately 5% decrease to approximately 95% decrease, approximately 5% decrease to approximately 90% decrease, approximately 5% decrease to approximately 85% decrease, approximately 5% decrease to approximately 80% decrease, approximately 5% decrease to approximately 75% decrease, approximately 5% to approximately 70% decrease, approximately 5% decrease to approximately 65% ​​decrease, approximately 5% decrease to approximately 6 0% decrease, approx. 5% decrease ~ approx. 55% decrease, approx. 5% decrease ~ approx. 50% decrease, approx. 5% decrease ~ approx. 45% decrease, approx. 5% decrease ~ approx. 40% decrease, approx. 5% decrease to approx. 10% decrease, approx. 10% decrease to approx. 99% decrease, approx. 10% decrease to approx. 95% decrease, approx. 10% decrease to approx. 90% decrease, approx. 10% decrease to approx. 0% decrease ~ 60% decrease, 10% decrease ~ 55% decrease, 10% decrease ~ 50% decrease, 10% decrease ~ 45% decrease, 10% decrease ~ 40% decrease, 10% decrease ~ 35% decrease, 10% decrease ~ 30% decrease, 10% decrease ~ 25% decrease, 10% decrease ~ 20% decrease, Approximately 10% decrease ~ approx. 15% decrease, approx. 15% decrease ~ approx. 99% decrease, approx. 15% decrease ~ approx. 95% decrease, approx. 15% decrease ~ approx. 90% decrease, approx. 15% decrease ~ approx. 85% decrease, approx. 15% decrease ~ approx.Approximately 15% decrease to approximately 60% decrease, approximately 15% decrease to approximately 55% decrease, approximately 15% decrease to approximately 50% decrease, approximately 15% decrease to approximately 45% decrease, approximately 15% decrease to approximately 40% decrease, approximately 15% decrease to approximately 35% decrease, approximately 15% decrease to approximately 30% decrease, approximately 15% decrease to approximately 25% decrease, approximately 15% decrease to approximately 20% decrease, approximately 20% decrease to approximately 99% decrease, approximately 20% decrease to approximately 95% decrease, approximately 20% decrease to approximately 90% decrease, approximately 20% decrease to approximately 85% decrease, approximately 20% decrease to approximately 80% decrease, approximately 20% decrease to approximately 75% decrease, approximately 20% decrease to approximately 70% decrease, approximately 20% decrease to approximately 65% ​​decrease, approximately 20% decrease to approximately 60% decrease. Approximately 20% decrease to approximately 55% decrease, approximately 20% decrease to approximately 50% decrease, approximately 20% decrease to approximately 45% decrease, approximately 20% decrease to approximately 40% decrease, approximately 20% decrease to approximately 35% decrease, approximately 20% decrease to approximately 30% decrease, approximately 20% decrease to approximately 25% decrease, approximately 25% decrease to approximately 99% decrease, approximately 25% decrease to approximately 95% decrease, approximately 25% decrease to approximately 90% decrease, approximately 25% decrease to approximately 85% decrease, approximately 25% decrease to approximately 80% decrease, approximately 25% decrease to approximately 75% decrease, approximately 25% decrease to approximately 70% decrease, approximately 25% decrease to approximately 65% ​​decrease, approximately 25% decrease to approximately 60% decrease, approximately 25% decrease to approximately 55% decrease, approximately 25% decrease to approximately 50% decrease. Approximately 25% decrease to approximately 45% decrease, approximately 25% decrease to approximately 40% decrease, approximately 25% decrease to approximately 35% decrease, approximately 25% decrease to approximately 30% decrease, approximately 30% decrease to approximately 99% decrease, approximately 30% decrease to approximately 95% decrease, approximately 30% decrease to approximately 90% decrease, approximately 30% decrease to approximately 85% decrease, approximately 30% decrease to approximately 80% decrease, approximately 30% decrease to approximately 75% decrease, approximately 30% decrease to approximately 70% decrease, approximately 30% decrease to approximately 65% ​​decrease, approximately 30% decrease to approximately 60% decrease, approximately 30% decrease to approximately 55% decrease, approximately 30% decrease to approximately 50% decrease, approximately 30% decrease to approximately 45% decrease, approximately 30% decrease to approximately 40% decrease, approximately 30% decrease to approximately 35% decrease. Approximately 35% reduction ~ approximately 99% reduction, approximately 35% reduction ~ approximately 95% reduction, approximately 35% reduction ~ approximately 90% reduction, approximately 35% reduction ~ approximately 85% reduction, approximately 35% reduction ~ approximately 80% reduction, approximately 35% reduction ~ approximately 75% reduction, approximately 35% reduction ~ approximately 70% reduction, approximately 35% reduction ~ approximately 65% ​​reduction, approximately 35% reduction ~ approximately 60% reduction, approximately 35% reduction ~ approximately 55% reduction, approximately 35% reduction ~ approximately 50% reduction, approximately 35% reduction ~ approximately 45% reduction, approximately 35% reduction ~ approximately 40% reduction, approximately 40% reduction ~ approximately 99% reduction, approximately 40% reduction ~ approximately 95% reduction, approximately 40% reduction ~ approximately 90% reduction, approximately 40% reduction ~ approximately 85% reduction, approximately 40% reduction ~ approximately 80% reduction.Approximately 40% decrease to approximately 75% decrease, approximately 40% decrease to approximately 70% decrease, approximately 40% decrease to approximately 65% ​​decrease, approximately 40% decrease to approximately 60% decrease, approximately 40% decrease to approximately 55% decrease, approximately 40% decrease to approximately 50% decrease, approximately 40% decrease to approximately 45% decrease, approximately 45% decrease to approximately 99% decrease, approximately 45% decrease to approximately 95% decrease, approximately 45% decrease to approximately 90% decrease, approximately 45% decrease to approximately 85% decrease, approximately 45% decrease to approximately 80% decrease, approximately 45% decrease to approximately 75% decrease, approximately 45% decrease to approximately 70% decrease, approximately 45% decrease to approximately 65% ​​decrease, approximately 45% decrease to approximately 60% decrease, approximately 45% decrease to approximately 55% decrease, approximately 45% decrease to approximately 50% decrease, approximately 50% reduction ~ approximately 99% reduction, approximately 50% reduction ~ approximately 95% reduction, approximately 50% reduction ~ approximately 90% reduction, approximately 50% reduction ~ approximately 85% reduction, approximately 50% reduction ~ approximately 80% reduction, approximately 50% reduction ~ approximately 75% reduction, approximately 50% reduction ~ approximately 70% reduction, approximately 50% reduction ~ approximately 65% ​​reduction, approximately 50% reduction ~ approximately 60% reduction, approximately 50% reduction ~ approximately 55% reduction, approximately 55% reduction ~ approximately 99% reduction, approximately 55% reduction ~ approximately 95% reduction, approximately 55% reduction ~ approximately 90% reduction, approximately 55% reduction ~ approximately 85% reduction, approximately 55% reduction ~ approximately 80% reduction, approximately 55% reduction ~ approximately 75% reduction, approximately 55% reduction ~ approximately 70% reduction, approximately 55% reduction ~ approximately 65% ​​reduction, approximately 5 5% reduction ~ approximately 60% reduction, approximately 60% reduction ~ approximately 99% reduction, approximately 60% reduction ~ approximately 95% reduction, approximately 60% reduction ~ approximately 90% reduction, approximately 60% reduction ~ approximately 85% reduction, approximately 60% reduction ~ approximately 80% reduction, approximately 60% reduction ~ approximately 75% reduction, approximately 60% reduction ~ approximately 70% reduction, approximately 60% reduction ~ approximately 65% ​​reduction, approximately 65% ​​reduction ~ approximately 99% reduction, approximately 65% ​​reduction ~ approximately 95% reduction, approximately 65% ​​reduction ~ approximately 80% reduction, approximately 65% ​​reduction ~ approximately 75% reduction, approximately 65% ​​reduction ~ approximately 70% reduction, approximately 70% reduction ~ approximately 99% reduction, approximately 70% reduction ~ approximately 95% reduction, approximately 70% reduction % decrease ~ approximately 90% decrease, approximately 70% decrease ~ approximately 85% decrease, approximately 70% decrease ~ approximately 80% decrease, approximately 70% decrease ~ approximately 75% decrease, approximately 75% decrease ~ approximately 99% decrease, approximately 75% decrease ~ approximately 95% decrease, approximately 75% decrease ~ approximately 90% decrease, approximately 75% decrease ~ approximately 85% decrease, approximately 75% decrease ~ approximately 80% decrease, approximately 80% decrease ~ approximately 99% decrease, approximately 80% decrease ~ approximately 95% decrease, approximately 80% decrease ~ approximately 95% decrease, approximately 85% decrease ~ approximately 95% decrease, approximately 85% decrease ~ approximately 90% decrease, approximately 90% decrease ~ approximately 99% decrease, approximately 90% decrease ~ approximately 95% decrease, approximately 90% decrease ~ approximately 95% decrease,Alternatively, a decrease of approximately 95% to 99% could result.

[0062] The term “subject” refers to any mammal. In some embodiments, the subject or “subject requiring treatment” may be a canid (e.g., dog), a feline (e.g., cat), an equid (e.g., horse), a sheep, a cow, a pig, a goat, a primate (e.g., simian, monkey (e.g., marmoset, baboon), or an ape (e.g., gorilla, chimpanzee, orangutan, or gibbon), or a human, or a rodent (e.g., mouse, guinea pig, hamster, or rat). In some embodiments, the subject or “subject requiring treatment” may be a non-human mammal, and in particular mammals that have been conventionally used as models to demonstrate therapeutic effects in humans (e.g., mouse, lapine, pig, dog, or primate) may be used.

[0063] Treg cells In some embodiments, Treg cells may be administered to a subject. In some embodiments, the Treg cells administered to the subject may be autologous Treg cells, haplotype-matched Treg cells, or allogeneic Treg cells isolated from peripheral blood or umbilical cord blood. In some embodiments, the method described herein may further include isolating Treg cells from a subject, culturing the isolated Treg cells in a liquid culture medium, and returning the Treg cells to the subject. In some embodiments, isolating Treg cells from a subject includes obtaining a sample containing Treg cells from the subject and isolating Treg cells from the sample using an antibody or ligand capable of binding to CD39. In some embodiments, the step of isolating Treg cells from a sample includes mixing the sample with an antibody or ligand capable of binding to CD39 under conditions that allow the antibody of ligand to bind to CD39-expressing Treg cells, and separating the antibody or ligand-bound Treg cells from other components in the sample, thereby isolating the Treg cells. In some embodiments, the antibody is a mouse, humanized, or human antibody, or an antigen-binding fragment thereof, and / or the antibody or ligand is labeled with at least one of biotin, avidin, streptavidin, or a fluorescent dye, or is conjugated to particles, beads, resin, or a solid support. In some embodiments, separation includes the use of flow cytometry, fluorescent cell sorting (FACS), centrifugation, or column, plate, particle, or bead-based methods. In some embodiments, fresh or frozen peripheral blood, umbilical cord blood, peripheral blood mononuclear cells, lymphocytes, CD4 + These are autologous Treg cells, haplotype-matched Treg cells, or allogeneic Treg cells isolated from a sample containing T cells or Treg cells. In some embodiments, the Treg cells are CD4 + CD25 + Foxp3 + These are cells. In some embodiments, Treg cells are CD4 + CD25 + CD127dim -These are cells. In some embodiments, Treg cells are immunosuppressive in vitro and in vivo.

[0064] In some embodiments, Treg cells are prepared using a commercially available kit (e.g., EasySep® Human CD4). + CD127 low CD25 + Regulatory T cell isolation kit or Dynabeads CD4 + CD25 + They can be isolated using a regulatory T cell kit (see reference). In some embodiments, the liquid culture medium may contain one or more single-chain chimeric polypeptides (e.g., any of the exemplary single-chain chimeric polypeptides described herein, e.g., 2t2 or 3t28). In some embodiments, the liquid culture medium may include the use of beads having CD3 and CD28 and recombinant IL-2 or 2t2 on their surface.

[0065] In some embodiments, Treg cells may contain a chimeric antigen receptor (e.g., a chimeric antigen receptor including an extracellular domain that specifically binds to tissue factor or CD36). A non-limiting example of an extracellular domain capable of binding to tissue factor or CD36 is scFv. Non-limiting examples of anti-CD36 antibodies are commercially available from Invitrogen, Abcam, GeneTex, Novus Biologicals, Proteintech, and EMD Millipore. Non-limiting examples of anti-tissue factor heavy chain variable domains and light chain variable domains are described in U.S. Patents 7,968,094 and 8,007,795. The chimeric antigen receptor includes a transmembrane domain, a costimulatory domain (e.g., an intracellular CD28 domain), and a CD3 zeta signaling domain. For example, the transmembrane domain may contain sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to sequence number 1 (FWVLVVVGGVLACYSLLVTVAFIIFWV). For example, the co-stimulatory domain may contain sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to sequence number 2 (RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS). For example, the CD3 zeta signaling domain may contain a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to Sequence ID No. 3 (RVKFSRSADAPAYQQGQNQLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR).

[0066] Treg cell activator In some embodiments, one or more Treg cell activators can be administered to a subject. In some embodiments, the Treg cell activator can be a single-chain chimeric polypeptide (e.g., any of the exemplary single-chain chimeric polypeptides described herein), an anti-tissue factor antibody (e.g., the anti-tissue factor antibodies described in U.S. Patent No. 7,968,094 and U.S. Patent No. 8,007,795), a soluble RAGE protein, or an anti-CD36 antibody.

[0067] The soluble RAGE protein can have a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 4 or SEQ ID NO: 5. Soluble human RAGE variant 1 (SEQ ID NO: 4) TIFF2026053343000002.tif17145 Soluble human RAGE variant 2 (SEQ ID NO: 5) TIFF2026053343000003.tif17145

[0068] In some instances, the soluble RAGE protein is encoded by a nucleic acid having a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. Soluble human RAGE variant 1 cDNA (SEQ ID NO: 6) TIFF2026053343000004.tif48145 Mouse RAGE cDNA (SEQ ID NO: 7) TIFF2026053343000005.tif38145

[0069] As can be appreciated by those skilled in the art, substitutions / mutations made at positions that are not conserved across different species are less likely to adversely affect the activity of the protein / nucleic acid, while substitutions / mutations made at positions that are conserved across species are more likely to adversely affect the activity of the protein / nucleic acid.

[0070] NK cells In some embodiments, NK cells may be administered to a subject. In some embodiments, the NK cells administered to the subject may be autologous NK cells, haplotype-matched NK cells, or allogeneic NK cells isolated from peripheral blood, isolated from umbilical cord blood, or isolated and differentiated from iPSCs. In some embodiments, the method described herein may further include isolating NK cells from a subject, culturing the isolated NK cells in a liquid culture medium, and returning the NK cells to the subject. In some embodiments, NK cells may be isolated using a commercially available kit (see, for example, EasySep® Human NK Cell Isolation Kit, MojoSort Human NK Cell Isolation Kit, and Novus Biologicals Human NK Cell Isolation Kit). In some embodiments, the liquid culture medium may contain one or more polychain chimeric polypeptides (for example, any of the exemplary polychain chimeric polypeptides described herein, e.g., 18t15-12s and / or 7t15-21s).

[0071] In some embodiments, NK cells may include a chimeric antigen receptor (e.g., a chimeric antigen receptor including an extracellular domain that specifically binds to tissue factor or CD26). A non-limiting example of an extracellular domain capable of binding to tissue factor or CD26 is scFv. Non-limiting examples of anti-CD26 antibodies are those commercially available from Abcam, Invitrogen, and GeneTex. Non-limiting examples of anti-tissue factor heavy chain variable domains and light chain variable domains are described in U.S. Patents 7,968,094 and 8,007,795. The chimeric antigen receptor includes a transmembrane domain, a costimulatory domain (e.g., an intracellular CD28 domain), and a CD3 zeta signaling domain. For example, the transmembrane domain may contain a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to Sequence ID No. 1. For example, the co-stimulatory domain may contain sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to sequence number 2. For example, the CD3 zeta signaling domain may contain sequences that are at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to sequence number 3.

[0072] NK cell activator In some embodiments, one or more NK cell activators may be administered to the subject. In some embodiments, the NK cell activators may be one or more polychain chimeric polypeptides (e.g., any of the exemplary polychain chimeric polypeptides described herein), anti-tissue factor antibodies (e.g., anti-tissue factor antibodies described in U.S. Patent Nos. 7,968,094 and 8,007,795), anti-CD36 antibodies (e.g., anti-CD36 antibodies commercially available from Invitrogen, Abcam, GeneTex, Novus Biologicals, Proteintech, and EMD Millipore), or anti-CD26 antibodies (e.g., anti-CD26 antibodies commercially available from Abcam, Invitrogen, and GeneTex). NK cell activators, such as cytokine-based substances, can act by directing the activation of NK cells or by enhancing NK cell activity, such as antibody-mediated antibody-dependent cytotoxicity (ADCC) of NK cells.

[0073] Polychain chimeric polypeptide In some embodiments of the polychain chimeric polypeptides described herein, the first target-binding domain (e.g., any of the first target-binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of the polychain chimeric polypeptides described herein, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the first target-binding domain (e.g., any of the exemplary first target-binding domains described herein) and the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) within the first chimeric polypeptide.

[0074] In some embodiments of the polychain chimeric polypeptides described herein, a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein) are directly adjacent to each other within the first chimeric polypeptide. In some embodiments of the polychain chimeric polypeptides described herein, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of the exemplary pair of affinity domains described herein) within the first chimeric polypeptide.

[0075] In some embodiments of the polychain chimeric polypeptides described herein, the second domain of a pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pair of affinity domains described herein) and the second target-binding domain (e.g., any of the exemplary second target-binding domains described herein) are directly adjacent to each other within the second chimeric polypeptide. In some embodiments of the polychain chimeric polypeptides described herein, the second chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between the second domain of a pair of affinity domains (e.g., any of the exemplary second domains of any of the exemplary pair of affinity domains described herein) and the second target-binding domain (e.g., any of the exemplary second target-binding domains described herein).

[0076] tissue factor Human tissue factor is a 263-amino acid transmembrane protein comprising the following three domains: (1) a 219-amino acid N-terminal extracellular domain (residues 1-219), (2) a 22-amino acid transmembrane domain (residues 220-242), and (3) a 21-amino acid cytoplasmic C-terminal tail (residues 242-263) (UniProtKB identifier number: P13726). The cytoplasmic tail contains two phosphorylation sites at Ser253 and Ser258, and one S-palmitoylation site at Cys245. No deletion or mutation of the cytoplasmic domain was found to affect tissue factor coagulation activity. Tissue factor has one S-palmitoylation site within the intracellular domain of the protein at Cys245. Cys245 is located at the amino acid terminus of the intracellular domain, near the membrane surface. The tissue factor transmembrane domain consists of a single transmembrane α-helix.

[0077] The extracellular domain of tissue factor, composed of two fibronectin type III domains, is linked to the transmembrane domain via a 6-amino acid linker. This linker provides conformational mobility to separate the tissue factor extracellular domain from its transmembrane and cytoplasmic domains. Each tissue factor fibronectin type III module consists of two overlapping β-sheets; the upper sheet domain contains three antiparallel β-strands, and the lower sheet contains four β-strands. The β-strands are linked by β-loops between strands βA and βB, βC and βD, and βE and βF, and all of these conformations are conserved within the two modules. Three short α-helix segments are present linking the β-strands. A unique feature of tissue factor is the 17-amino acid β-hairpin between strands β10 and β11, which is not a common element of the fibronectin superfamily. The N-terminal domain also contains a 12-amino acid loop between β6F and β7G, which is 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 family folding and is conserved among a wide variety of extracellular proteins.

[0078] Zymogen FVII is rapidly converted to FVIIa by limited protein degradation once it binds to tissue and forms an active tissue factor-FVIIa complex. FVIIa, which circulates as an enzyme at a concentration of approximately 0.1 nM (1% of plasma FVII), can also directly bind to tissue factor. Allosteric interactions between tissue factor and FVIIa on the tissue factor-FVIIa complex significantly increase the enzymatic activity of FVIIa, increasing the hydrolysis rate of small chromogenic peptidyl substrates by approximately 20 to 100 times and the activation rate of the natural polymer substrates FIX and FX by almost 1 million times. In conjunction with the allosteric activation of the active site of FVIIa upon binding to tissue factor, the formation of the tissue factor-FVIIa complex on the phospholipid bilayer (i.e., upon exposure of phosphatidyl-L-serine on the membrane surface) is linked to Ca 2+ It further increases the activation rate of FIX or FX by a factor of 1,000 in a dependent manner. The overall increase of approximately 1 million times in FX activation by the tissue factor-FVIIa-phospholipid complex compared to free FVIIa represents a critical regulatory point in the coagulation cascade.

[0079] FVII is a single-chain polypeptide of approximately 50 kDa, consisting of 406 amino acid residues, and has 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 has an Ile- 154 -Arg 152 It is activated to FVIIa by specific proteolytic cleavage of the binding. This cleavage causes the light and heavy chains to break down into Cys 135 and Cys 262 They are held together by a single disulfide bond. FVIIa is then connected to Ca via its N-terminal GLA domain. 2+ It binds to the phospholipid membrane in a dependent manner. Immediately to the C-terminus of the GLA domain are an aromatic stack and two EGF domains. The aromatic stack connects the GLA domain to a single Ca 2+It binds to the EGF1 domain that binds to ions. 2+ Occupation of the binding site increases FVIIa amide degradation activity and tissue factor association. The catalytic triad is His 193 Asp 242 , and Ser 344 It consists of a single Ca within the FVIIa protease domain. 2+ Ionic bonding is essential for its catalytic activity. The proteolytic activity of FVII to FVIIa is due to Ile 153 The newly formed amino terminus is released, folded back, and inserted into the activation pocket, as shown by Asp 343 It forms a salt bridge with the carboxylate salt of FVIIa, generating an oxyanion hole. This salt bridge formation is essential for FVIIa activity. However, oxyanion hole formation does not occur in free FVIIa during proteolytic activation. As a result, FVIIa circulates in a zymogen-like state that is largely unrecognized by plasma protease inhibitors, allowing it to circulate with a half-life of approximately 90 minutes.

[0080] Tissue factor-mediated positioning of the FVIIa active site on the membrane surface is crucial for FVIIa relative to its congener substrates. Free FVIIa adopts a stable, extended structure upon binding to the membrane, with its active site positioned approximately 80 Å above the membrane surface. Upon binding of FVIIa to tissue factor, the FVa active site is repositioned approximately 6 Å closer to the membrane. This regulation may facilitate proper alignment of the FVIIa catalytic triad with the target substrate cleavage site. Using GLA domain-less FVIIa, it has been shown that the active site remains positioned at a similar distance from the membrane, demonstrating that tissue factor can fully support FVIIa active site positioning even in the absence of FVIIa-membrane interactions. Additional data show that tissue factor supports complete FVIIa proteolytic activity as long as the tissue factor extracellular domain is somehow anchored to the membrane surface. However, raising the active site of FVIIa above 80 Å from the membrane surface significantly reduced the tissue factor-FVIIa complex's ability to activate FX, but did not reduce tissue factor-FVIIa amid degradation activity.

[0081] Alanine scanning mutagenesis has been used to evaluate the role of specific amino acid side chains in the extracellular domain of tissue factor for interaction with FVIIa (Gibbs et al., Biochemistry 33(47):14003-14010, 1994; Schullek et al., J Biol Chem 269(30):19399-19403, 1994). Alanine substitution identified a limited number of residue locations where alanine substitution results in a 5- to 10-fold lower affinity for FVIIa binding. Most of these residue side chains were found to be well-exposed to the solvent in the crystal structure, in conjunction with polymeric ligand interactions. The FVIIa ligand binding site is located across a broad region of the boundary between the two modules. In the C-module, the residue Arg is located on the protruding BC loop. 135 and Phe 140 This provides independent contact with FVIIa. Leu 133 It is located at the base of the finger-like structure and is packed into the gap between the two modules. This allows Lys 20 , Thr 60 Asp 58 , and Ile 22 This provides continuity to the major clusters of important binding residues consisting of Thr. 60 It is only partially exposed to the solvent and may play a local structural role rather than making significant contact with the ligand. The binding site is Glu 24 and Gln 110 , as well as potentially more distal residues Val 207 It extends to the concave side of the intermodule angle, including the . The coupling region is from Asp58 to Lys 48 Lys 46 , Gln 37 Asp 44 , and Trp 45 It extends into the convex region formed by Trp. 45 and Asp 44 It does not interact with FVIIa independently, and Trp 45 The mutation effect at the position is adjacent to Asp44 and Gln 37 This suggests that the structural importance of this side chain for local packing of the side chain may be reflected. The interaction region consists of two surface-exposed aromatic residues Phe that form part of a hydrophobic cluster within the N-module. 76 and Tyr 78 It also includes.

[0082] Known physiological substrates of tissue factor-FVIIa include FVII, FIX, and FX, as well as certain proteinase-activated receptors. Mutation analysis has identified several residues that, when mutated, support complete FVIIa amide degradation activity for small peptidyl substrates but lack the ability to support activation of high molecular weight 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 within or adjacent to this mobile loop, have been shown to be essential for the proteolytic activity of the tissue factor-FVIIa complex. This defines the proposed substrate-binding exocyte region of tissue factor, which is considerably distant from the FVIIa active site. Substitution of the glycine residue with a slightly bulkier alanine residue significantly impairs the proteolytic activity of tissue factor-FVIIa. This suggests that the mobility provided by glycine is essential for the residue 159–165 loop for tissue factor macromolecular substrate recognition.

[0083] Residue Lys 165 and Lys 166 It has also been demonstrated that these are important for substrate recognition and binding. Mutation of any of these residues to alanine significantly reduces tissue factor cofactor function. Lys 165 and Lys166 are directed outward from each other, Lys 165 refers to FVIIa within most of the tissue factor - FVIIa structure, Lys 166 refers to the substrate - binding exosite region within the crystal structure. The Lys of FVIIa 165 and the Gla of FVIIa 35 The putative salt - bridge formation between them would support the concept that the interaction of the GLA domain of FVIIa with tissue factor regulates substrate recognition. These results suggest that the C - terminal portion of the tissue factor extracellular domain interacts directly with the GLA domains of FIX and FX and the potentially adjacent EGF1 domain, and the presence of the FVIIa GLA domain can regulate these interactions either directly or indirectly.

[0084] Soluble tissue factor domain In some embodiments of any of the polypeptides described herein, the soluble tissue factor domain can be a wild - type tissue factor polypeptide lacking a signal sequence, transmembrane domain, and intracellular domain. In some examples, the soluble tissue factor domain can be a tissue factor variant, where the wild - type tissue factor polypeptide lacks a signal sequence, transmembrane domain, and intracellular domain and is further modified with selected amino acids. In some examples, the soluble tissue factor domain can be a soluble human tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble mouse tissue factor domain. In some examples, the soluble tissue factor domain can be a soluble rat tissue factor domain. Non - limiting examples of soluble human tissue factor domains, soluble mouse tissue factor domains, soluble rat tissue factor domains, and variant soluble tissue factor domains are shown below. Exemplary soluble human tissue factor domain (SEQ ID NO: 8) SGTTNTVAAYNLTWKSTNFKTILEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECDLTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVRRNNTFLSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary nucleic acids encoding a soluble human tissue factor domain (SEQ ID NO: 9) AGCGGCACAACCAACACAGTCGCTGCCTATAACCTCACTTGGAAGAGCACCAACTTCAAAACCATCCTCGAATGGGAACCCAAACCCGTTAACCAAGTTTACACCGTGCAGATCAGCACCAAGTCCGGCGACTGGAAGTCCAAATGTTTCTATACCACCGACAC CGAGTGCGATCTCACCGATGAGATCGTGAAAGATGTGAAACAGACCTACCTCGCCCGGGTGTTTAGCTACCCCGCCGGCAATGTGGAGAGCACTGGTTCCGCTGGCGAGCCTTTATACGAGAACAGCCCCGAATTTACCCCTTACCTCGAGACCAATTTAGGAC AGCCCACCATCCAAAGCTTTGAGCAAGTTGGCACAAAGGTGAATGTGACAGTGGAGGACGAGCGGACTTTAGTGCGGCGGAACAACACCTTTCTCAGCCTCCGGGATGTGTTCGGCAAAGATTTAATCTACACACTGTATTACTGGAAGTCCTCTTCCTCCGGC AAGAAGACAGCTAAAACCAACACAAACGAGTTTTTAATCGACGTGGATAAAGGCGAAAACTACTGTTTCAGCGTGCAAGCTGTGATCCCCTCCCGGACCGTGAATAGGAAAAGCACCGATAGCCCCGTTGAGTGCATGGGCCAAGAAAAGGGCGAGTTCCGGGAG Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 10) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDWKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLYENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary mutant soluble human tissue factor domain (SEQ ID NO: 11) SGTTNTVAAYNLTWKSTNFATALEWEPKPVNQVYTVQISTKSGDAKSKCFYTTDTECALTDEIVKDVKQTYLARVFSYPAGNVESTGSAGEPLAENSPEFTPYLETNLG QPTIQSFEQVGTKVNVTVEDERTLVARNNTALSLRDVFGKDLIYTLYYWKSSSSGKKTAKTNTNEFLIDVDKGENYCFSVQAVIPSRTVNRKSTDSPVECMGQEKGEFRE Exemplary soluble mouse tissue factor domain (SEQ ID NO: 12) agipekafnltwistdfktilewqpkptnytytvqisdrsrnwknkcfsttdtecdltdeivkdvtwayeakvlsvprrnsvhgdgdqlvihgeeppftnapkflpyrdtn lgqpviqqfeqdgrklnvvvkdsltlvrkngtfltlrqvfgkdlgyiityrkgsstgkktnitntnefsidveegvsycffvqamifsrktnqnspgsstvcteqwksflge Exemplary soluble rat tissue factor domain (SEQ ID NO: 13) agtppgkafnltwistdfktilewqpkptnytytvqisdrsrnwkykctgttdtecdltdeivkdvnwtyearvlsvpwrnsthgketlfgthgeeppftnarkflpyrdtk igqpviqkyeqggtklkvtvkdsftlvrkngtfltlrqvfgndlgyiltyrkdsstgrktntthtneflidvekgvsycffaqavifsrktnhkspesitkcteqwksvlge

[0085] In some versions, the soluble tissue factor domain may contain sequences that are at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to sequence number 8, 10, 11, 12, or 13. In some embodiments, the soluble tissue factor domain may include the sequence of SEQ ID NOs. 8, 10, 11, 12, or 13, with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its N-terminus and / or with 1 to 20 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) removed from its C-terminus.

[0086] As can be understood in the art, a person skilled in the art will understand that amino acid mutations conserved between different mammalian species are likely to reduce the activity and / or structural stability of a protein, while amino acid mutations not conserved between different mammalian species are less likely to reduce the activity and / or structural stability of a protein.

[0087] In some examples of the single-chain or multi-chain chimeric polypeptides described herein, the soluble tissue factor domain cannot bind to factor VIIa. In some examples of the single-chain or multi-chain chimeric polypeptides described herein, the soluble tissue factor domain does not convert inactive factor X to factor Xa. In some embodiments of the single-chain or multi-chain chimeric polypeptides described herein, the single-chain or multi-chain chimeric polypeptides do not stimulate blood coagulation in mammals.

[0088] In some examples, 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.

[0089] In some examples, the soluble tissue factor domain does not contain one or more (e.g., 2, 3, 4, 5, 6, or 7) of the following amino acids: lysine at the position corresponding to amino acid position 20 of the mature wild-type human tissue factor protein, isoleucine at the position corresponding to amino acid position 22 of the mature wild-type human tissue factor protein, tryptophan at the position corresponding to amino acid position 45 of the mature wild-type human tissue factor protein, aspartic acid at the position corresponding to amino acid position 58 of the mature wild-type human tissue factor protein, tyrosine at the position corresponding to amino acid position 94 of the mature wild-type human tissue factor protein, arginine at the position corresponding to amino acid position 135 of the mature wild-type human tissue factor protein, and phenylalanine at the position corresponding to amino acid position 140 of the mature wild-type human tissue factor protein. In some embodiments, the mutant soluble tissue factor has the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11.

[0090] In some cases, the soluble tissue factor domain may be encoded by nucleic acids containing sequences that are at least 70% identical, at least 72% identical, at least 74% identical, at least 76% identical, at least 78% identical, at least 80% identical, at least 82% identical, at least 84% identical, at least 86% identical, at least 88% identical, at least 90% identical, at least 92% identical, at least 94% identical, at least 96% identical, at least 98% identical, at least 99% identical, or 100% identical to Sequence ID No. 9.

[0091] Linker array In some embodiments, the linker sequence may be a mobile linker sequence. Non-limiting examples of linker sequences that can be used are described in Klein et al., Protein Engineering, Design & Selection 27(10):325-330, 2014 and Priyanka et al., Protein Sci. 22(2):153-167, 2013. In some examples, the linker sequence is a synthetic linker sequence.

[0092] In some embodiments of the polychain chimeric polypeptides described herein, the first chimeric polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art). In some embodiments of the polychain chimeric polypeptides described herein, the second chimeric polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker sequences (e.g., the same or different linker sequences, e.g., any of the exemplary linker sequences described herein or known in the art).

[0093] In some embodiments, the linker sequence is 1 amino acid to approximately 100 amino acids, 1 amino acid to approximately 90 amino acids, 1 amino acid to approximately 80 amino acids, 1 amino acid to approximately 70 amino acids, 1 amino acid to approximately 60 amino acids, 1 amino acid to approximately 50 amino acids, 1 amino acid to approximately 45 amino acids, 1 amino acid to approximately 40 amino acids, 1 amino acid to approximately 35 amino acids, 1 amino acid to approximately 30 amino acids, 1 amino acid to approximately 25 amino acids, 1 amino acid to approximately 24 amino acids, 1 amino acid to approximately 22 amino acids, 1 amino acid to approximately 20 amino acids, 1 amino acid to approximately 18 amino acids, 1 amino acid to approximately 16 amino acids, 1 amino acid Acid ~ approximately 14 amino acids, 1 amino acid ~ approximately 12 amino acids, 1 amino acid ~ approximately 10 amino acids, 1 amino acid ~ approximately 8 amino acids, 1 amino acid ~ approximately 6 amino acids, 1 amino acid ~ approximately 4 amino acids, approximately 2 amino acids ~ approximately 100 amino acids, approximately 2 amino acids ~ approximately 90 amino acids, approximately 2 amino acids ~ approximately 80 amino acids, approximately 2 amino acids ~ approximately 70 amino acids, approximately 2 amino acids ~ approximately 60 amino acids, approximately 2 amino acids ~ approximately 50 amino acids, approximately 2 amino acids ~ approximately 45 amino acids, approximately 2 amino acids ~ approximately 40 amino acids, approximately 2 amino acids ~ approximately 35 amino acids, approximately 2 amino acids ~ approximately 30 amino acids, approximately 2 amino acids ~ approximately 25 amino acids, approximately 2 amino acids ~ Approximately 24 amino acids, approximately 2 to approximately 22 amino acids, approximately 2 to approximately 20 amino acids, approximately 2 to approximately 18 amino acids, approximately 2 to approximately 16 amino acids, approximately 2 to approximately 14 amino acids, approximately 2 to approximately 12 amino acids, approximately 2 to approximately 10 amino acids, approximately 2 to approximately 8 amino acids, approximately 2 to approximately 6 amino acids, approximately 2 to approximately 4 amino acids, approximately 4 to approximately 100 amino acids, approximately 4 to approximately 90 amino acids, approximately 4 to approximately 80 amino acids, approximately 4 to approximately 70 amino acids, approximately 4 to approximately 60 amino acids, approximately 4 to approximately 50 amino acids, approximately 4 amino acids 0 amino acids ~ approximately 45 amino acids, approximately 4 amino acids ~ approximately 40 amino acids, approximately 4 amino acids ~ approximately 35 amino acids, approximately 4 amino acids ~ approximately 30 amino acids, approximately 4 amino acids ~ approximately 25 amino acids, approximately 4 amino acids ~ approximately 24 amino acids, approximately 4 amino acids ~ approximately 22 amino acids, approximately 4 amino acids ~ approximately 20 amino acids, approximately 4 amino acids ~ approximately 18 amino acids, approximately 4 amino acids ~ approximately 16 amino acids, approximately 4 amino acids ~ approximately 14 amino acids, approximately 4 amino acids ~ approximately 12 amino acids, approximately 4 amino acids ~ approximately 10 amino acids, approximately 4 amino acids ~ approximately 8 amino acids, approximately 4 amino acids ~ approximately 6 amino acids, approximately 6 amino acids ~ approximately 100 amino acids, approximately 6 amino acids ~ approximately 90 amino acids,Approximately 6 amino acids to approximately 80 amino acids, approximately 6 amino acids to approximately 70 amino acids, approximately 6 amino acids to approximately 60 amino acids, approximately 6 amino acids to approximately 50 amino acids, approximately 6 amino acids to approximately 45 amino acids, approximately 6 amino acids to approximately 40 amino acids, approximately 6 amino acids to approximately 35 amino acids, approximately 6 amino acids to approximately 30 amino acids, approximately 6 amino acids to approximately 25 amino acids, approximately 6 amino acids to approximately 24 amino acids, approximately 6 amino acids to approximately 22 amino acids, approximately 6 amino acids to approximately 20 amino acids, approximately 6 amino acids to approximately 18 amino acids, approximately 6 amino acids to approximately 16 amino acids, approximately 6 amino acids to approximately 14 amino acids, approximately 6 amino acids to approximately 12 amino acids, approximately 6 amino acids to Approximately 10 amino acids, approximately 6 to approximately 8 amino acids, approximately 8 to approximately 100 amino acids, approximately 8 to approximately 90 amino acids, approximately 8 to approximately 80 amino acids, approximately 8 to approximately 70 amino acids, approximately 8 to approximately 60 amino acids, approximately 8 to approximately 50 amino acids, approximately 8 to approximately 45 amino acids, approximately 8 to approximately 40 amino acids, approximately 8 to approximately 35 amino acids, approximately 8 to approximately 30 amino acids, approximately 8 to approximately 25 amino acids, approximately 8 to approximately 24 amino acids, approximately 8 to approximately 22 amino acids, approximately 8 to approximately 20 amino acids, approximately 8 to approximately 18 amino acids Approximately 8 to 16 amino acids, approximately 8 to 14 amino acids, approximately 8 to 12 amino acids, approximately 8 to 10 amino acids, approximately 10 to 100 amino acids, approximately 10 to 90 amino acids, approximately 10 to 80 amino acids, approximately 10 to 70 amino acids, approximately 10 to 60 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 40 amino acids 24 amino acids, approximately 10 to 22 amino acids, approximately 10 to 20 amino acids, approximately 10 to 18 amino acids, approximately 10 to 16 amino acids, approximately 10 to 14 amino acids, approximately 10 to 12 amino acids, approximately 12 to 100 amino acids, approximately 12 to 90 amino acids, approximately 12 to 80 amino acids, approximately 12 to 70 amino acids, approximately 12 to 60 amino acids, approximately 12 to 50 amino acids, approximately 12 to 45 amino acids, approximately 12 to 40 amino acids, approximately 12 to 35 amino acids,Approximately 12 amino acids to approximately 30 amino acids, approximately 12 amino acids to approximately 25 amino acids, approximately 12 amino acids to approximately 24 amino acids, approximately 12 amino acids to approximately 22 amino acids, approximately 12 amino acids to approximately 20 amino acids, approximately 12 amino acids to approximately 18 amino acids, approximately 12 amino acids to approximately 16 amino acids, approximately 12 amino acids to approximately 14 amino acids, approximately 14 amino acids to approximately 100 amino acids, approximately 14 amino acids to approximately 90 amino acids, approximately 14 amino acids to approximately 80 amino acids, approximately 14 amino acids to approximately 70 amino acids, approximately 14 amino acids to approximately 60 amino acids, approximately 14 amino acids to approximately 50 amino acids, approximately 14 amino acids to approximately 45 amino acids, approximately 14 amino acids to Approximately 40 amino acids, approximately 14 to approximately 35 amino acids, approximately 14 to approximately 30 amino acids, approximately 14 to approximately 25 amino acids, approximately 14 to approximately 24 amino acids, approximately 14 to approximately 22 amino acids, approximately 14 to approximately 20 amino acids, approximately 14 to approximately 18 amino acids, approximately 14 to approximately 16 amino acids, approximately 16 to approximately 100 amino acids, approximately 16 to approximately 90 amino acids, approximately 16 to approximately 80 amino acids, approximately 16 to approximately 70 amino acids, approximately 16 to approximately 60 amino acids, approximately 16 to approximately 50 amino acids, approximately 16 to approximately 45 amino acids, Approximately 16 amino acids to approximately 40 amino acids, approximately 16 amino acids to approximately 35 amino acids, approximately 16 amino acids to approximately 30 amino acids, approximately 16 amino acids to approximately 25 amino acids, approximately 16 amino acids to approximately 24 amino acids, approximately 16 amino acids to approximately 22 amino acids, approximately 16 amino acids to approximately 20 amino acids, approximately 16 amino acids to approximately 18 amino acids, approximately 18 amino acids to approximately 100 amino acids, approximately 18 amino acids to approximately 90 amino acids, approximately 18 amino acids to approximately 80 amino acids, approximately 18 amino acids to approximately 70 amino acids, approximately 18 amino acids to approximately 60 amino acids, approximately 18 amino acids to approximately 50 amino acids, approximately 18 amino acids to approximately 45 amino acids, approximately 18 amino acids to Approximately 40 amino acids, approximately 18 to approximately 35 amino acids, approximately 18 to approximately 30 amino acids, approximately 18 to approximately 25 amino acids, approximately 18 to approximately 24 amino acids, approximately 18 to approximately 22 amino acids, approximately 18 to approximately 20 amino acids, approximately 20 to approximately 100 amino acids, approximately 20 to approximately 90 amino acids, approximately 20 to approximately 80 amino acids, approximately 20 to approximately 70 amino acids, approximately 20 to approximately 60 amino acids, approximately 20 to approximately 50 amino acids, approximately 20 to approximately 45 amino acids, approximately 20 to approximately 40 amino acids, approximately 20 to approximately 35 amino acids,Approximately 20 to 30 amino acids, approximately 20 to 25 amino acids, approximately 20 to 24 amino acids, approximately 20 to 22 amino acids, approximately 22 to 100 amino acids, approximately 22 to 90 amino acids, approximately 22 to 80 amino acids, approximately 22 to 70 amino acids, approximately 22 to 60 amino acids, approximately 22 to 50 amino acids, approximately 22 to 45 amino acids, approximately 22 to 40 amino acids, approximately 22 to 35 amino acids, approximately 22 to 30 amino acids, approximately 22 to 25 amino acids, approximately 22 to approx 24 amino acids, approximately 25 to approximately 100 amino acids, approximately 25 to approximately 90 amino acids, approximately 25 to approximately 80 amino acids, approximately 25 to approximately 70 amino acids, approximately 25 to approximately 60 amino acids, approximately 25 to approximately 50 amino acids, approximately 25 to approximately 45 amino acids, approximately 25 to approximately 40 amino acids, approximately 25 to approximately 35 amino acids, approximately 25 to approximately 30 amino acids, approximately 30 to approximately 100 amino acids, approximately 30 to approximately 90 amino acids, approximately 30 to approximately 80 amino acids, approximately 30 to approximately 70 amino acids, approximately 30 to approximately 60 amino acids, approximately 30 amino acids to approximately 50 amino acids, approximately 30 amino acids to approximately 45 amino acids, approximately 30 amino acids to approximately 40 amino acids, approximately 30 amino acids to approximately 35 amino acids, approximately 35 amino acids to approximately 100 amino acids, approximately 35 amino acids to approximately 90 amino acids, approximately 35 amino acids to approximately 80 amino acids, approximately 35 amino acids to approximately 70 amino acids, approximately 35 amino acids to approximately 60 amino acids, approximately 35 amino acids to approximately 50 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 100 amino acids, approximately 40 amino acids to approximately 90 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 70 amino acids, approximately 40 to 60 amino acids, approximately 40 to 50 amino acids, approximately 40 to 45 amino acids, approximately 45 to 100 amino acids, approximately 45 to 90 amino acids, approximately 45 to 80 amino acids, approximately 45 to 70 amino acids, approximately 45 to 60 amino acids, approximately 45 to 50 amino acids, approximately 50 to 100 amino acids, approximately 50 to 90 amino acids, approximately 50 to 80 amino acids, approximately 50 to 70 amino acids, approximately 50 to 60 amino acids, approximately 60 to 100 amino acids,It may have a total length of approximately 60 to 90 amino acids, approximately 60 to 80 amino acids, approximately 60 to 70 amino acids, approximately 70 to 100 amino acids, approximately 70 to 90 amino acids, approximately 70 to 80 amino acids, approximately 80 to 100 amino acids, approximately 80 to 90 amino acids, or approximately 90 to 100 amino acids.

[0094] In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in both glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more GGSG sequences.

[0095] In some embodiments, the linker sequence may include or consist of GGGGSGGGGSGGGGS (SEQ ID NO: 14). In some embodiments, the linker sequence may be encoded by nucleic acids including or consisting of GGCGGTGGAGGATCCGGAGGAGGTGGCTCCGGCGGCGGAGGATCT (SEQ ID NO: 15). In some embodiments, the linker sequence may include or consist of GGGSGGGS (SEQ ID NO: 16).

[0096] Target-binding domain In some embodiments of any single-chain or 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 an antigen-binding domain (e.g., any of the exemplary antigen-binding domains described herein or known in the art), a soluble interleukin or cytokine protein (e.g., any of the exemplary soluble interleukin proteins or soluble cytokine proteins described herein), and a soluble interleukin or cytokine receptor (e.g., any of the exemplary soluble interleukin receptors or soluble cytokine receptors described herein).

[0097] In some embodiments of the single-chain or multi-chain chimeric polypeptides described herein, one or more of the first target-binding domain (e.g., any of the exemplary first target-binding domains described herein or known in the art), the second target-binding domain (e.g., any of the exemplary second target-binding domains described herein or known in the art), and one or more additional target-binding domains can each independently bind specifically to targets selected from the following group: CD16a, CD28, CD3 (e.g., one or more of CD3α, CD3β, CD3δ, CD3M, and CD3γ), CD33, CD20, CD19, CD22, CD123, IL-1R, IL-1, VEGF, IL-6R, IL-4 , IL-10, PDL-1, TIGIT, PD-1, TIM3, CTLA4, MICA, MICB, IL-6, IL-8, TNFα, CD26a, CD36, ULBP2, CD30, CD200, IGF -1R, MUC4AC, MUC5AC, Trop-2, CMET, EGFR, HER1, HER2, HER3, PSMA, CEA, B7H3, EPCAM, BCMA, P-cadherin, CEACAM5, U L16 binding proteins (e.g., ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6), HLA-DR, DLL4, TYRO3, AXL, MER, CD122, CD155, PDGF-DD, TGF-β receptor II (TGF-βRII) ligand, TGF-βRIII ligand, DNAM-1 ligand, NKp46 ligand, NKp44 ligand, NKG2D ligand, NK P 30 ligands, scMHCI ligand, scMHCII ligand, scTCR ligand, IL-1 receptor, IL-2 receptor, IL-3 receptor, IL-7 receptor, IL-8 receptor, IL-10 receptor, IL-12 receptor, IL-15 receptor, IL-17 receptor, IL-18 receptor, IL-21 receptor, PDGF-DD receptor, stem cell factor (SCF) receptor, stem cell-like tyrosine kinase 3 ligand (FLT3L) receptor, MICA receptor, MICB receptor, ULP16-binding protein receptor, CD155 receptor, CD122 receptor, and CD28 receptor.

[0098] In some embodiments of any single-chain or multi-chain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and / or one or more additional target-binding domains each independently bind to approximately 5 to 1000 amino acids, approximately 5 to 950 amino acids, approximately 5 to 900 amino acids, approximately 5 to 850 amino acids, approximately 5 to 800 amino acids, approximately 5 to 750 amino acids, approximately 5 to 700 amino acids, approximately 5 to 650 amino acids, approximately 5 to 600 amino acids, and approximately 5 amino acids. 5 amino acids ~ approximately 550 amino acids, approximately 5 amino acids ~ approximately 500 amino acids, approximately 5 amino acids ~ approximately 450 amino acids, approximately 5 amino acids ~ approximately 400 amino acids, approximately 5 amino acids ~ approximately 350 amino acids, approximately 5 amino acids ~ approximately 300 amino acids, approximately 5 amino acids ~ approximately 280 amino acids, approximately 5 amino acids ~ approximately 260 amino acids, approximately 5 amino acids ~ approximately 240 amino acids, approximately 5 amino acids ~ approximately 220 amino acids, approximately 5 amino acids ~ approximately 200 amino acids, approximately 5 amino acids ~ approximately 195 amino acids, approximately 5 amino acids ~ approximately 190 amino acids, approximately 5 amino acids ~ approximately 185 amino acids, approximately 5 amino acids ~ approximately 180 amino acids, approximately 5 amino acids ~ approximately 175 amino acids 5 amino acids, approximately 5 amino acids to approximately 170 amino acids, approximately 5 amino acids to approximately 165 amino acids, approximately 5 amino acids to approximately 160 amino acids, approximately 5 amino acids to approximately 155 amino acids, approximately 5 amino acids to approximately 150 amino acids, approximately 5 amino acids to approximately 145 amino acids, approximately 5 amino acids to approximately 140 amino acids, approximately 5 amino acids to approximately 135 amino acids, approximately 5 amino acids to approximately 130 amino acids, approximately 5 amino acids to approximately 125 amino acids, approximately 5 amino acids to approximately 120 amino acids, approximately 5 amino acids to approximately 115 amino acids, approximately 5 amino acids to approximately 110 amino acids, approximately 5 amino acids to approximately 105 amino acids, approximately 5 amino acids to approximately 100 amino acids, approximately 5 amino acids ~95 amino acids, approximately 5 amino acids to approximately 90 amino acids, approximately 5 amino acids to approximately 85 amino acids, approximately 5 amino acids to approximately 80 amino acids, approximately 5 amino acids to approximately 75 amino acids, approximately 5 amino acids to approximately 70 amino acids, approximately 5 amino acids to approximately 65 amino acids, approximately 5 amino acids to approximately 60 amino acids, approximately 5 amino acids to approximately 55 amino acids, approximately 5 amino acids to approximately 50 amino acids, approximately 5 amino acids to approximately 45 amino acids, approximately 5 amino acids to approximately 40 amino acids, approximately 5 amino acids to approximately 35 amino acids, approximately 5 amino acids to approximately 30 amino acids, approximately 5 amino acids to approximately 25 amino acids, approximately 5 amino acids to approximately 20 amino acids, approximately 5 amino acids to approximately 15 amino acids,Approximately 5 amino acids to approximately 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, approximately 10 amino acids to approximately 150 amino acids, approximately 10 amino acids to approximately 145 amino acids, approximately 10 amino acids to approximately 140 amino acids, approximately 10 amino acids to approximately 135 amino acids, approximately 10 amino acids to approximately 130 amino acids, approximately 10 amino acids to approximately 125 amino acids, approximately 10 amino acids to approximately 120 amino acids, approximately 10 amino acids to approximately 115 amino acids, approximately 10 amino acids to approximately 110 amino acids, approximately 10 amino acids to approximately 105 amino acids, approximately 10 amino acids to approximately 100 amino acids, approximately 10 amino acids to approximately 95 amino acids, approximately 10 amino acids to approximately 90 amino acids, approximately 10 amino acids to approximately 85 Amino acids, approximately 10 to 80 amino acids, 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, approximately 15 to 1000 amino acids,Approximately 15 amino acids to approximately 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, approximately 15 amino acids to approximately 240 amino acids, approximately 15 amino acids to approximately 220 amino acids, approximately 15 amino acids to approximately 200 amino acids, approximately 15 amino acids to approximately 195 amino acids, approximately 15 amino acids to approximately 190 amino acids, approximately 15 amino acids to approximately 185 amino acids, approximately 15 amino acids to approximately 180 amino acids, approximately 15 amino acids to approximately 175 amino acids, approximately 15 amino acids to approximately 170 amino acids, approximately 15 amino acids to approximately 165 amino acids, approximately 15 amino acids to approximately 160 amino acids, approximately 15 amino acids to approximately 155 amino acids, approximately 15 amino acids to approximately 150 amino acids, approximately 15 amino acids to approximately 145 amino acids, approximately 15 amino acids to approximately 140 amino acids, approximately 15 amino acids to approximately 135 amino acids, approximately 15 amino acids to approximately 130 amino acids, approximately 15 amino acids to approximately 125 amino acids, approximately 15 amino acids to approximately 120 amino acids, approximately 15 amino acids to approximately 115 amino acids, approximately 15 amino acids to approximately 110 amino acids, approximately 15 amino acids to approximately 105 amino acids, approximately 15 amino acids to approximately 100 amino acids, approximately 15 amino acids to approximately 95 amino acids, approximately 15 amino acids to approximately 90 amino acids, approximately 15 amino acids to approximately 85 amino acids, approximately 15 amino acids to approximately 80 amino acids, approximately 15 amino acids to approximately 75 amino acids Acid, approximately 15 amino acids to approximately 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 to approximately 200 amino acids, approximately 20 to approximately 195 amino acids, approximately 20 to approximately 190 amino acids, approximately 20 to approximately 185 amino acids, approximately 20 to approximately 180 amino acids, approximately 20 to approximately 175 amino acids, approximately 20 to approximately 170 amino acids, approximately 20 to approximately 165 amino acids, approximately 20 to approximately 160 amino acids, approximately 20 to approximately 155 amino acids, approximately 20 to approximately 150 amino acids, approximately 20 to approximately 145 amino acids, approximately 20 to approximately 140 amino acids, approximately 20 to approximately 135 amino acids, approximately 20 amino acids to approximately 130 amino acids, approximately 20 amino acids to approximately 125 amino acids, approximately 20 amino acids to approximately 120 amino acids, approximately 20 amino acids to approximately 115 amino acids, approximately 20 amino acids to approximately 110 amino acids, approximately 20 amino acids to approximately 105 amino acids, approximately 20 amino acids to approximately 100 amino acids, approximately 20 amino acids to approximately 95 amino acids, approximately 20 amino acids to approximately 90 amino acids, approximately 20 amino acids to approximately 85 amino acids, approximately 20 amino acids to approximately 80 amino acids, approximately 20 amino acids to approximately 75 amino acids, approximately 20 amino acids to approximately 70 amino acids, approximately 20 amino acids to approximately 65 amino acids, approximately 20 amino acids to approximately 60 amino acids, approximately 20 amino acids to approximately 55 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 25 amino acids to approximately 1000 amino acids, approximately 25 amino acids to approximately 950 amino acids, approximately 25 amino acids to approximately 900 amino acids, approximately 25 amino acids to approximately 850 amino acids, approximately 25 amino acids to approximately 800 amino acids, approximately 25 amino acids to approximately 750 amino acids, approximately 25 amino acids to approximately 700 amino acids, approximately 25 amino acids to approximately 650 amino acids,Approximately 25 amino acids to approximately 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, approximately 25 amino acids to approximately 175 amino acids, approximately 25 amino acids to approximately 170 amino acids, approximately 25 amino acids to approximately 165 amino acids, approximately 25 amino acids to approximately 160 amino acids, approximately 25 amino acids to approximately 155 amino acids, approximately 25 amino acids to approximately 150 amino acids, approximately 25 amino acids to approximately 145 amino acids, approximately 25 amino acids to approximately 140 amino acids, approximately 25 amino acids to approximately 135 amino acids, approximately 25 amino acids to approximately 130 amino acids, approximately 25 amino acids to approximately 125 amino acids, approximately 25 amino acids to approximately 120 amino acids, approximately 25 amino acids to approximately 115 amino acids, approximately 2 5 amino acids to approximately 110 amino acids, approximately 25 amino acids to approximately 105 amino acids, approximately 25 amino acids to approximately 100 amino acids, approximately 25 amino acids to approximately 95 amino acids, approximately 25 amino acids to approximately 90 amino acids, approximately 25 amino acids to approximately 85 amino acids, approximately 25 amino acids to approximately 80 amino acids, approximately 25 amino acids to approximately 75 amino acids, approximately 25 amino acids to approximately 70 amino acids, approximately 25 amino acids to approximately 65 amino acids, approximately 25 amino acids to approximately 60 amino acids, approximately 25 amino acids to approximately 55 amino acids, approximately 25 amino acids to approximately 50 amino acids, approximately 25 amino acids to approximately 45 amino acids, approximately 25 amino acids to approximately 40 amino acids, approximately 25 amino acids Acid ~ approximately 35 amino acids, approximately 25 amino acids ~ approximately 30 amino acids, approximately 30 amino acids ~ approximately 1000 amino acids, approximately 30 amino acids ~ approximately 950 amino acids, approximately 30 amino acids ~ approximately 900 amino acids, approximately 30 amino acids ~ approximately 850 amino acids, approximately 30 amino acids ~ approximately 800 amino acids, approximately 30 amino acids ~ approximately 750 amino acids, approximately 30 amino acids ~ approximately 700 amino acids, approximately 30 amino acids ~ approximately 650 amino acids, approximately 30 amino acids ~ approximately 600 amino acids, approximately 30 amino acids ~ approximately 550 amino acids, approximately 30 amino acids ~ approximately 500 amino acids, approximately 30 amino acids ~ approximately 450 amino acids, approximately 30 amino acids ~ approximately 400 amino acids,Approximately 30 amino acids to approximately 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, about 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, approximately 30 amino acids to approximately 150 amino acids, approximately 30 amino acids to approximately 145 amino acids, approximately 30 amino acids to approximately 140 amino acids, approximately 30 amino acids to approximately 135 amino acids, approximately 30 amino acids to approximately 130 amino acids, approximately 30 amino acids to approximately 125 amino acids, approximately 30 amino acids to approximately 120 amino acids, approximately 30 amino acids to approximately 115 amino acids, approximately 30 amino acids to approximately 1 10 amino acids, approximately 30 to 105 amino acids, approximately 30 to 100 amino acids, approximately 30 to 95 amino acids, approximately 30 to 90 amino acids, approximately 30 to 85 amino acids, approximately 30 to 80 amino acids, approximately 30 to 75 amino acids, approximately 30 to 70 amino acids, approximately 30 to 65 amino acids, approximately 30 to 65 amino acids, approximately 30 to 55 amino acids, approximately 30 to 50 amino acids, approximately 30 to 45 amino acids, approximately 30 to 40 amino acids, approximately 30 to 35 amino acids Approximately 35 amino acids to approximately 1000 amino acids, approximately 35 amino acids to approximately 950 amino acids, approximately 35 amino acids to approximately 900 amino acids, approximately 35 amino acids to approximately 850 amino acids, approximately 35 amino acids to approximately 800 amino acids, approximately 35 amino acids to 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 ~approximately 300 amino acids, approximately 35 amino acids~approximately 280 amino acids, approximately 35 amino acids~approximately 260 amino acids, approximately 35 amino acids~approximately 240 amino acids, approximately 35 amino acids~approximately 220 amino acids, approximately 35 amino acids~approximately 200 amino acids, approximately 35 amino acids~approximately 195 amino acids, approximately 35 amino acids~approximately 190 amino acids, approximately 35 amino acids~approximately 185 amino acids, approximately 35 amino acids~approximately 180 amino acids, approximately 35 amino acids~approximately 175 amino acids, approximately 35 amino acids~approximately 170 amino acids, approximately 35 amino acids~approximately 165 amino acids, approximately 35 amino acids~approximately 160 amino acids, approximately 35 amino acids~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 125 amino acids, approximately 35 amino acids to approximately 120 amino acids, approximately 35 amino acids to approximately 115 amino acids, approximately 35 amino acids to approximately 110 amino acids, approximately 35 amino acids to approximately 105 amino acids, approximately 35 amino acids to approximately 100 amino acids, approximately 35 amino acids to approximately 95 amino acids, approximately 35 amino acids to approximately 90 amino acids, approximately 35 amino acids to approximately 85 amino acids, approximately 35 amino acids to approximately 80 amino acids 0 amino acids, approximately 35 to 75 amino acids, approximately 35 to 70 amino acids, approximately 35 to 65 amino acids, approximately 35 to 60 amino acids, approximately 35 to 55 amino acids, approximately 35 to 50 amino acids, approximately 35 to 45 amino acids, approximately 35 to 40 amino acids, approximately 40 to 1000 amino acids, approximately 40 to 950 amino acids, approximately 40 to 900 amino acids, approximately 40 to 850 amino acids, approximately 40 to 800 amino acids, approximately 40 to 750 amino acids, approximately 40 to 700 amino acids 0 amino acids, approximately 40 to 650 amino acids, approximately 40 to 600 amino acids, approximately 40 to 550 amino acids, approximately 40 to 500 amino acids, approximately 40 to 450 amino acids, approximately 40 to 400 amino acids, approximately 40 to 350 amino acids, approximately 40 to 300 amino acids, approximately 40 to 280 amino acids, approximately 40 to 260 amino acids, approximately 40 to 240 amino acids, approximately 40 to 220 amino acids, approximately 40 to 200 amino acids, approximately 40 to 195 amino acids, approximately 40 amino acids ~approximately 190 amino acids, approximately 40 amino acids~approximately 185 amino acids, approximately 40 amino acids~approximately 180 amino acids, approximately 40 amino acids~approximately 175 amino acids, approximately 40 amino acids~approximately 170 amino acids, approximately 40 amino acids~approximately 165 amino acids, approximately 40 amino acids~approximately 160 amino acids, approximately 40 amino acids~approximately 155 amino acids, approximately 40 amino acids~approximately 150 amino acids, approximately 40 amino acids~approximately 145 amino acids, approximately 40 amino acids~approximately 140 amino acids, approximately 40 amino acids~approximately 135 amino acids, approximately 40 amino acids~approximately 130 amino acids, approximately 40 amino acids~approximately 125 amino acids, approximately 40 amino acids~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 amino acids, approximately 40 amino acids to approximately 85 amino acids, approximately 40 amino acids to approximately 80 amino acids, approximately 40 amino acids to approximately 75 amino acids, approximately 40 amino acids to approximately 70 amino acids, approximately 40 amino acids to approximately 65 amino acids, approximately 40 amino acids to approximately 65 amino acids, approximately 40 amino acids to approximately 55 amino acids, approximately 40 amino acids to approximately 50 amino acids, approximately 40 amino acids to approximately 45 amino acids, approximately 45 Amino acids ~ approximately 1000 amino acids, approximately 45 amino acids ~ approximately 950 amino acids, approximately 45 amino acids ~ approximately 900 amino acids, approximately 45 amino acids ~ approximately 850 amino acids, approximately 45 amino acids ~ approximately 800 amino acids, approximately 45 amino acids ~ approximately 750 amino acids, approximately 45 amino acids ~ approximately 700 amino acids, approximately 45 amino acids ~ approximately 650 amino acids, approximately 45 amino acids ~ approximately 600 amino acids, approximately 45 amino acids ~ approximately 550 amino acids, approximately 45 amino acids ~ approximately 500 amino acids, approximately 45 amino acids ~ approximately 450 amino acids, approximately 45 amino acids ~ approximately 400 amino acids, approximately 45 amino acids ~ approximately 350 amino acids, approximately 45 amino acids ~ approximately 30 0 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 acids to approximately 195 amino acids, approximately 45 amino acids to approximately 190 amino acids, approximately 45 amino acids to approximately 185 amino acids, approximately 45 amino acids to approximately 180 amino acids, approximately 45 amino acids to approximately 175 amino acids, approximately 45 amino acids to approximately 170 amino acids, approximately 45 amino acids to approximately 165 amino acids, approximately 45 amino acids to approximately 160 amino acids, approximately 45 amino acids to approximately 155 amino acids, approximately 45 Amino acids ~ approximately 150 amino acids, approximately 45 amino acids ~ approximately 145 amino acids, approximately 45 amino acids ~ approximately 140 amino acids, approximately 45 amino acids ~ approximately 135 amino acids, approximately 45 amino acids ~ approximately 130 amino acids, approximately 45 amino acids ~ approximately 125 amino acids, approximately 45 amino acids ~ approximately 120 amino acids, approximately 45 amino acids ~ approximately 115 amino acids, approximately 45 amino acids ~ approximately 110 amino acids, approximately 45 amino acids ~ approximately 105 amino acids, approximately 45 amino acids ~ approximately 100 amino acids, approximately 45 amino acids ~ approximately 95 amino acids, approximately 45 amino acids ~ approximately 90 amino acids, approximately 45 amino acids ~ approximately 85 amino acids, approximately 45 amino acids ~ 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 50 amino acids to approximately 1000 amino acids, approximately 50 amino acids to approximately 950 amino acids, approximately 50 amino acids to approximately 900 amino acids, approximately 50 amino acids to approximately 850 amino acids, approximately 50 amino acids to approximately 800 amino acids, approximately 50 amino acids to approximately 750 amino acids, approximately 50 amino acids to approximately 700 amino acids, approximately 50 amino acids to approximately 650 amino acids, approximately 50 amino acids to approximately 600 amino acids 50 amino acids, approximately 50 to 550 amino acids, approximately 50 to 500 amino acids, approximately 50 to 450 amino acids, approximately 50 to 400 amino acids, approximately 50 to 350 amino acids, approximately 50 to 300 amino acids, approximately 50 to 280 amino acids, approximately 50 to 260 amino acids, approximately 50 to 240 amino acids, approximately 50 to 220 amino acids, approximately 50 to 200 amino acids, approximately 50 to 195 amino acids, approximately 50 to 190 amino acids, approximately 50 to 185 amino acids, approximately 50 amino acids ~180 amino acids, ~50 amino acids~175 amino acids, ~50 amino acids~170 amino acids, ~50 amino acids~165 amino acids, ~50 amino acids~160 amino acids, ~50 amino acids~155 amino acids, ~50 amino acids~150 amino acids, ~50 amino acids~145 amino acids, ~50 amino acids~140 amino acids, ~50 amino acids~135 amino acids, ~50 amino acids~130 amino acids, ~50 amino acids~125 amino acids, ~50 amino acids~120 amino acids, ~50 amino acids~115 amino acids, ~50 amino acids~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, 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, 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 22 0 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 to approximately 175 amino acids, approximately 55 amino acids to approximately 170 amino acids, approximately 55 amino acids to approximately 165 amino acids, approximately 55 amino acids to approximately 160 amino acids, approximately 55 amino acids to approximately 155 amino acids, approximately 55 amino acids to approximately 150 amino acids, approximately 55 amino acids to approximately 145 amino acids, approximately 55 amino acids to approximately 140 amino acids, approximately 55 amino acids to approximately 135 amino acids, approximately 55 amino acids 55 amino acids - approximately 130 amino acids, 55 amino acids - approximately 125 amino acids, 55 amino acids - approximately 120 amino acids, 55 amino acids - approximately 115 amino acids, 55 amino acids - approximately 110 amino acids, 55 amino acids - approximately 105 amino acids, 55 amino acids - approximately 100 amino acids, 55 amino acids - approximately 95 amino acids, 55 amino acids - approximately 90 amino acids, 55 amino acids - approximately 85 amino acids, 55 amino acids - approximately 80 amino acids, 55 amino acids - approximately 75 amino acids, 55 amino acids - approximately 70 amino acids, 55 amino acids - approximately 65 amino acids, 55 amino acids - approximately 60 amino acids, approximately 60 amino acids ~1000 amino acids, approximately 60 amino acids~approximately 950 amino acids, approximately 60 amino acids~approximately 900 amino acids, approximately 60 amino acids~approximately 850 amino acids, approximately 60 amino acids~approximately 800 amino acids, approximately 60 amino acids~approximately 750 amino acids, approximately 60 amino acids~approximately 700 amino acids, approximately 60 amino acids~approximately 650 amino acids, approximately 60 amino acids~approximately 600 amino acids, approximately 60 amino acids~approximately 550 amino acids, approximately 60 amino acids~approximately 500 amino acids, approximately 60 amino acids~approximately 450 amino acids, approximately 60 amino acids~approximately 400 amino acids, approximately 60 amino acids~approximately 350 amino acids, approximately 60 amino acids~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 195 amino acids, approximately 60 amino acids to approximately 190 amino acids, approximately 60 amino acids, Mino acids ~ approximately 185 amino acids, approximately 60 amino acids ~ approximately 180 amino acids, approximately 60 amino acids ~ approximately 17 5 amino acids, approximately 60 to 170 amino acids, approximately 60 to 165 amino acids, approximately 60 to 160 amino acids, approximately 60 to 155 amino acids, approximately 60 to 150 amino acids, approximately 60 to 145 amino acids, approximately 60 to 140 amino acids, approximately 60 to 135 amino acids, approximately 60 to 130 amino acids, approximately 60 to 125 amino acids, approximately 60 to 120 amino acids, approximately 60 to 115 amino acids, approximately 60 to 110 amino acids, approximately 60 to 105 amino acids, approximately 6 0 amino acids to approximately 100 amino acids, approximately 60 amino acids to approximately 95 amino acids, approximately 60 amino acids to approximately 90 amino acids, approximately 60 amino acids to approximately 85 amino acids, 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 60 amino acids to approximately 65 amino acids, approximately 65 amino acids to approximately 1000 amino acids, approximately 65 amino acids to approximately 950 amino acids, approximately 65 amino acids to approximately 900 amino acids, approximately 65 amino acids to approximately 850 amino acids, approximately 65 amino acids to approximately 800 amino acids, approximately 65 amino acids to approximately 750 amino acids, approximately 65 amino acids to approximately 700 amino acids, Approximately 65 amino acids to approximately 650 amino acids, approximately 65 amino acids to approximately 600 amino acids, approximately 65 amino acids to approximately 550 amino acids, approximately 65 amino acids to approximately 500 amino acids, approximately 65 amino acids to approximately 450 amino acids, approximately 65 amino acids to approximately 400 amino acids, approximately 65 amino acids to approximately 350 amino acids, approximately 65 amino acids to approximately 300 amino acids, approximately 65 amino acids to approximately 280 amino acids, approximately 65 amino acids to approximately 260 amino acids, approximately 65 amino acids to 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 to approximately 185 amino acids, approximately 65 to approximately 180 amino acids, approximately 65 to approximately 175 amino acids, approximately 65 to approximately 170 amino acids, approximately 65 to approximately 165 amino acids, approximately 65 to approximately 160 amino acids, approximately 65 to approximately 155 amino acids, approximately 65 to approximately 150 amino acids, approximately 65 to approximately 145 amino acids, approximately 65 to approximately 140 amino acids, approximately 65 to approximately 135 amino acids, approximately 65 to approximately 130 amino acids, approximately 65 to approximately 125 amino acids, approximately 65 to approximately 120 amino acids,Approximately 65 amino acids to approximately 115 amino acids, approximately 65 amino acids to approximately 110 amino acids, approximately 65 amino acids to approximately 105 amino acids, approximately 65 amino acids to approximately 100 amino acids, approximately 65 amino acids to approximately 95 amino acids, approximately 65 amino acids to approximately 90 amino acids, approximately 65 amino acids to approximately 85 amino acids, approximately 65 amino acids to approximately 80 amino acids, approximately 65 amino acids to approximately 75 amino acids, approximately 65 amino acids to approximately 70 amino acids, approximately 70 amino acids to approximately 1000 amino acids, approximately 70 amino acids to approximately 950 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 Acid, 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 to approximately 260 amino acids, approximately 70 amino acids to approximately 240 amino acids, approximately 70 amino acids to approximately 220 amino acids, approximately 70 amino acids to approximately 200 amino acids, approximately 70 to 195 amino acids, approximately 70 to 190 amino acids, approximately 70 to 185 amino acids, approximately 70 to 180 amino acids, approximately 70 to 175 amino acids, approximately 70 to 170 amino acids, approximately 70 to 165 amino acids, approximately 70 to 160 amino acids, approximately 70 to 155 amino acids, approximately 70 to 150 amino acids, approximately 70 to 145 amino acids, approximately 70 to 140 amino acids, approximately 70 to 135 amino acids, approximately 70 to 130 amino acids, approximately 7 0 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, 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, approximately 75 amino acids to approximately 260 amino acids, approximately 75 amino acids to approximately 240 amino acids, approximately 75 amino acids to approximately 220 Amino acids, approximately 75 amino acids to approximately 200 amino acids, approximately 75 amino acids to approximately 195 amino acids, approximately 75 amino acids to approximately 190 amino acids, approximately 75 amino acids to approximately 185 amino acids, approximately 75 amino acids to approximately 180 amino acids, approximately 75 amino acids to approximately 175 amino acids, approximately 75 amino acids to approximately 170 amino acids, approximately 75 amino acids to approximately 165 amino acids, approximately 75 amino acids to approximately 160 amino acids, approximately 75 amino acids to approximately 155 amino acids, approximately 75 amino acids to approximately 150 amino acids, approximately 75 amino acids to approximately 145 amino acids, approximately 75 amino acids to approximately 140 amino acids, approximately 75 amino acids to approximately 135 amino acids, approximately 75 amino acids ~Approximately 130 amino acids, approximately 75 amino acids~Approximately 125 amino acids, approximately 75 amino acids~Approximately 120 amino acids, approximately 75 amino acids~Approximately 115 amino acids, approximately 75 amino acids~Approximately 110 amino acids, approximately 75 amino acids~Approximately 105 amino acids, approximately 75 amino acids~Approximately 100 amino acids, approximately 75 amino acids~Approximately 95 amino acids, approximately 75 amino acids~Approximately 90 amino acids, approximately 75 amino acids~Approximately 85 amino acids, approximately 75 amino acids~Approximately 80 amino acids, approximately 80 amino acids~Approximately 1000 amino acids, approximately 80 amino acids~Approximately 950 amino acids, approximately 80 amino acids~Approximately 900 amino acids, approximately 80 amino acids~Approximately 850 amino acids, approximately 80 amino acids 0 amino acids ~ approximately 800 amino acids, approximately 80 amino acids ~ approximately 750 amino acids, approximately 80 amino acids ~ approximately 700 amino acids, approximately 80 amino acids ~ approximately 650 amino acids, approximately 80 amino acids ~ approximately 600 amino acids, approximately 80 amino acids ~ approximately 550 amino acids, approximately 80 amino acids ~ approximately 500 amino acids, approximately 80 amino acids ~ approximately 450 amino acids, approximately 80 amino acids ~ approximately 400 amino acids, approximately 80 amino acids ~ approximately 350 amino acids, approximately 80 amino acids ~ approximately 300 amino acids, approximately 80 amino acids ~ approximately 280 amino acids, approximately 80 amino acids ~ approximately 260 amino acids, approximately 80 amino acids ~ approximately 240 amino acids, approximately 80 amino acids ~ approximately 220 amino acids,Approximately 80 amino acids to approximately 200 amino acids, approximately 80 amino acids to approximately 195 amino acids, approximately 80 amino acids to approximately 190 amino acids, approximately 80 amino acids to approximately 185 amino acids, approximately 80 amino acids to approximately 180 amino acids, approximately 80 amino acids to approximately 175 amino acids, approximately 80 amino acids to approximately 170 amino acids, approximately 80 amino acids to approximately 165 amino acids, approximately 80 amino acids to approximately 160 amino acids, approximately 80 amino acids to approximately 155 amino acids, approximately 80 amino acids to approximately 150 amino acids, approximately 80 amino acids to approximately 145 amino acids, approximately 80 amino acids to approximately 140 amino acids, approximately 80 amino acids to approximately 135 amino acids, approximately 80 amino acids to approximately 130 Amino acids, approximately 80 amino acids to approximately 125 amino acids, approximately 80 amino acids to approximately 120 amino acids, approximately 80 amino acids to approximately 115 amino acids, approximately 80 amino acids to approximately 110 amino acids, approximately 80 amino acids to approximately 105 amino acids, approximately 80 amino acids to approximately 100 amino acids, approximately 80 amino acids to approximately 95 amino acids, approximately 80 amino acids to approximately 90 amino acids, approximately 80 amino acids to approximately 85 amino acids, approximately 85 amino acids to approximately 1000 amino acids, approximately 85 amino acids to approximately 950 amino acids, approximately 85 amino acids to approximately 900 amino acids, approximately 85 amino acids to approximately 850 amino acids, approximately 85 amino acids to approximately 800 amino acids, approximately 85 amino acids to approximately 750 amino acids, approximately 85 amino acids to approximately 700 amino acids, approximately 85 amino acids to approximately 650 amino acids, approximately 85 amino acids to approximately 600 amino acids, approximately 85 amino acids to approximately 550 amino acids, approximately 85 amino acids to approximately 500 amino acids, approximately 85 amino acids to approximately 450 amino acids, approximately 85 amino acids to approximately 400 amino acids, approximately 85 amino acids to approximately 350 amino acids, approximately 85 amino acids to approximately 300 amino acids, approximately 85 amino acids to approximately 280 amino acids, approximately 85 amino acids to approximately 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 Mino acids ~ approximately 195 amino acids, approximately 85 amino acids ~ approximately 190 amino acids, approximately 85 amino acids ~ approximately 185 amino acids, approximately 85 amino acids ~ approximately 180 amino acids, approximately 85 amino acids ~ approximately 175 amino acids, approximately 85 amino acids ~ approximately 170 amino acids, approximately 85 amino acids ~ approximately 165 amino acids, approximately 85 amino acids ~ approximately 160 amino acids, approximately 85 amino acids ~ approximately 155 amino acids, approximately 85 amino acids ~ approximately 150 amino acids, approximately 85 amino acids ~ approximately 145 amino acids, approximately 85 amino acids ~ approximately 140 amino acids, approximately 85 amino acids ~ approximately 135 amino acids, approximately 85 amino acids ~ approximately 130 amino acids, approximately 85 amino acids ~ 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 to approximately 110 amino acids, approximately 90 to approximately 105 amino acids, approximately 90 to approximately 100 amino acids, approximately 90 to approximately 95 amino acids, approximately 95 to approximately 1000 amino acids, approximately 95 to approximately 950 amino acids, approximately 95 to approximately 900 amino acids, approximately 95 to approximately 850 amino acids, approximately 95 to approximately 800 amino acids, approximately 95 to approximately 750 amino acids, approximately 95 to approximately 700 amino acids, approximately 95 to approximately 650 amino acids, approximately 95 to approximately 600 amino acids, approximately 95 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, Amino acids, approximately 95 amino acids to approximately 240 amino acids, approximately 95 amino acids to approximately 220 amino acids, approximately 9 5 amino acids to approximately 200 amino acids, approximately 95 amino acids to approximately 195 amino acids, approximately 95 amino acids to approximately 190 amino acids, approximately 95 amino acids to approximately 185 amino acids, approximately 95 amino acids to approximately 180 amino acids, approximately 95 amino acids to approximately 175 amino acids, approximately 95 amino acids to approximately 170 amino acids, approximately 95 amino acids to approximately 165 amino acids, approximately 95 amino acids to approximately 160 amino acids, approximately 95 amino acids to approximately 155 amino acids, approximately 95 amino acids to approximately 150 amino acids, approximately 95 amino acids to approximately 145 amino acids, approximately 95 amino acids to approximately 140 amino acids, approximately 95 amino acids to approximately 135 amino acids, approximately 95 amino acids to approximately 1 30 amino acids, approximately 95 to 125 amino acids, approximately 95 to 120 amino acids, approximately 95 to 115 amino acids, approximately 95 to 110 amino acids, approximately 95 to 105 amino acids, approximately 95 to 100 amino acids, approximately 100 to 1000 amino acids, approximately 100 to 950 amino acids, approximately 100 to 900 amino acids, approximately 100 to 850 amino acids, approximately 100 to 800 amino acids, approximately 100 to 750 amino acids, approximately 100 to 700 amino acids, approximately 100 to 6 50 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 Mino acids ~ approximately 190 amino acids, approximately 100 amino acids ~ approximately 185 amino acids, approximately 100 amino acids ~ approximately 180 amino acids, approximately 100 amino acids ~ approximately 175 amino acids, approximately 100 amino acids ~ approximately 170 amino acids, approximately 100 amino acids ~ approximately 165 amino acids, approximately 100 amino acids ~ approximately 160 amino acids, approximately 100 amino acids ~ approximately 155 amino acids, approximately 100 amino acids ~ approximately 150 amino acids, approximately 100 amino acids ~ approximately 145 amino acids, approximately 100 amino acids ~ approximately 140 amino acids, approximately 100 amino acids ~ approximately 135 amino acids, approximately 100 amino acids ~ approximately 130 amino acids, approximately 100 amino acids ~ approximately 125 amino acids,Approximately 100-120 amino acids, approximately 100-115 amino acids, approximately 100-110 amino acids, approximately 100-105 amino acids, approximately 105-1000 amino acids, approximately 105-950 amino acids, approximately 105-900 amino acids, approximately 105-850 amino acids, approximately 105-800 amino acids, approximately 105-750 amino acids, approximately 105-700 amino acids, approximately 105-650 amino acids, approximately 105-600 amino acids, approximately 105-550 amino acids Amino acids, approximately 105 amino acids to approximately 500 amino acids, approximately 105 amino acids to approximately 450 amino acids, approximately 105 amino acids to approximately 400 amino acids, approximately 105 amino acids to approximately 350 amino acids, approximately 105 amino acids to approximately 300 amino acids, approximately 105 amino acids to approximately 280 amino acids, approximately 105 amino acids to approximately 260 amino acids, approximately 105 amino acids to approximately 240 amino acids, approximately 105 amino acids to approximately 220 amino acids, approximately 105 amino acids to approximately 200 amino acids, approximately 105 amino acids to approximately 195 amino acids, approximately 105 amino acids to approximately 190 amino acids, approximately 105 amino acids to approximately 185 amino acids, approximately 105 amino acids to approximately 195 amino acids, approximately 105 amino acids to approximately 185 amino acids, approximately 105 amino acids to Approximately 180 amino acids, approximately 105 to approximately 175 amino acids, approximately 105 to approximately 170 amino acids, approximately 105 to approximately 165 amino acids, approximately 105 to approximately 160 amino acids, approximately 105 to approximately 155 amino acids, approximately 105 to approximately 150 amino acids, approximately 105 to approximately 145 amino acids, approximately 105 to approximately 140 amino acids, approximately 105 to approximately 135 amino acids, approximately 105 to approximately 130 amino acids, approximately 105 to approximately 125 amino acids, approximately 105 to approximately 120 amino acids, approximately 105 to approximately 115 amino acids, approximately 105 amino acids Mino acids ~ approximately 110 amino acids, approximately 110 amino acids ~ approximately 1000 amino acids, approximately 110 amino acids ~ approximately 950 amino acids, approximately 110 amino acids ~ approximately 900 amino acids, approximately 110 amino acids ~ approximately 850 amino acids, approximately 110 amino acids ~ approximately 800 amino acids, approximately 110 amino acids ~ approximately 750 amino acids, approximately 110 amino acids ~ approximately 700 amino acids, approximately 110 amino acids ~ approximately 650 amino acids, approximately 110 amino acids ~ approximately 600 amino acids, approximately 110 amino acids ~ approximately 550 amino acids, approximately 110 amino acids ~ approximately 500 amino acids, approximately 110 amino acids ~ approximately 450 amino acids, approximately 110 amino acids ~ approximately 400 amino acids,Approximately 110 amino acids to approximately 350 amino acids, approximately 110 amino acids to approximately 300 amino acids, approximately 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 approximately 150 amino acids, approximately 110 amino acids to approximately 145 amino acids, approximately 110 amino acids to approximately 140 amino acids, approximately 110 amino acids to approximately 135 amino acids, approximately 110 amino acids to approximately 130 amino acids, approximately 110 amino acids to approximately 125 amino acids, approximately 110 amino acids to approximately 120 amino acids, approximately 110 amino acids to approximately 115 amino acids, approximately 115 amino acids to approximately 1000 amino acids, approximately 115 amino acids to approximately 950 amino acids, approximately 115 amino acids to approximately 900 amino acids, approximately 115 amino acids ~Approximately 850 amino acids, approximately 115 amino acids~Approximately 800 amino acids, approximately 115 amino acids~Approximately 750 amino acids, approximately 115 amino acids~Approximately 700 amino acids, approximately 115 amino acids~Approximately 650 amino acids, approximately 115 amino acids~Approximately 600 amino acids, approximately 115 amino acids~Approximately 550 amino acids, approximately 115 amino acids~Approximately 500 amino acids, approximately 115 amino acids~Approximately 450 amino acids, approximately 115 amino acids~Approximately 400 amino acids, approximately 115 amino acids~Approximately 350 amino acids, approximately 115 amino acids~Approximately 300 amino acids, approximately 115 amino acids~Approximately 280 amino acids, approximately 115 amino acids~Approximately 260 amino acids, approximately 115 Amino acids ~ approximately 240 amino acids, approximately 115 amino acids ~ approximately 220 amino acids, approximately 115 amino acids ~ approximately 200 amino acids, approximately 115 amino acids ~ approximately 195 amino acids, approximately 115 amino acids ~ approximately 190 amino acids, approximately 115 amino acids ~ approximately 185 amino acids, approximately 115 amino acids ~ approximately 180 amino acids, approximately 115 amino acids ~ approximately 175 amino acids, approximately 115 amino acids ~ approximately 170 amino acids, approximately 115 amino acids ~ approximately 165 amino acids, approximately 115 amino acids ~ approximately 160 amino acids, approximately 115 amino acids ~ approximately 155 amino acids, approximately 115 amino acids ~ approximately 150 amino acids, approximately 115 amino acids ~ approximately 145 amino acids,Approximately 115 amino acids to approximately 140 amino acids, approximately 115 amino acids to approximately 135 amino acids, approximately 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 amino acids to approximately 450 amino acids, approximately 120 amino acids to approximately 400 amino acids, approximately 120 amino acids to approximately 350 amino acids, approximately 120 amino acids to approximately 300 amino acids, approximately 120 amino acids to approximately 280 amino acids, approximately 120 amino acids to approximately 260 amino acids, approximately 120 amino acids to approximately 240 amino acids, approximately 120 amino acids to approximately 220 amino acids, approximately 120 amino acids to approximately 200 amino acids, approximately 120 amino acids to approximately 195 amino acids, approximately 120 amino acids to approximately 190 amino acids, approximately 120 amino acids to approximately 120 amino acids Approximately 185 amino acids, approximately 120 to 180 amino acids, approximately 120 to 175 amino acids, approximately 120 to 170 amino acids, approximately 120 to 165 amino acids, approximately 120 to 160 amino acids, approximately 120 to 155 amino acids, approximately 120 to 150 amino acids, approximately 120 to 145 amino acids, approximately 120 to 140 amino acids, approximately 120 to 135 amino acids, approximately 120 to 130 amino acids, approximately 120 to 125 amino acids, approximately 125 to 1000 amino acids, approximately 125 Amino acids ~ approximately 950 amino acids, approximately 125 amino acids ~ approximately 900 amino acids, approximately 125 amino acids ~ approximately 850 amino acids, approximately 125 amino acids ~ approximately 800 amino acids, approximately 125 amino acids ~ approximately 750 amino acids, approximately 125 amino acids ~ approximately 700 amino acids, approximately 125 amino acids ~ approximately 650 amino acids, approximately 125 amino acids ~ approximately 600 amino acids, approximately 125 amino acids ~ approximately 550 amino acids, approximately 125 amino acids ~ approximately 500 amino acids, approximately 125 amino acids ~ approximately 450 amino acids, approximately 125 amino acids ~ approximately 400 amino acids, approximately 125 amino acids ~ approximately 350 amino acids, approximately 125 amino acids ~ approximately 300 amino acids,Approximately 125 amino acids to approximately 280 amino acids, approximately 125 amino acids to approximately 260 amino acids, approximately 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 Mino acids, 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 1000 amino acids, approximately 130 amino acids to approximately 950 amino acids, approximately 130 amino acids to approximately 900 amino acids, approximately 130 amino acids to approximately 850 amino acids, approximately 130 amino acids to approximately 800 amino acids, approximately 130 amino acids to approximately 750 amino acids, approximately 130 amino acids to approximately 700 amino acids, approximately 130 amino acids to approximately 650 amino acids, approximately 130 amino acids to approximately 130 amino acids Approximately 600 amino acids, approximately 130 amino acids to approximately 550 amino acids, approximately 130 amino acids to approximately 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 Mino acids ~ approximately 185 amino acids, approximately 130 amino acids ~ approximately 180 amino acids, approximately 130 amino acids ~ approximately 175 amino acids, approximately 130 amino acids ~ approximately 170 amino acids, approximately 130 amino acids ~ approximately 165 amino acids, approximately 130 amino acids ~ approximately 160 amino acids, approximately 130 amino acids ~ approximately 155 amino acids, approximately 130 amino acids ~ approximately 150 amino acids, approximately 130 amino acids ~ approximately 145 amino acids, approximately 130 amino acids ~ approximately 140 amino acids, approximately 130 amino acids ~ approximately 135 amino acids, approximately 135 amino acids ~ approximately 1000 amino acids, approximately 135 amino acids ~ approximately 950 amino acids, approximately 135 amino acids ~ 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 0.0 amino acids ~ approximately 400 amino acids, approximately 135 amino acids ~ approximately 350 amino acids, approximately 135 amino acids ~ approximately 300 amino acids, approximately 135 amino acids ~ approximately 280 amino acids, approximately 135 amino acids ~ approximately 260 amino acids, approximately 135 amino acids ~ approximately 240 amino acids, approximately 135 amino acids ~ approximately 220 amino acids, approximately 135 amino acids ~ approximately 200 amino acids, approximately 135 amino acids ~ approximately 195 amino acids, approximately 135 amino acids ~ approximately 190 amino acids, approximately 135 amino acids ~ approximately 185 amino acids, approximately 135 amino acids ~ approximately 180 amino acids, approximately 135 amino acids ~ approximately 175 amino acids, approximately 135 amino acids ~ 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 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 65 0 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, approximately 140 amino acids to approximately 280 amino acids, approximately 140 amino acids to approximately 260 amino acids, approximately 140 amino acids to approximately 240 amino acids, approximately 140 amino acids to approximately 220 amino acids, approximately 140 amino acids to approximately 200 amino acids, approximately 140 amino acids to approximately 195 amino acids, approximately 140 amino acids 0 amino acids ~ approximately 190 amino acids, approximately 140 amino acids ~ approximately 185 amino acids, approximately 140 amino acids ~ approximately 180 amino acids, approximately 140 amino acids ~ approximately 175 amino acids, approximately 140 amino acids ~ approximately 170 amino acids, approximately 140 amino acids ~ approximately 165 amino acids, approximately 140 amino acids ~ approximately 160 amino acids, approximately 140 amino acids ~ approximately 155 amino acids, approximately 140 amino acids ~ approximately 150 amino acids, approximately 140 amino acids ~ approximately 145 amino acids, approximately 145 amino acids ~ approximately 1000 amino acids, approximately 145 amino acids ~ approximately 950 amino acids, approximately 145 amino acids ~ approximately 900 amino acids, approximately 145 amino acids ~ 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 amino acids to approximately 450 amino acids, approximately 145 amino acids to approximately 400 amino acids, approximately 145 amino acids to approximately 350 amino acids, approximately 145 amino acids to approximately 300 amino acids, approximately 145 amino acids to approximately 280 amino acids, approximately 145 amino acids to approximately 260 amino acids, approximately 145 amino acids to approximately 240 Amino acids, approximately 145 amino acids to approximately 220 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, 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 ~950 amino acids, approximately 150 amino acids~approximately 900 amino acids, approximately 150 amino acids~approximately 850 amino acids, approximately 150 amino acids~approximately 800 amino acids, approximately 150 amino acids~approximately 750 amino acids, approximately 150 amino acids~approximately 700 amino acids, approximately 150 amino acids~approximately 650 amino acids, approximately 150 amino acids~approximately 600 amino acids, approximately 150 amino acids~approximately 550 amino acids, approximately 150 amino acids~approximately 500 amino acids, approximately 150 amino acids~approximately 450 amino acids, approximately 150 amino acids~approximately 400 amino acids, approximately 150 amino acids~approximately 350 amino acids, approximately 150 amino acids~approximately 300 amino acids, approximately 150 Amino acids ~ approximately 280 amino acids, approximately 150 amino acids ~ approximately 260 amino acids, approximately 150 amino acids ~ approximately 240 amino acids, approximately 150 amino acids ~ approximately 220 amino acids, approximately 150 amino acids ~ approximately 200 amino acids, approximately 150 amino acids ~ approximately 195 amino acids, approximately 150 amino acids ~ approximately 190 amino acids, approximately 150 amino acids ~ approximately 185 amino acids, approximately 150 amino acids ~ approximately 180 amino acids, approximately 150 amino acids ~ approximately 175 amino acids, approximately 150 amino acids ~ approximately 170 amino acids, approximately 150 amino acids ~ approximately 165 amino acids, approximately 150 amino acids ~ approximately 160 amino acids, approximately 150 amino acids ~ 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 amino acids to approximately 650 amino acids, approximately 155 amino acids to approximately 600 amino acids, approximately 155 amino acids to approximately 550 amino acids, approximately 155 amino acids to approximately 500 amino acids, approximately 155 amino acids to approximately 450 amino acids, approximately 155 amino acids to approximately 400 amino acids, approximately 155 amino acids to approximately 350 Amino acids, approximately 155 amino acids to approximately 300 amino acids, approximately 155 amino acids to approximately 280 amino acids, approximately 155 amino acids to approximately 260 amino acids, approximately 155 amino acids to approximately 240 amino acids, approximately 155 amino acids to approximately 220 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 amino acids to approximately 165 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, approximately 160 amino acids to approximately 650 amino acids, approximately 160 amino acids to approximately 600 amino acids, approximately 160 amino acids to approximately 550 amino acids, approximately 160 amino acids to approximately 500 amino acids, approximately 160 amino acids to approximately 450 amino acids, approximately 160 amino acids to approximately 400 amino acids, approximately 160 Amino acids ~ approximately 350 amino acids, approximately 160 amino acids ~ approximately 300 amino acids, approximately 160 amino acids ~ approximately 280 amino acids, approximately 160 amino acids ~ approximately 260 amino acids, approximately 160 amino acids ~ approximately 240 amino acids, approximately 160 amino acids ~ approximately 220 amino acids, approximately 160 amino acids ~ approximately 200 amino acids, approximately 160 amino acids ~ approximately 195 amino acids, approximately 160 amino acids ~ approximately 190 amino acids, approximately 160 amino acids ~ approximately 185 amino acids, approximately 160 amino acids ~ approximately 180 amino acids, approximately 160 amino acids ~ approximately 175 amino acids, approximately 160 amino acids ~ approximately 170 amino acids, approximately 160 amino acids ~ 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 amino acids to approximately 650 amino acids, approximately 165 amino acids to approximately 600 amino acids, approximately 165 amino acids to approximately 550 amino acids, approximately 165 amino acids to approximately 500 amino acids, approximately 165 amino acids to approximately 450 amino acids, approximately 165 amino acids to approximately 400 amino acids, approximately 165 amino acids to approximately 350 Amino acids, approximately 165 amino acids to approximately 300 amino acids, approximately 165 amino acids to approximately 280 amino acids, approximately 165 amino acids to approximately 260 amino acids, approximately 165 amino acids to approximately 240 amino acids, approximately 165 amino acids to approximately 220 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 1000 amino acids, approximately 170 amino acids ~950 amino acids, approximately 170 amino acids~approximately 900 amino acids, approximately 170 amino acids~approximately 850 amino acids, approximately 170 amino acids~approximately 800 amino acids, approximately 170 amino acids~approximately 750 amino acids, approximately 170 amino acids~approximately 700 amino acids, approximately 170 amino acids~approximately 650 amino acids, approximately 170 amino acids~approximately 600 amino acids, approximately 170 amino acids~approximately 550 amino acids, approximately 170 amino acids~approximately 500 amino acids, approximately 170 amino acids~approximately 450 amino acids, approximately 170 amino acids~approximately 400 amino acids, approximately 170 amino acids~approximately 350 amino acids, approximately 170 amino acids~approximately 300 amino acids, approximately 170 Amino acids ~ approximately 280 amino acids, approximately 170 amino acids ~ approximately 260 amino acids, approximately 170 amino acids ~ approximately 240 amino acids, approximately 170 amino acids ~ approximately 220 amino acids, approximately 170 amino acids ~ approximately 200 amino acids, approximately 170 amino acids ~ approximately 195 amino acids, approximately 170 amino acids ~ approximately 190 amino acids, approximately 170 amino acids ~ approximately 185 amino acids, approximately 170 amino acids ~ approximately 180 amino acids, approximately 170 amino acids ~ approximately 175 amino acids, approximately 175 amino acids ~ approximately 1000 amino acids, approximately 175 amino acids ~ approximately 950 amino acids, approximately 175 amino acids ~ approximately 900 amino acids, approximately 175 amino acids ~ 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 amino acids to approximately 450 amino acids, approximately 175 amino acids to approximately 400 amino acids, approximately 175 amino acids to approximately 350 amino acids, approximately 175 amino acids to approximately 300 amino acids, approximately 175 amino acids to approximately 280 amino acids, approximately 175 amino acids to approximately 260 amino acids, approximately 175 amino acids to approximately 240 amino acids Mino acids, approximately 175 amino acids to approximately 220 amino acids, approximately 175 amino acids to approximately 200 amino acids, approximately 175 amino acids to approximately 195 amino acids, approximately 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 1000 amino acids, 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 180 amino acids 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, 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 Mino acids ~ approximately 190 amino acids, approximately 180 amino acids ~ approximately 185 amino acids, approximately 185 amino acids ~ approximately 1000 amino acids, approximately 185 amino acids ~ approximately 950 amino acids, approximately 185 amino acids ~ approximately 900 amino acids, approximately 185 amino acids ~ approximately 850 amino acids, approximately 185 amino acids ~ approximately 800 amino acids, approximately 185 amino acids ~ approximately 750 amino acids, approximately 185 amino acids ~ approximately 700 amino acids, approximately 185 amino acids ~ approximately 650 amino acids, approximately 185 amino acids ~ approximately 600 amino acids, approximately 185 amino acids ~ approximately 550 amino acids, approximately 185 amino acids ~ approximately 500 amino acids, approximately 185 amino acids ~ 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 approximately 200 amino acids, approximately 185 amino acids to approximately 195 amino acids, approximately 185 amino acids Mino acids ~ approximately 190 amino acids, approximately 190 amino acids ~ approximately 1000 amino acids, approximately 190 amino acids ~ approximately 950 amino acids, approximately 190 amino acids ~ approximately 900 amino acids, approximately 190 amino acids ~ approximately 850 amino acids, approximately 190 amino acids ~ approximately 800 amino acids, approximately 190 amino acids ~ approximately 750 amino acids, approximately 190 amino acids ~ approximately 700 amino acids, approximately 190 amino acids ~ approximately 650 amino acids, approximately 190 amino acids ~ approximately 600 amino acids, approximately 190 amino acids ~ approximately 550 amino acids, approximately 190 amino acids ~ approximately 500 amino acids, approximately 190 amino acids ~ approximately 450 amino acids, approximately 190 amino acids ~ 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, approximately 195 amino acids to approximately 800 amino acids, approximately 195 amino acids to approximately 7 50 amino acids, approximately 195 amino acids to approximately 700 amino acids, approximately 195 amino acids to approximately 650 amino acids, approximately 195 amino acids to approximately 600 amino acids, approximately 195 amino acids to approximately 550 amino acids, approximately 195 amino acids to approximately 500 amino acids, approximately 195 amino acids to approximately 450 amino acids, approximately 195 amino acids to approximately 400 amino acids, approximately 195 amino acids to approximately 350 amino acids, approximately 195 amino acids to approximately 300 amino acids, approximately 195 amino acids to approximately 280 amino acids, approximately 195 amino acids to approximately 260 amino acids, approximately 195 amino acids to approximately 240 amino acids, approximately 195 amino acids to approximately 220 amino acids, approximately 195 amino acids 200 amino acids, 200 amino acids to 1000 amino acids, 200 amino acids to 950 amino acids, 200 amino acids to 900 amino acids, 200 amino acids to 850 amino acids, 200 amino acids to 800 amino acids, 200 amino acids to 750 amino acids, 200 amino acids to 700 amino acids, 200 amino acids to 650 amino acids, 200 amino acids to 600 amino acids, 200 amino acids to 550 amino acids, 200 amino acids to 500 amino acids, 200 amino acids to 450 amino acids, 200 amino acids to 400 amino acidsApproximately 200 to 350 amino acids, approximately 200 to 300 amino acids, approximately 200 to 280 amino acids, approximately 200 to 260 amino acids, approximately 200 to 240 amino acids, approximately 200 to 220 amino acids, approximately 220 to 1000 amino acids, approximately 220 to 950 amino acids, approximately 220 to 900 amino acids, approximately 220 to 850 amino acids, approximately 220 to 800 amino acids, approximately 220 to 750 amino acids, approximately 220 to 700 amino acids, approximately 220 to 650 amino acids 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, approximately 240 amino acids to approximately 900 amino acids, approximately 240 amino acids ~approximately 850 amino acids, approximately 240 amino acids~approximately 800 amino acids, approximately 240 amino acids~approximately 750 amino acids, approximately 240 amino acids~approximately 700 amino acids, approximately 240 amino acids~approximately 650 amino acids, approximately 240 amino acids~approximately 600 amino acids, approximately 240 amino acids~approximately 550 amino acids, approximately 240 amino acids~approximately 500 amino acids, approximately 240 amino acids~approximately 450 amino acids, approximately 240 amino acids~approximately 400 amino acids, approximately 240 amino acids~approximately 350 amino acids, approximately 240 amino acids~approximately 300 amino acids, approximately 240 amino acids~approximately 280 amino acids, approximately 240 amino acids~approximately 260 amino acids, approximately 260 Amino acids ~ approximately 1000 amino acids, approximately 260 amino acids ~ approximately 950 amino acids, approximately 260 amino acids ~ approximately 900 amino acids, approximately 260 amino acids ~ approximately 850 amino acids, approximately 260 amino acids ~ approximately 800 amino acids, approximately 260 amino acids ~ approximately 750 amino acids, approximately 260 amino acids ~ approximately 700 amino acids, approximately 260 amino acids ~ approximately 650 amino acids, approximately 260 amino acids ~ approximately 600 amino acids, approximately 260 amino acids ~ approximately 550 amino acids, approximately 260 amino acids ~ approximately 500 amino acids, approximately 260 amino acids ~ approximately 450 amino acids, approximately 260 amino acids ~ approximately 400 amino acids, approximately 260 amino acids ~ approximately 350 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 1000 amino acids, approximately 280 amino acids to approximately 950 amino acids, approximately 280 amino acids to approximately 900 amino acids, approximately 280 amino acids to approximately 850 amino acids, approximately 280 amino acids to approximately 800 amino acids, approximately 280 amino acids to approximately 750 amino acids, approximately 280 amino acids to approximately 700 amino acids, approximately 280 amino acids to approximately 650 amino acids, approximately 280 amino acids to approximately 600 amino acids, approximately 280 amino acids to approximately 550 amino acids, 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, approximately 300 amino acids to approximately 550 amino acids, approximately 300 amino acids to approximately 300 amino acids Approximately 500 amino acids, approximately 300 to approximately 450 amino acids, approximately 300 to approximately 400 amino acids, approximately 300 to approximately 350 amino acids, approximately 350 to approximately 1000 amino acids, approximately 350 to approximately 950 amino acids, approximately 350 to approximately 900 amino acids, approximately 350 to approximately 850 amino acids, approximately 350 to approximately 800 amino acids, approximately 350 to approximately 750 amino acids, approximately 350 to approximately 700 amino acids, approximately 350 to approximately 650 amino acids, approximately 350 to approximately 600 amino acids, approximately 350 to approximately 550 amino acids, approximately 350 Amino acids ~ approximately 500 amino acids, approximately 350 amino acids ~ approximately 450 amino acids, approximately 350 amino acids ~ approximately 400 amino acids, approximately 400 amino acids ~ approximately 1000 amino acids, approximately 400 amino acids ~ approximately 950 amino acids, approximately 400 amino acids ~ approximately 900 amino acids, approximately 400 amino acids ~ approximately 850 amino acids, approximately 400 amino acids ~ approximately 800 amino acids, approximately 400 amino acids ~ approximately 750 amino acids, approximately 400 amino acids ~ approximately 700 amino acids, approximately 400 amino acids ~ approximately 650 amino acids, approximately 400 amino acids ~ approximately 600 amino acids, approximately 400 amino acids ~ approximately 550 amino acids, approximately 400 amino acids ~ approximately 500 amino acids,Approximately 400-450 amino acids, approximately 450-1000 amino acids, approximately 450-950 amino acids, approximately 450-900 amino acids, approximately 450-850 amino acids, approximately 450-800 amino acids, approximately 450-750 amino acids, approximately 450-700 amino acids, approximately 450-650 amino acids, approximately 450-600 amino acids, approximately 450-550 amino acids, approximately 450-500 amino acids, approximately 500-1000 amino acids, approximately 500-950 amino acids 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 to approximately 700 amino acids, approximately 550 to approximately 650 amino acids, approximately 550 to approximately 600 amino acids, approximately 600 to approximately 1000 amino acids, approximately 600 to approximately 950 amino acids, approximately 600 to approximately 900 amino acids, approximately 600 to approximately 850 amino acids, approximately 600 to approximately 800 amino acids, approximately 600 to approximately 750 amino acids, approximately 600 to approximately 700 amino acids, approximately 600 to approximately 650 amino acids, approximately 650 to approximately 1000 amino acids, approximately 650 to approximately 950 amino acids, approximately 650 Amino acids ~ approximately 900 amino acids, approximately 650 amino acids ~ approximately 850 amino acids, approximately 650 amino acids ~ approximately 800 amino acids, approximately 650 amino acids ~ approximately 750 amino acids, approximately 650 amino acids ~ approximately 700 amino acids, approximately 700 amino acids ~ approximately 1000 amino acids, approximately 700 amino acids ~ approximately 950 amino acids, approximately 700 amino acids ~ approximately 900 amino acids, approximately 700 amino acids ~ approximately 850 amino acids, approximately 700 amino acids ~ approximately 800 amino acids, approximately 700 amino acids ~ approximately 750 amino acids, approximately 750 amino acids ~ approximately 1000 amino acids, approximately 750 amino acids ~ approximately 950 amino acids, approximately 750 amino acids ~ approximately 900 amino acids,It may have a total amino acid number of about 750 amino acids to about 850 amino acids, about 750 amino acids to about 800 amino acids, about 800 amino acids to about 1000 amino acids, about 800 amino acids to about 950 amino acids, about 800 amino acids to about 900 amino acids, about 800 amino acids to about 850 amino acids, about 850 amino acids to about 1000 amino acids, about 850 amino acids to about 950 amino acids, about 850 amino acids to about 900 amino acids, about 900 amino acids to about 1000 amino acids, about 900 amino acids to about 950 amino acids, or about 950 amino acids to about 1000 amino acids.,

[0099] Any of the target-binding domains described herein has a dissociation equilibrium constant (K -7 less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M, less than 1×10 -11 M, less than 1×10 -12 M, or less than 1×10 -13 M and can bind to its target with a dissociation equilibrium constant (K D ). In some embodiments, the antigen-binding protein constructs provided herein have a K -3 from 1×10 -5 M to about 1×10 -4 M, from about 1×10 -6 M to about 1×10 -5 M, from about 1×10 -7 M to about 1×10 -6 M, from about 1×10 -8 M to about 1×10 -7 M, from about 1×10 -9 M to about 1×10 -8 M, from about 1×10 -10 M to about 1×10 -9 M, or from about 1×10 -11 M to about 1×10 D M (including its upper and lower limits) and can bind to a specific antigen.,

[0100] Any of the target-binding domains described herein is approximately 1 pM to approximately 30 nM (for example, approximately 1 pM to approximately 25 nM, approximately 1 pM to approximately 20 nM, approximately 1 pM to approximately 15 nM, approximately 1 pM to approximately 10 nM, approximately 1 pM to approximately 5 nM, approximately 1 pM to approximately 2 nM, approximately 1 pM to approximately 1 nM, approximately 1 pM to approximately 950 pM, approximately 1 pM to approximately 900 pM, approximately 1 pM to approximately 850 pM, approximately 1 pM to approximately 800 pM, approximately 1 pM to approximately 750 pM, approximately 1 pM to approximately 700 pM, approximately 1 pM to approximately 650 pM, approximately 1 pM to approximately 600 pM, approximately 1 pM to approximately 550 pM, approximately 1 pM to approximately 500 pM, approximately 1 pM to approximately 450 pM, approximately 1 p M ~ about 400pM, about 1pM - about 350pM, about 1pM - about 300pM, about 1pM - about 250pM, about 1pM - about 200pM, about 1pM - Approximately 150pM, approximately 1pM to approximately 100pM, approximately 1pM to approximately 90pM, approximately 1pM to approximately 80pM, approximately 1pM to approximately 70pM, approximately 1pM to approximately 60pM , 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 5pM, about 1pM to about 4pM, about 1pM to about 3pM, about 1pM to about 2pM, about 2pM to about 30nM, about 2pM to about 25nM, about 2pM to about 2 0nM, about 2pM to about 15nM, about 2pM to about 10nM, about 2pM to about 5nM, about 2pM to about 2nM, about 2pM to about 1nM, about 2pM to about 950pM, about 2pM to about 900pM, about 2pM to about 850pM, about 2pM to about 800pM, about 2pM to about 750pM, about 2pM to about 70 0pM, about 2pM to about 650pM, about 2pM to about 600pM, about 2pM to about 550pM, about 2pM to about 500pM, about 2pM to about 450p M, about 2 pM to about 400 pM, about 2 pM to about 350 pM, about 2 pM to about 300 pM, about 2 pM to about 250 pM, about 2 pM to about 200 pM, about 2pM to about 150pM, about 2pM to about 100pM, about 2pM to about 90pM, about 2pM to about 80pM, about 2pM to about 70pM, about 2pM to about 60pM, about 2pM to about 50pM, about 2pM to about 40pM, about 2pM to about 30pM, about 2pM to about 20pM, about 2pM to about 10pM, about 2 pM to about 5 pM, about 2 pM to about 4 pM, about 2 pM to about 3 pM, about 5 pM to about 30 nM, about 5 pM to about 25 nM, about 5 pM to about 20 nM, about 5pM to about 15nM, about 5pM to about 10nM, about 5pM to about 5nM, about 5pM to about 2nM, about 5pM to about 1nM, about 5pM to about 950pM,Approximately 5pM to approximately 900pM, approximately 5pM to approximately 850pM, approximately 5pM to approximately 800pM, approximately 5pM to approximately 750pM, approximately 5pM to approximately 700pM, approximately 5pM to approximately 650pM, approximately 5pM to approximately 600pM, approximately 5pM to approximately 550pM, approximately 5pM to approximately 500pM, approximately 5pM to approximately 450pM, approximately 5pM~ Approximately 400pM, approximately 5pM to approximately 350pM, approximately 5pM to approximately 300pM, approximately 5pM to approximately 250pM, approximately 5pM to approximately 200pM, approximately 5pM to approximately 150pM, approximately 5pM to approximately 100pM, approximately 5pM to approximately 90pM, approximately 5pM to approximately 80pM, approximately 5pM to approximately 70pM, approximately 5pM to approximately 60pM, approximately 5 pM ~ approximately 50 pM, approximately 5 pM ~ approximately 40 pM, approximately 5 pM ~ approximately 30 pM, approximately 5 pM ~ approximately 20 pM, approximately 5 pM ~ approximately 10 pM, approximately 10 pM ~ approximately 30 nM, approximately 10 pM ~ approximately 25 nM, approximately 10 pM ~ approximately 20 nM, approximately 10 pM ~ approximately 15 nM, approximately 10 pM ~ approximately 10 nM, approximately 10 pM ~ approximately 5 nM, approximately 10pM to approximately 2nm, approximately 10pM to approximately 1nm, approximately 10pM to approximately 950pM, approximately 10pM to approximately 900pM, approximately 10pM to approximately 850pM, approximately 10pM to approximately 800pM, approximately 10pM to approximately 750pM, approximately 10pM to approximately 700pM, approximately 10pM to approximately 650pM, approximately 10pM to approximately 600pM Approximately 10 pM to approximately 550 pM, approximately 10 pM 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 1 0pM, approximately 10pM to approximately 90pM, approximately 10pM to approximately 80pM, approximately 10pM to approximately 70pM, approximately 10pM to approximately 60pM, approximately 10pM to approximately 50pM, approximately 10pM to approximately 40pM, approximately 10pM to approximately 30pM, approximately 10pM to approximately 20pM, approximately 15pM to approximately 30nm, approximately 15pM to approximately 25nm. Approximately 15 pM to approximately 20 nm, approximately 15 pM to approximately 15 nm, approximately 15 pM to approximately 10 nm, approximately 15 pM to approximately 5 nm, approximately 15 pM to approximately 2 nm, approximately 15 pM to approximately 1 nm, 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 p M~approx. 750pM, approx. 15pM~approx. 700pM, approx. 15pM~approx. 650pM, approx. 15pM~approx. 600pM, approx. 15pM~approx. 550pM, approx. 15pM~approx. 500pM, approx. 15pM~approx. 450pM, approx. 15pM~approx. 400pM, approx. 15pM~approx. 350pM, approx. 15pM~approx. 300pMApproximately 15pM to approximately 250pM, approximately 15pM to approximately 200pM, approximately 15pM to approximately 150pM, approximately 15pM to approximately 100pM, approximately 15pM to approximately 90pM, approximately 15pM to approximately 80pM, approximately 15pM to approximately 70pM, approximately 15pM to approximately 60pM, approximately 15pM to approximately 50pM, approximately 15pM to approximately 40pM. Approximately 15 pM to approximately 30 pM, approximately 15 pM to approximately 20 pM, approximately 20 pM to approximately 30 nmM, approximately 20 pM to approximately 25 nmM, approximately 20 pM to approximately 20 nmM, approximately 20 pM to approximately 15 nmM, approximately 20 pM to approximately 10 nmM, approximately 20 pM to approximately 5 nmM, approximately 20 pM to approximately 2 nmM, approximately 20 pM to approximately 1 nmM, approximately 20 pM to approximately 950pM, approximately 20pM 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 M, approximately 20pM to approximately 70pM, approximately 20pM to approximately 60pM, approximately 20pM to approximately 50pM, approximately 20pM to approximately 40pM, approximately 20pM to approximately 30pM, approximately 30pM to approximately 30nM, approximately 30pM to approximately 25nM, approximately 30pM to approximately 30nM, approximately 30pM to approximately 15nM, approximately 30pM to approximately 10nM, approximately 3 0pM to approximately 5nm, approximately 30pM to approximately 2nm, approximately 30pM to approximately 1nm, approximately 30pM to approximately 950pM, approximately 30pM to approximately 900pM, approximately 30pM to approximately 850pM, approximately 30pM to approximately 800pM, approximately 30pM to approximately 750pM, approximately 30pM to approximately 700pM, approximately 30pM to approximately 650pM, approximately 3 0pM~approx. 600pM, approx. 30pM~approx. 550pM, approx. 30pM~approx. 500pM, approx. 30pM~approx. 450pM, approx. 30pM~approx. 400pM, approx. 30pM~approx. 350pM, approx. 30pM~approx. 300pM, approx. 30pM~approx. 250pM, approx. 30pM~approx. 200pM, approx. 30pM~approx. 150pM 0pM, 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 40 pM to approximately 15 nm, 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 40pM to approximately 700pM, approximately 40pM to approximately 650pM, approximately 40pM to approximately 600pM, approximately 40pM to approximately 550pM, approximately 40pM to approximately 500pM, approximately 40pM to approximately 450pM, approximately 40pM to approximately 400pM, approximately 40pM to approximately 350pM, approximately 40pM to approximately 300pM, approximately 40pM to approximately 250pM M, approximately 40pM 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 50 pM to approximately 30 nm, approximately 50 pM to approximately 15 nm, approximately 50 pM to approximately 10 nm, approximately 50 pM to approximately 5 nm, approximately 50 pM to approximately 2 nm, approximately 50 pM to approximately 1 nm, approximately 50 pM to approximately 950 pM, approximately 50 pM to approximately 900 pM, approximately 50 pM to approximately 850 pM, approximately 50 pM to approximately 800 pM, approximately 50 p M~approx. 750pM, approx. 50pM~approx. 700pM, approx. 50pM~approx. 650pM, approx. 50pM~approx. 600pM, approx. 50pM~approx. 550pM, approx. 50pM~approx. 500pM, approx. 50pM~approx. 450pM, approx. 50pM~approx. 400pM, approx. 50pM~approx. 350pM, approx. 50pM~approx. 300pM 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 approximately 30 nm, approximately 60 pM to approximately 15 nm, approximately 60 pM to approximately 10 nm, approximately 60 pM to approximately 5 nm, approximately 60 pM to approximately 2 nm, approximately 60 pM to approximately 1 nm, approximately 60 pM to approximately 950 pM, approximately 60 pM to approximately 900 pM, approximately 60 pM to approximately 850 pM, approximately 60 pM to approximately 800 pM, approximately 60 p M~approx. 750pM, approx. 60pM~approx. 700pM, approx. 60pM~approx. 650pM, approx. 60pM~approx. 600pM, approx. 60pM~approx. 550pM, approx. 60pM~approx. 500pM, approx. 60pM~approx. 450pM, approx. 60pM~approx. 400pM, approx. 60pM~approx. 350pM, approx. 60pM~approx. 300pMApproximately 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 70pM ~ approximately 15nM, approximately 70pM ~ approximately 10nM, approximately 70pM ~ approximately 5nM, approximately 70pM ~ approximately 2nM, approximately 70pM ~ approximately 1nM, approximately 70pM ~ approximately 950pM, approximately 70pM ~ approximately 900pM, approximately 70pM ~ approximately 850pM, approximately 70pM ~ approximately 800pM, approximately 70pM ~ approximately 750pM, approximately 70pM M~approx. 700pM, 70pM~approx. 650pM, 70pM~approx. 600pM, 70pM~approx. 550pM, 70pM~approx. 500pM, 70pM~approx. 450pM, 70pM~approx. 400pM, 70pM~approx. 350pM, 70pM~approx. 300pM, 70pM~approx. 250pM 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 ~ approximately 5nm, approximately 80pM ~ approximately 2nm, approximately 80pM ~ approximately 1nm, approximately 80pM ~ approximately 950pM, approximately 80pM ~ approximately 900pM, approximately 80pM ~ approximately 850pM, approximately 80pM ~ approximately 800pM, approximately 80pM ~ approximately 750pM, approximately 80pM ~ approximately 700pM, approximately 80pM ~ approximately 650pM, approximately 8 0pM~approx. 600pM, approx. 80pM~approx. 550pM, approx. 80pM~approx. 500pM, approx. 80pM~approx. 450pM, approx. 80pM~approx. 400pM, approx. 80pM~approx. 350pM, approx. 80pM~approx. 300pM, approx. 80pM~approx. 250pM, approx. 80pM~approx. 200pM, approx. 80pM~approx. 150pM pM, approximately 80pM to approximately 100pM, approximately 80pM to approximately 90pM, approximately 90pM to approximately 30nM, approximately 90pM to approximately 25nM, approximately 90pM to approximately 30nM, approximately 90pM to approximately 15nM, approximately 90pM to approximately 10nM, approximately 90pM to approximately 5nM, approximately 90pM to approximately 2nM, approximately 90pM to approximately 1nM, approximately 90pM M~approx. 950pM, approx. 90pM~approx. 900pM, approx. 90pM~approx. 850pM, approx. 90pM~approx. 800pM, approx. 90pM~approx. 750pM, approx. 90pM~approx. 700pM, approx. 90pM~approx. 650pM, approx. 90pM~approx. 600pM, approx. 90pM~approx. 550pM, approx. 90pM~approx. 500pMApproximately 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 2... 5 nM, approximately 100pM to approximately 30nM, approximately 100pM to approximately 15nM, approximately 100pM to approximately 10nM, approximately 100pM to approximately 5nM, approximately 100pM to approximately 2nM, approximately 100pM to approximately 1nM, approximately 100pM to approximately 950pM, approximately 100pM to approximately 900pM, approximately 100pM to approximately 850pM, approximately 10 0pM~approx. 800pM, approx. 100pM~approx. 750pM, approx. 100pM~approx. 700pM, approx. 100pM~approx. 650pM, approx. 100pM~approx. 600pM, approx. 100pM~approx. 550pM, approx. 100pM~approx. 500pM, approx. 100pM~approx. 450pM, approx. 100pM~approx. 400pM, approx. 100pM~approx. 350pM, approx. 100pM~approx. 300pM, approx. 100pM~approx. 250pM, approx. 100pM~approx. 200pM, approx. 100pM~approx. 150pM, approx. 150pM~approx. 300pM, approx. 150pM~approx. 150pM, approx. 150pM~approx. 150pM, approx. 150pM~approx. 150pM 0pM ~ approximately 10nM, approximately 150pM ~ approximately 5nM, approximately 150pM ~ approximately 2nM, approximately 150pM ~ approximately 1nM, approximately 150pM ~ approximately 950pM, approximately 150pM ~ approximately 900pM, approximately 150pM ~ approximately 850pM, approximately 150pM ~ approximately 800pM, approximately 150pM ~ approximately 750pM, approximately 150pM ~ approximately 700pM, approximately 150pM to approximately 650pM, approximately 150pM to approximately 600pM, approximately 150pM to approximately 550pM, approximately 150pM to approximately 500pM, approximately 150pM to approximately 450pM, approximately 150pM to approximately 400pM, approximately 150pM to approximately 350pM, approximately 150pM to approximately 300pM, approximately 150pM ~approximately 250 pM, approximately 150 pM ~ approximately 200 pM, approximately 200 pM ~ approximately 30 nm, approximately 200 pM ~ approximately 25 nm, approximately 200 pM ~ approximately 30 nm, approximately 200 pM ~ approximately 15 nm, approximately 200 pM ~ approximately 10 nm, approximately 200 pM ~ approximately 5 nm, approximately 200 pM ~ approximately 2 nm, approximately 200 pM ~ approximately 1 nm, approximately 200pM to approximately 950pM, approximately 200pM to approximately 900pM, approximately 200pM to approximately 850pM, approximately 200pM to approximately 800pM, approximately 200pM to approximately 750pM, approximately 200pM to approximately 700pM, approximately 200pM to approximately 650pM, approximately 200pM to approximately 600pM, approximately 200pM to approximately 550pM Approximately 200pM to 500pM, approximately 200pM to 450pM, approximately 200pM to 400pM, approximately 200pM to 350pM, approximately 200pM to 300pM, approximately 200pM to 250pM, approximately 300pM to 30nm, approximately 300pM to 25nm, approximately 300pM to 30nm.Approximately 300pM to approximately 15nm, approximately 300pM to approximately 10nm, approximately 300pM to approximately 5nm, approximately 300pM to approximately 2nm, approximately 300pM to approximately 1nm, approximately 300pM to approximately 950pM, approximately 300pM to approximately 900pM, approximately 300pM to approximately 850pM, approximately 300pM to approximately 800pM, approximately 300pM~ Approximately 750pM, approximately 300pM to approximately 700pM, approximately 300pM to approximately 650pM, approximately 300pM to approximately 600pM, approximately 300pM to approximately 550pM, approximately 300pM to approximately 500pM, approximately 300pM to approximately 450pM, approximately 300pM to approximately 400pM, approximately 300pM to approximately 350pM, approximately 400pM ~approx. 30 nm, approx. 400 pM ~ approx. 25 nm, approx. 400 pM ~ approx. 30 nm, approx. 400 pM ~ approx. 15 nm, approx. 400 pM ~ approx. 10 nm, approx. 400 pM ~ approx. 5 nm, approx. 400 pM ~ approx. 2 nm, approx. 400 pM ~ approx. 1 nm, approx. 400 pM ~ approx. 950 pM, approx. 400 pM ~ approx. 900 pM, approx. 4 00pM~approx. 850pM, approx. 400pM~approx. 800pM, approx. 400pM~approx. 750pM, approx. 400pM~approx. 700pM, approx. 400pM~approx. 650pM, approx. 400pM~approx. 600pM, approx. 400pM~approx. 550pM, approx. 400pM~approx. 500pM, approx. 500pM~approx. 30nm, approx. 5 00pM ~ approximately 25nM, approximately 500pM ~ approximately 30nM, approximately 500pM ~ approximately 15nM, approximately 500pM ~ approximately 10nM, approximately 500pM ~ approximately 5nM, approximately 500pM ~ approximately 2nM, approximately 500pM ~ approximately 1nM, approximately 500pM ~ approximately 950pM, approximately 500pM ~ approximately 900pM, approximately 500pM ~ approximately 850pM pM, approximately 500pM to approximately 800pM, approximately 500pM to approximately 750pM, approximately 500pM to approximately 700pM, approximately 500pM to approximately 650pM, approximately 500pM to approximately 600pM, approximately 500pM to approximately 550pM, approximately 600pM to approximately 30nM, approximately 600pM to approximately 25nM, approximately 600pM to approximately 30nM Approximately 600pM to approximately 15nm, approximately 600pM to approximately 10nm, approximately 600pM to approximately 5nm, approximately 600pM to approximately 2nm, approximately 600pM to approximately 1nm, approximately 600pM to approximately 950pM, approximately 600pM to approximately 900pM, approximately 600pM to approximately 850pM, approximately 600pM to approximately 800pM, approximately 600pM ~750pM, approximately 600pM~approx. 700pM, approximately 600pM~approx. 650pM, approximately 700pM~approx. 30nM, approximately 700pM~approx. 25nM, approximately 700pM~approx. 30nM, approximately 700pM~approx. 15nM, approximately 700pM~approx. 10nM, approximately 700pM~approx. 5nM, approximately 700pM~approx. 2nMApproximately 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 750 pM, approximately 800 pM to approximately 30 nm, approximately 800 pM to approximately 25 nm, approximately 800 pM to approximately 30 nm, approximately 800 pM to approximately 15 nm, approximately 800 pM to approximately 10 nm, approximately 800 pM to approximately 5 nm, approximately 800 pM to approximately 2 nm, approximately 800 pM M ~ approximately 1 nmM, approximately 800 pM ~ approximately 950 pM, approximately 800 pM ~ approximately 900 pM, approximately 800 pM ~ approximately 850 pM, approximately 900 pM ~ approximately 30 nmM, approximately 900 pM ~ approximately 25 nmM, approximately 900 pM ~ approximately 30 nmM, approximately 900 pM ~ approximately 15 nmM, approximately 900 pM ~ approximately 10 nmM, approximately 900 pM ~ approximately 5 nmM, approximately 900 pM ~ approximately 2 nmM, approximately 900 pM ~ approximately 1 nmM, approximately 900 pM ~ approximately 950 pM, approximately 1 nmM ~ approximately 30 nmM, approximately 1 nM ~ approximately 25 nM, approximately 1 nM ~ approximately 20 nM, approximately 1 nM ~ approximately 15 nM, approximately 1 nM ~ approximately 10 nM, approximately 1 nM ~ approximately 5 nM, approximately 2 nM ~ approximately 30 nM, approximately 2 nM ~ approximately 25 nM, approximately 2 nM ~ approximately 20 nM, approximately 2 nM ~ approximately 15 nM, approximately 2 nM ~ approximately 10 nM, approximately 2 nM ~ approximately 5 nM, approximately 4 nM ~ approximately 30 nM, approximately 4 nM ~ approximately 25 nM, approximately 4 nM ~ approximately 20 nM, approximately 4 nM ~ approximately 15 nM, approximately 4 nM ~ approximately 10 nM, approximately 4 n M~approx. 5nM, approx. 5nM~approx. 30nM, approx. 5nM~approx. 25nM, approx. 5nM~approx. 20nM, approx. 5nM~approx. 15nM, approx. 5nM~approx. 10nM, approx. 10nM~approx. 30nM, approx. 15nM~approx. 25nM, approx. 15nM~approx. 20nM, approx. 20nM~approx. 30nM, approx. 20nM~approx. 30nM, approx. 20nM~approx. 25nM) K, D The combination of the でその standard and the することができる.

[0101] Among the target binding domains described herein, all have a range of approximately 1nM to 10nM (for example, approximately 1nM to 9nM, approximately 1nM to 8nM, approximately 1nM to 7nM, approximately 1nM to 6nM, approximately 1nM to 5nM, approximately 1nM to 4nM, approximately 1nM to 3nM, approximately 1nM to 2nM, approximately 2nM to 10nM, approximately 2nM to 9nM, approximately 2nM to 8nM, approximately 2nM to 7nM, approximately 2nM to 6nM, approximately 2nM to 5nM, approximately 2nM to 4nM, approximately 2nM to 3nM, approximately 3nM to 10nM, approximately 3nM to 9nM, approximately 3nM to 8nM, approximately 3nM to 7nM, approximately 3nM). ~about 6nM, about 3nM to about 5nM, about 3nM to about 4nM, about 4nM to about 10nM, about 4nM to about 9nM, about 4nM to about 8nM, about 4nM ~about 7nM, about 4nM to about 6nM, about 4nM to about 5nM, about 5nM to about 10nM, about 5nM to about 9nM, about 5nM to about 8nM, about 5nM Approximately 7nM, approximately 5nM to approximately 6nM, approximately 6nM to approximately 10nM, approximately 6nM to approximately 9nM, approximately 6nM to approximately 8nM, approximately 6nM to approximately 7nM, approximately 7nM to approximately K of 10nM, about 7nM to about 9nM, about 7nM to about 8nM, about 8nM to about 10nM, about 8nM to about 9nM, and about 9nM to about 10nM) D It can then bind to its target.

[0102] Using various different methods known in the art, any K of the polypeptides described herein D The values ​​can also be determined (e.g., electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecular binding reaction kinetic assays).

[0103] antigen-binding domain In some embodiments of the single-chain or multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain specifically bind to the same antigen. In some embodiments of these single-chain or multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain specifically bind to the same epitope. In some embodiments of these single-chain or multi-chain chimeric polypeptides, the first target-binding domain and the second target-binding domain contain the same amino acid sequence.

[0104] In some embodiments of the single-chain or multi-chain chimeric polypeptides described herein, the first target-binding domain and the second target-binding domain specifically bind to different antigens.

[0105] In some embodiments of the single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain are antigen-binding domains.

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

[0107] In some examples, the antigen-binding domain (e.g., any of the antigen-binding domains described herein) is CD16a (e.g., see U.S. Patent No. 9,035,026), CD28 (e.g., see U.S. Patent No. 7,723,482), CD3 (e.g., see U.S. Patent No. 9,226,962), CD33 (e.g., see U.S. Patent No. 8,759,494), CD20 (e.g., see WO2014 / 026054), CD19 (e.g., see U.S. Patent No. 9,701,758), CD22 (e.g., see WO2003 / 104425). See also), CD123 (see, for example, WO2014 / 130635), IL-1R (see, for example, U.S. Patent No. 8,741,604), IL-1 (see, for example, WO2014 / 095808), VEGF (see, for example, U.S. Patent No. 9,090,684), IL-6R (see, for example, U.S. Patent No. 7,482,436), IL-4 (see, for example, U.S. Patent Application Publication No. 2012 / 0171197), IL-10 (see, for example, U.S. Patent Application Publication No. 2016 / 0340413), PDL-1 (see, for example, Dress See, for example, Gejima et al., Protein Express. Purif. 94:60-66, 2014), TIGIT (see, for example, U.S. Patent Application Publication No. 2017 / 0198042), PD-1 (see, for example, U.S. Patent No. 7,488,802), TIM3 (see, for example, U.S. Patent No. 8,552,156), CTLA4 (see, for example, WO2012 / 120125), MICA (see, for example, WO2016 / 154585), MICB (see, for example, U.S. Patent No. 8,753,640), IL-6 (see, for example, Gejima et al.See Human Antibodies 11(4):121-129, 2002), IL-8 (see, for example, U.S. Patent No. 6,117,980), TNFα (see, for example, Geng et al., Immunol. Res. 62(3):377-385, 2015), CD26a (see, for example, WO2017 / 189526), ​​CD36 (see, for example, U.S. Patent Application Publication No. 2015 / 0259429), ULBP2 (see, for example, U.S. Patent No. 9,273,136), CD30 (see, for example, Homac et al. See, for example, al.,Scand.J.Immunol.48(5):497-501,1998), CD200 (see, for example, U.S. Patent No. 9,085,623), IGF-1R (see, for example, U.S. Patent Publication No. 2017 / 0051063), MUC4AC (see, for example, WO2012 / 170470), MUC5AC (see, for example, U.S. Patent No. 9,238,084), Trop-2 (see, for example, WO2013 / 068946), CMET (see, for example, Edwardraja et al., Biotechnol.Bioeng.106(3):367-375,2010), EGFR (see, for example, Akbari et al.,Protein See Expr.Purif.127:8-15, 2016), HER1 (see, for example, U.S. Patent Application Publication No. 2013 / 0274446), HER2 (see, for example, Cao et al., Biotechnol. Lett. 37(7):1347-1354, 2015), HER3 (see, for example, U.S. Patent No. 9,505,843), PSMA (see, for example, Parker et al., Protein Expr.Purif.See also 89(2):136-145,2013), CEA (see, for example, WO1995 / 015341), B7H3 (see, for example, U.S. Patent No. 9,371,395), EPCAM (see, for example, WO2014 / 159531), BCMA (see, for example, Smith et al., Mol.Ther. 26(6):1447-1456,2018), P-cadherin (see, for example, U.S. Patent No. 7,452,537), CEACAM5 (see, for example, U.S. Patent No. 9,617,345), UL16 binding protein (see, for example, WO2017 / 083612), HLA-DR (see, for example, Pistillo et al.) It can specifically bind to any one of the following: al., Exp. Clin. Immunogenet. 14(2):123-130, 1997; DLL4 (e.g., see WO2014 / 007513); TYRO3 (e.g., see WO2016 / 166348); AXL (e.g., see WO2012 / 175692); MER (e.g., see WO2016 / 106221); CD122 (e.g., see U.S. Patent Application Publication No. 2016 / 0367664); CD155 (e.g., see WO2017 / 149538); or PDGF-DD (e.g., see U.S. Patent No. 9,441,034).

[0108] The antigen-binding domains present in any of the single-chain or multi-chain chimeric polypeptides described herein are each independently selected from the group consisting of VHH domains, VNAR domains, and scFv. In some embodiments, any of the antigen-binding domains described herein is BiTe, (scFv)2, nanobody, nanobody-HSA, DART, TandAb, scDiabody, scDiabody-CH3, scFv-CH-CL-scFv, HSAbody, scDiabody-HAS, or tandem-scFv. Additional examples of antigen-binding domains that can be used in either single-chain or multi-chain chimeric polypeptides are known in the art.

[0109] The VHH domain is a monomeric variable antibody domain that can be found in camelids. The VNAR domain is a monomeric variable antibody domain that can be found in cartilaginous fish. VHH domain and V NARNon-limiting aspects of the domain include, for example, Cromie et al., Curr.Top.Med.Chem.15:2543-2557,2016, De Genst et al., Dev.Comp.Immunol.30:187-198,2006, De Meyer et al., Trends Biotechnol.32:263-270,2014, Kijanka et al., Nanomedicine 10:161-174,2015, Kovaleva et al., Expert.Opin.Biol.Ther.14:1527-1539,2014, Krah et al., Immunopharmacol.Immunotoxicol.38:21-28,2016, and Mujic-Delic et al., Trends Pharmacol.Sci.35:247-255,2014, Muyldermans,J.Biotechnol.74:277-302,2001, Muyldermans et al.,Trends Biochem.Sci.26:230-235,2001, Muyldermans,Ann.Rev.Biochem.82:775-797,2013, Rahbarizadeh et al.,Immunol.Invest.40:299-338,2011, Van Audenhove et al.,EBioMedicine 8:40-48,2016, Van Bockstaele et al.,Curr.Opin.Investig.Drugs 10:1212-1224,2009, Vincke et al.,Methods This is described in Mol.Biol.911:15-26,2012, and Wesolowski et al., Med.Microbiol.Immunol.198:157-174,2009.

[0110] In some embodiments, each antigen-binding domain in the single-chain or multi-chain chimeric polypeptide described herein is either a VHH domain or at least one antigen-binding domain is a VHH domain. In some embodiments, each antigen-binding domain in the single-chain or multi-chain chimeric polypeptide described herein is either a VNAR domain or at least one antigen-binding domain is a VNAR domain. In some embodiments, each antigen-binding domain in the single-chain or multi-chain chimeric polypeptide described herein is either an scFv domain or at least one antigen-binding domain is an scFv domain.

[0111] In some embodiments, two or more polypeptides present in a polychain chimeric polypeptide assemble (e.g., non-covalently) to form one of the antigen-binding domains described herein, e.g., an antigen-binding fragment of an antibody (e.g., one of the antigen-binding fragments of an antibody described herein), VHH-scAb, VHH-Fab, double scFab, F(ab')2, diabody, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knob-in-hole common light chain, knob-in-hole aggregate, charge pair, Fab arm exchange, SEEDbody, LUZ-Y, Fcab, γλ-body, orthogonal Fab, DVD-IgG, IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-IgG, Diabody-CH3, Triplebody, Mino antibody, Minibody, TriBi Minibody, scFv-CH3 KIH, Fab-scFv, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, Tetravalent HCAb, scDiabody-Fc, Diabody-Fc, Tandem scFv-Fc, Intrabody, Dock and Lock, lmmTAC, IgG-IgG conjugate, Cov-X-Body, and scFv1-PEG-scFv2 can be formed. For a description of these elements, see, for example, Spiess et al., Mol.Immunol. 67:95-106, 2015, which is incorporated in whole herein. Non-limiting examples of antibody antigen-binding fragments include the Fv fragment, Fab fragment, F(ab')2 fragment, and Fab' fragment.Examples of adding antigen-binding fragments to antibodies include antigen-binding fragments for IgG (e.g., antigen-binding fragments of IgG1, IgG2, IgG3, or IgG4) (e.g., human or humanized IgG, e.g., antigen-binding fragments of human or humanized IgG1, IgG2, IgG3, or IgG4), antigen-binding fragments for IgA (e.g., antigen-binding fragments of IgA1 or IgA2) (e.g., human or humanized IgA, e.g., antigen-binding fragments of human or humanized IgA1 or IgA2), antigen-binding fragments for IgD (e.g., antigen-binding fragments of human or humanized IgD), antigen-binding fragments for IgE (e.g., antigen-binding fragments of human or humanized IgE), or antigen-binding fragments for IgM (e.g., antigen-binding fragments of human or humanized IgM).

[0112] The "Fv" fragment contains a non-covalent dimer having one heavy chain variable domain and one light chain variable domain.

[0113] The "Fab" fragment, in addition to the heavy and light chain variable domains of the Fv fragment, also contains a constant domain of the light chain and a first constant domain of the heavy chain (C H1 ) includes.

[0114] The "F(ab')2" fragment contains two Fab fragments linked by disulfide bonds near the hinge region.

[0115] "Dual variable domain immunoglobulin" or "DVD-Ig" refers to the multivalent and multispecific binding proteins described in, for example, DiGiammarino et al., Methods Mol. Biol. 899:145-156, 2012, Jakob et al., MABs 5:358-363, 2013, and U.S. Patents 7,612,181, 8,258,268, 8,586,714, 8,716,450, 8,722,855, 8,735,546, and 8,822,645, which are incorporated in their entirety by reference.

[0116] DART is described, for example, in Garber, Nature Reviews Drug Discovery 13:799-801, 2014.

[0117] In some embodiments of the antigen-binding domains described herein, the antigens can bind to antigens selected from the group consisting of proteins, carbohydrates, lipids, and combinations thereof.

[0118] Examples and embodiments of the addition of antigen-binding domains are known in the art.

[0119] Soluble interleukins or cytokine proteins In some embodiments of any single-chain or multi-chain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain may be a soluble interleukin protein or a soluble cytokine protein. In some embodiments, the soluble interleukin or soluble cytokine protein is selected from the group IL-2, IL-3, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L. Non-limiting examples of soluble IL-2, IL-3, IL-7, IL-8, IL-10, IL-15, IL-17, IL-18, IL-21, PDGF-DD, SCF, and FLT3L are provided below. Human soluble IL-2 (SEQ ID NO: 17) TIFF2026053343000006.tif20145 Human soluble IL-3 (SEQ ID NO: 18) TIFF2026053343000007.tif20145 Human soluble IL-7 (SEQ ID NO: 19) TIFF2026053343000008.tif19145 Human soluble IL-8 (SEQ ID NO: 20) TIFF2026053343000009.tif13145 Human soluble IL-10 (SEQ ID NO: 21) TIFF2026053343000010.tif27145 Human soluble IL-15 (SEQ ID NO: 22) TIFF2026053343000011.tif21145 Human soluble IL-17 (SEQ ID NO: 23) TIFF2026053343000012.tif21145 Human soluble IL-18 (SEQ ID NO: 24) TIFF2026053343000013.tif19145 Human soluble PDGF-DD (SEQ ID NO: 25) TIFF2026053343000014.tif50145 Human soluble SCF (SEQ ID NO: 26) TIFF2026053343000015.tif33145 Human soluble FLT3L (SEQ ID NO: 27) TIFF2026053343000016.tif33145

[0120] Non-limiting examples of soluble MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6 are provided below. Human soluble MICA (SEQ ID NO: 28) TIFF2026053343000017.tif58145 Human soluble MICB (SEQ ID NO: 29) TIFF2026053343000018.tif58145 Human soluble ULBP1 (SEQ ID NO: 30) TIFF2026053343000019.tif27145 Human soluble ULBP2 (SEQ ID NO: 31) TIFF2026053343000020.tif26145 Human soluble ULBP3 (SEQ ID NO: 32) TIFF2026053343000021.tif28145 Human soluble ULBP4 (SEQ ID NO: 33) TIFF2026053343000022.tif36145 Human soluble ULBP5 (SEQ ID NO: 34) TIFF2026053343000023.tif29145 Human soluble ULBP6 (SEQ ID NO: 35) TIFF2026053343000024.tif29145

[0121] Examples of the addition of soluble interleukin proteins and soluble cytokine proteins are known in the art.

[0122] Soluble receptors In some embodiments of the polychain chimeric polypeptides described herein, one or both of the first target-binding domain and the second target-binding domain are soluble interleukin receptors, soluble cytokine receptors, or ligand receptors. In some embodiments, the soluble receptor is soluble TGF-β receptor II (TGF-β RII) (see, e.g., Yung et al., Am.J.Resp.Crit.Care Med. 194(9):1140-1151, 2016), soluble TGF-β RIII (see, e.g., Heng et al., Placenta 57:320, 2017), soluble NKG2D (see, e.g., Cosman et al., Immunity 14(2):123-133, 2001, Costa et al., Front.Immunol., Vol.9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp30 (see, e.g., Costa et al. See Costa et al., Front.Immunol., Vol.9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp44 (see, for example, Costa et al., Front.Immunol., Vol.9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble NKp46 (see, for example, Mandelboim et al., Nature 409:1055-1060, 2001, Costa et al., Front.Immunol., Vol.9, Article 1150, May See May 29, 2018, doi:10.3389 / fimmu.2018.01150), soluble DNAM-1 (see, for example, Costa et al., Front.Immunol., Vol.9, Article 1150, May 29, 2018, doi:10.3389 / fimmu.2018.01150), scMHCI (see, for example, Washburn et al.See PLoS One 6(3):e18439, 2011), scMHCII (e.g., see Bishwajit et al., Cellular Immunol. 170(1):25-33, 1996), scTCR (e.g., see Weber et al., Nature 356(6372):793-796, 1992), soluble CD155 (e.g., see Tahara-Hanaoka et al., Int.Immunol. 16(4):533-538, 2004), or soluble CD28 (e.g., see Hebbar et al., Clin.Exp.Immunol. 136:388-392, 2004).

[0123] Examples of the addition of soluble interleukin receptors and soluble cytokine receptors are known in the art.

[0124] Additional antigen-binding domains In some embodiments of the single-chain chimeric polypeptide, the first chimeric polypeptide further comprises one or more additional target-binding domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) (e.g., any of the exemplary target-binding domains described herein or known in the art). In some embodiments of the polychain chimeric polypeptide, at least one of the one or more additional antigen-binding domains may be positioned between a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein). In some embodiments, the first chimeric polypeptide may further include a linker sequence (e.g., one of the exemplary linker sequences described herein or known in the art) between a soluble tissue factor domain (e.g., one of the exemplary soluble tissue factor domains described herein) and at least one of one or more additional target-binding domains (e.g., one of the exemplary target-binding domains described herein or known in the art), and / or a linker sequence (e.g., one of the exemplary linker sequences described herein or known in the art) between at least one of one or more additional target-binding domains (e.g., one of the exemplary target-binding domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., one of the exemplary first domains described herein or one of the exemplary pair of affinity domains described herein).

[0125] In some embodiments of the polychain chimeric polypeptides described herein, the first chimeric polypeptide further includes one or more additional target-binding domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N-terminus and / or C-terminus of the first chimeric polypeptide. In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is directly adjacent to the first domain of a pair of affinity domains in the first chimeric polypeptide (e.g., any of the exemplary first domains described herein or known in the art). In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein or known in the art). In some embodiments, at least one of one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is directly adjacent to the first target-binding domain in the first chimeric polypeptide (e.g., any of the exemplary target-binding domains described herein or known in the art). In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) between at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art).

[0126] In some embodiments of any of the polychain chimeric polypeptides described herein, at least one of one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is located at the N-terminus and / or C-terminus of the first chimeric polypeptide, and at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is located between a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein or known in the art) and a first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein). In some embodiments, at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) located at the N-terminus is directly adjacent to the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein or any of the exemplary pair of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any of the linker sequences described herein or known in the art) located between the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains described herein or any of the exemplary pair of affinity domains described herein).In some embodiments, at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) located at the C-terminus is directly adjacent to the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein) in the first chimeric polypeptide. In some embodiments, the first chimeric polypeptide further includes a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) located between the at least one additional target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) and the first target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art) or the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein). In some embodiments, at least one of one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) located between a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factor domains described herein) and a first domain of a pair of affinity domains (e.g., any of the first domains described herein or any of the exemplary pair of affinity domains described herein) is directly adjacent to the soluble tissue factor domain and / or the first domain of the pair of affinity domains.In some embodiments, the first chimeric polypeptide further comprises (i) a soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein) and at least one of one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) positioned between the soluble tissue factor domain (e.g., any of the exemplary soluble tissue factors described herein) and the first domain of a pair of affinity domains (e.g., any of the exemplary first domains of any of the exemplary pair of affinity domains described herein), and / or (ii) a linker sequence (e.g., any of the exemplary linker sequences described herein or known in the art) positioned between the first domain of a pair of affinity domains and at least one of one or more additional target-binding domains positioned between the soluble tissue factor domain and the first domain of a pair of affinity domains.

[0127] In some embodiments of the polychain chimeric polypeptides described herein, the second chimeric polypeptide further comprises one or more additional target-binding domains (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) at the N-terminus and / or C-terminus of the second chimeric polypeptide (e.g., any of the exemplary target-binding domains described herein or known in the art). In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is directly adjacent to the second domain of a pair of affinity domains in the second chimeric polypeptide (e.g., any of the exemplary second domains of any of the exemplary pair of affinity domains described herein). In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., one of the exemplary linker sequences described herein or known in the art) between at least one of one or more additional target-binding domains within the second chimeric polypeptide (e.g., any of the exemplary target-binding domains described herein or known in the art) and a second domain of a pair of affinity domains (e.g., any of the exemplary second domains described herein or known in the art). In some embodiments, at least one of the one or more additional target-binding domains (e.g., any of the exemplary target-binding domains described herein or known in the art) is directly adjacent to the second target-binding domain within the second chimeric polypeptide (e.g., any of the target-binding domains described herein or known in the art).In some embodiments, the second chimeric polypeptide further includes a linker sequence (e.g., one of the example linker sequences described herein or known in the art) between at least one of one or more additional target-binding domains in the second chimeric polypeptide (e.g., any of the exemplary target-binding domains described herein or known in the art) and the second target-binding domain (e.g., any of the exemplary target-binding domains described herein or known in the art).

[0128] In some embodiments of any of the polychain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and two or more of the one or more additional target-binding domains (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) specifically bind to the same antigen. In some embodiments, the first target-binding domain, the second target-binding domain, and two or more of the one or more additional target-binding domains (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) specifically bind to the same epitope. In some embodiments, the first target-binding domain, the second target-binding domain, and two or more of the one or more additional target-binding domains (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) contain 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.

[0129] In some embodiments of the polychain chimeric polypeptides described herein, the first target-binding domain, the second target-binding domain, and one or more additional target-binding domains specifically bind to different antigens. In some embodiments of the polychain chimeric polypeptides described herein, one or more of the first target-binding domain, the second target-binding domain, and one or more target-binding domains (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or more) are antigen-binding domains. In some embodiments, the first target-binding domain, the second target-binding domain, and one or more additional target-binding domains are each antigen-binding domains (e.g., scFv or single-domain antibodies).

[0130] A pair of affinity domains In some embodiments, the polychain chimeric polypeptide comprises 1) a first chimeric polypeptide containing a first domain of a pair of affinity domains, and 2) a second chimeric polypeptide containing a second domain of a pair of affinity domains, thereby causing the first and second chimeric polypeptides to associate via the binding of the first and second domains of the pair of affinity domains. In some embodiments, the pair of affinity domains are a sucrose domain derived from the human IL-15 receptor alpha chain (IL15Rα) and soluble IL-15. Sucrose domains, also known as short consensus repeats or type 1 glycoprotein motifs, are common motifs for protein-protein interactions. Sucrose domains have been identified on several protein-binding molecules, including complement components C1r, C1s, factor H, C2m, and non-immunological molecules factor XIII and β2-glycoprotein. A typical cysteine ​​domain has approximately 60 amino acid residues and contains four cysteines (Ranganathan, Pac. Symp Biocomput. 2000:155-67). The first cysteine ​​can form a disulfide bond with the third cysteine, and the second cysteine ​​can form a disulfide bridge with the fourth cysteine. In some embodiments, one member of the affinity domain pair is soluble IL-15, where the soluble IL-15 has a D8N or D8A amino acid substitution. In some embodiments, one member of the affinity domain pair is human IL-15 receptor alpha chain (IL15Rα), where the human IL15Rα is mature full-length IL15Rα. In some embodiments, the affinity domain pair is barnase and barnster. In some embodiments, the affinity domain pair is PKA and AKAP.In some embodiments, the pair of affinity domains is an adapter / docking tag module based on a mutant RNase I fragment (Rossi, Proc Natl Acad Sci USA. 103:6841-6846, 2006; Sharkey et al., Cancer Res. 68:5282-5290, 2008; Rossi et al., Trends Pharmacol Sci. 33:474-481, 2012), or a SNARE module based on the interaction of protein syntaxin, synaptotagmin, synaptobrevinn, and SNAP25 (Deyev et al., Nat Biotechnol. 1486-1492, 2003).

[0131] In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide comprises a first domain of a pair of affinity domains, the second chimeric polypeptide of the polychain chimeric polypeptide comprises a second domain of a pair of affinity domains, and the first domain of a pair of affinity domains and the second domain of a pair of affinity domains comprise 1 × 10⁻¹⁶ -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, 1 x 10 -10 Less than M, 1 x 10 -11 Less than M, 1 x 10 -12 Less than M, or 1 × 10 -13 Dissociation equilibrium constant (K) less than M D They are bound to each other at ). In some embodiments, the first domain of the pair of affinity domains and the second domain of the pair of affinity domains are approximately 1 × 10 -4 M ~ approx. 1×10 -6 M, about 1 x 10 -5 M ~ approx. 1×10 -7 M, about 1 x 10 -6 M ~ approx. 1×10 -8 M, about 1 x 10 -7 M ~ approx. 1×10 -9 M, about 1 x 10 -8 M ~ approx. 1×10 -10 M, about 1 x 10 -9 M ~ approx. 1×10 -11 M, about 1 x 10 -10 M ~ approx. 1×10-12 M, about 1 x 10 -11 M ~ approx. 1×10 -13 M, about 1 x 10 -4 M ~ approx. 1×10 -5 M, about 1 x 10 -5 M ~ approx. 1×10 -6 M, about 1 x 10 -6 M ~ approx. 1×10 -7 M, about 1 x 10 -7 M ~ approx. 1×10 -8 M, about 1 x 10 -8 M ~ approx. 1×10 -9 M, about 1 x 10 -9 M ~ approx. 1×10 -10 M, about 1 x 10 -10 M ~ approx. 1×10 -11 M, about 1 x 10 -11 M ~ approx. 1×10 -12 M, or approximately 1 x 10 -12 M ~ approx. 1×10 -13 K of M (including its upper and lower limits) D They bind to each other. The binding of the first domain of the pair of affinity domains and the second domain of the pair of affinity domains is performed using one of the various different methods known in the art. D The values ​​can be determined (e.g., electrophoretic mobility shift assays, filter binding assays, surface plasmon resonance, and biomolecular binding reaction kinetic assays).

[0132] In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide comprises a first domain of a pair of affinity domains, and the second chimeric polypeptide of the polychain chimeric polypeptide comprises a second domain of a pair of affinity domains, with the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both being approximately 10 to 100 amino acids in length. For example, the first domain of a pair of affinity domains, the second domain of a pair of affinity domains, or both, are approximately 10-100 amino acid lengths, approximately 15-100 amino acid lengths, approximately 20-100 amino acid lengths, approximately 25-100 amino acid lengths, approximately 30-100 amino acid lengths, approximately 35-100 amino acid lengths, approximately 40-100 amino acid lengths, approximately 45-100 amino acid lengths, approximately 50-100 amino acid lengths, approximately 55-100 amino acid lengths, approximately 60-100 amino acid lengths, approximately 65-100 amino acid lengths, approximately 70-100 amino acid lengths, approximately 75-100 amino acid lengths, approximately 80-100 amino acid lengths, approximately 85-100 amino acid lengths, approximately 90-100 amino acid lengths, approximately 95-100 amino acid lengths, approximately 10-95 amino acid lengths, approximately 10-90 amino acid lengths, approximately 10-85 amino acid lengths, approximately 10-8 The amino acid length may be 0, approximately 10-75, approximately 10-70, approximately 10-65, approximately 10-60, approximately 10-55, approximately 10-50, approximately 10-45, approximately 10-40, approximately 10-35, approximately 10-30, approximately 10-25, approximately 10-20, approximately 10-15, approximately 20-30, approximately 30-40, approximately 40-50, approximately 50-60, approximately 60-70, approximately 70-80, approximately 80-90, approximately 90-100, approximately 20-90, approximately 30-80, approximately 40-70, approximately 50-60, or any range in between. In some embodiments, the first domain of the pair of affinity domains, the second domain of the pair of affinity domains, or both thereof, are approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids long.

[0133] In some embodiments, either of the first and / or second domains of a pair of affinity domains disclosed herein may contain one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more) at its N-terminus and / or C-terminus, provided that the function of the first and / or second domains of the pair of affinity domains remains unimpaired. For example, a sucrose domain derived from the human IL-15 receptor alpha chain (IL15Rα) may contain one or more additional amino acids at its N-terminus and / or C-terminus while still retaining its ability to bind to soluble IL-15. In addition, or alternatively, soluble IL-15 may contain one or more additional amino acids at its N-terminus and / or C-terminus while still retaining its ability to bind to a sucrose domain derived from the human IL-15 receptor alpha chain (IL15Rα).

[0134] Non-limiting examples of sucoid domains derived from the IL-15 receptor alpha chain (IL15Rα) may include sequences that are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR (SEQ ID NO: 36). In some embodiments, sucoid domains derived from the IL15Rα alpha chain may be encoded by nucleic acids including ATTACATGCCCCCCTCCCATGAGCGTGGAGCACGCCGACATCTGGGTGAAGAGCTATAGCCTCTACAGCCGGGAGAGGTATATCTGTAACAGCGGCTTCAAGAGGAAGGCCGGCACCAGCAGCCTCACCGAGTGCGTGCTGAATAAGGCTACCAACGTGGCTCACTGGACAACACCCTCTTTAAAGTGCATCCGG (SEQ ID NO: 37).

[0135] In some embodiments, soluble IL-15 may contain sequences that are at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 22). In some embodiments, soluble IL-15 may be encoded by a nucleic acid containing the sequence AACTGGGTGAACGTCATCAGCGATTTAAAGAAGATCGAAGATTTAATTCAGTCCATGCATATCGACGCCACTTTATACACAGAATCCGACGTGCACCCCTCTTGTAAGGTGACCGCCATGAAATGTTTTTTACTGGAGCTGCAAGTTATCTCTTTAGAGAGCGGAGACGCTAGCATCCACGACACCGTGGAGAATTTAATCATTTTAGCCAATAACTCTTTATCCAGCAACGGCAACGTGACAGAGTCCGGCTGCAAGGAGTGCGAAGAGCTGGAGGAGAAGAACATCAAGGAGTTTCTGCAATCCTTTGTGCACATTGTCCAGATGTTCATCAATACCTCC (Sequence ID 38).

[0136] signal sequence In some embodiments, a polychain chimeric polypeptide comprises a first chimeric polypeptide having a signal sequence at its N-terminus. In some embodiments, a polychain chimeric polypeptide comprises a second chimeric polypeptide having a signal sequence at its N-terminus. In some embodiments, both the first and second chimeric polypeptides of the polychain chimeric polypeptide contain a signal sequence. As will be understood by those skilled in the art, a signal sequence is an amino acid sequence present at the N-terminus of several endogenously produced proteins that direct the protein towards a secretory pathway (for example, a protein is directed to reside in a particular intracellular organelle, in the cell membrane, or to be secreted from the cell). Signal sequences are heterogeneous, and their primary amino acid sequences differ considerably. However, signal sequences are typically 16–30 amino acid long and include a hydrophilic, usually positively charged N-terminal region, a central hydrophobic domain, and a C-terminal region containing a cleavage site for signal peptidases.

[0137] In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, contains a signal sequence having the amino acid sequence MKWVTFISLLFLFSSAYS (SEQ ID NO: 39). In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, contains a nucleic acid sequence ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (Sequence No. 40), ATGAAGTGGGTCACATTTATCTCTTTACTGTTCCTCTTCTCCAGCGCCTACAGC (Sequence ID 41), or ATGAAATGGGTGACCTTTATTTCTTTACTGTTCCTCTTTAGCAGCGCCTACTCC (Sequence No. 42) Includes a signal array encoded by [the specified method].

[0138] In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, includes a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 43). In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, includes a signal sequence having the amino acid sequence MGQIVTMFEALPHIIDEVINIVIIVLIIITSIKAVYNFATCGILALVSFLFLAGRSCG (SEQ ID NO: 44). In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, includes a signal sequence having the amino acid sequence MPNHQSGSPTGSSDLLLSGKKQRPHLALRRKRRREMRKINRKVRRMNLAPIKEKTAWQHLQALISEAEEVLKTSQTPQNSLTLFLALLSVLGPPVTG (SEQ ID NO: 45). In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, contain a signal sequence having the amino acid sequence MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS (SEQ ID NO: 46). Those skilled in the art will recognize other signal sequences suitable for use in the first and / or second chimeric polypeptides of the polychain chimeric polypeptide described herein.

[0139] In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, contain a signal sequence approximately 10 to 100 amino acids long. For example, signal sequences are approximately 10-100 amino acid lengths, approximately 15-100 amino acid lengths, approximately 20-100 amino acid lengths, approximately 25-100 amino acid lengths, approximately 30-100 amino acid lengths, approximately 35-100 amino acid lengths, approximately 40-100 amino acid lengths, approximately 45-100 amino acid lengths, approximately 50-100 amino acid lengths, approximately 55-100 amino acid lengths, approximately 60-100 amino acid lengths, approximately 65-100 amino acid lengths, approximately 70-100 amino acid lengths, approximately 75-100 amino acid lengths, approximately 80-100 amino acid lengths, approximately 85-100 amino acid lengths, approximately 90-100 amino acid lengths, approximately 95-100 amino acid lengths, approximately 10-95 amino acid lengths, approximately 10-85 amino acid lengths, approximately 10-80 amino acid lengths, approximately 10-75 amino acid lengths, approximately The amino acid lengths may be 10-70, approximately 10-65, approximately 10-60, approximately 10-55, approximately 10-50, approximately 10-45, approximately 10-40, approximately 10-35, approximately 10-30, approximately 10-25, approximately 10-20, approximately 10-15, approximately 20-30, approximately 30-40, approximately 40-50, approximately 50-60, approximately 60-70, approximately 70-80, approximately 80-90, approximately 90-100, approximately 20-90, approximately 30-80, approximately 40-70, approximately 50-60, or any range in between. In some embodiments, the signal sequence is approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids long.

[0140] In some embodiments, any of the signal sequences disclosed herein may include one or more additional amino acids (e.g., 1, 2, 3, 5, 6, 7, 8, 9, 10, or more) at its N-terminus and / or C-terminus, provided that the function of the signal sequence is not impaired. For example, a signal sequence having the amino acid sequence MKCLLYLAFLFLGVNC (SEQ ID NO: 43) may include one or more additional amino acids at its N-terminus or C-terminus while still retaining the ability to direct the first chimeric polypeptide of a polychain chimeric polypeptide, the second chimeric polypeptide of a polychain chimeric polypeptide, or both, to the secretory pathway.

[0141] In some embodiments, the first chimeric polypeptide of the polychain chimeric polypeptide, the second chimeric polypeptide of the polychain chimeric polypeptide, or both thereof, contain a signal sequence that directs the polychain chimeric polypeptide to the extracellular space. Such embodiments are useful for generating polychain chimeric polypeptides that are relatively easy to isolate and / or purify.

[0142] Peptide tags In some embodiments, the polychain chimeric polypeptide comprises a first chimeric polypeptide containing a peptide tag (for example, at the N-terminus or C-terminus of the first chimeric polypeptide). In some embodiments, the polychain chimeric polypeptide comprises a second chimeric polypeptide containing a peptide tag (for example, at the N-terminus or C-terminus of the second chimeric polypeptide). In some embodiments, both the first and second chimeric polypeptides of the polychain chimeric polypeptide contain peptide tags. In some embodiments, the first chimeric polypeptide, the second chimeric polypeptide, or both of them contain two or more peptide tags.

[0143] Exemplary peptide tags that may be included in the first chimeric polypeptide, the second chimeric polypeptide, or both of the polychain chimeric polypeptides include AviTag (GLNDIFEAQKIEWHE, SEQ ID NO: 47), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL, SEQ ID NO: 48), polyglutamic acid tag (EEEEEE, SEQ ID NO: 49), E tag (GAPVPYPDPLEPR, SEQ ID NO: 50), FLAG tag (DYKDDDDK, SEQ ID NO: 51), HA tag, hemagglutinin-derived peptide (YPYDVPDYA, P / E) Column number 52), his tag (HHHHH (sequence number 53), HHHHHH (sequence number 54), HHHHHHH (sequence number 55), HHHHHHHH (sequence number 56), HHHHHHHHH (sequence number 57), or HHHHHHHHHH (sequence number 58)), myc tag (EQKLISEEDL, sequence n...

Claims

1. A method for treating age-related diseases or inflammatory diseases in a subject, wherein the subject (i) A therapeutically effective amount of NK cell activator and / or NK cells and / or monoclonal antibody, (ii) A therapeutically effective amount of a Treg cell activator and / or a Treg cell and / or a monoclonal antibody and / or an advanced glycation end product (AGE) inhibitor The method comprising administering the following.

2. The method according to claim 1, wherein the age-related disease is related to inflammatory aging.

3. The method according to claim 1 or 2, wherein (i) is administered to the subject substantially simultaneously with (ii).

4. The method according to claim 1 or 2, wherein (i) is administered to the subject before (ii) is administered to the subject.

5. The method according to claim 1 or 2, wherein (ii) is administered to the subject before (i) is administered to the subject.

6. The method according to any one of claims 1 to 5, comprising administering a therapeutically effective amount of NK cells to the subject.

7. The method according to claim 6, wherein the NK cells are autologous, haplotype-matched, or allogeneic NK cells isolated from peripheral blood, isolated from umbilical cord blood, or isolated and differentiated from iPSCs.

8. To isolate the NK cells from the subject, The isolated NK cells are cultured in a liquid culture medium under conditions sufficient to induce or increase the proliferation of the NK cells. It further includes, After the isolation step and the culture step, the NK cells are administered to the subject. The method according to claim 7.

9. The method according to claim 8, wherein the liquid culture medium contains a polychain chimeric polypeptide.

10. The method according to any one of claims 6 to 9, wherein the NK cells include a chimeric antigen receptor.

11. The method according to claim 10, wherein the chimeric antigen receptor includes an extracellular domain that specifically binds to tissue factor or CD26.

12. The method according to any one of claims 1 to 5, comprising administering a therapeutically effective amount of an NK cell activator and / or a monoclonal antibody to the subject.

13. The method according to claim 12, wherein the NK cell activating substance is one or more polychain chimeric polypeptides.

14. The method according to claim 12, wherein the monoclonal antibody is one or more of anti-tissue factor antibodies and / or anti-CD26 antibodies.

15. The method according to claim 12, wherein the NK cell activator comprises one or more polychain chimeric polypeptides, and the monoclonal antibody comprises one or more anti-tissue factor antibodies and / or anti-CD26 antibodies.

16. The method according to any one of claims 1 to 15, comprising administering a therapeutically effective amount of Treg cells to the subject.

17. The method according to claim 16, wherein the Treg cells are autologous Treg cells, haplotype-matched Treg cells, or allogeneic Treg cells isolated from peripheral blood or umbilical cord blood.

18. To isolate the Treg cells from the subject, The isolated Treg cells are cultured in a liquid culture medium under conditions sufficient to induce or increase the proliferation of the Treg cells. It further includes, After the isolation step and the culture step, the Treg cells are administered to the subject. The method according to claim 17.

19. The method according to claim 18, wherein the step of isolating the Treg cells from the subject comprises obtaining a sample containing Treg cells from the subject and isolating the Treg cells from the sample using an antibody or ligand capable of binding to CD39.

20. The step of isolating the Treg cells from the sample is, The sample is mixed with the antibody or ligand capable of binding to CD39 under conditions that enable the binding of the antibody to the ligand on Treg cells expressing CD39, The Treg cells bound to the antibody or ligand are separated from other components in the sample, thereby isolating the Treg cells. The method according to claim 19, including the method described in claim 19.

21. The antibody is a mouse, humanized, or human antibody, or an antigen-binding fragment thereof, and / or The antibody or ligand is labeled with at least one of biotin, avidin, streptavidin, or a fluorescent dye, or is confined to particles, beads, resin, or a solid support. The method according to claim 19 or 20.

22. The method according to claim 20, wherein the separation includes the use of flow cytometry, fluorescent cell sorting (FACS), centrifugation, or a column, plate, particle, or bead-based method.

23. Said T ｒｅｇ Cells include fresh or frozen peripheral blood, umbilical cord blood, peripheral blood mononuclear cells, lymphocytes, and CD4 cells. ＋ T cell, or T ｒｅｇ Autologous T cells isolated from a sample containing cells ｒｅｇ Cells, haplotype-matched T ｒｅｇ Cells, or allogeneic T cells ｒｅｇ The method according to any one of claims 18 to 22, wherein the cell is a cell.

24. The aforementioned T ｒｅｇ cells are CD4 ＋ CD25 ＋ Foxp3 ＋ cells, and the method according to any one of claims 18 to 23.

25. Said T ｒｅｇ Cells CD4 ＋ CD25 ＋ CD127 ｄｉｍ－ The method according to any one of claims 18 to 23, wherein the cell is a cell.

26. Said T ｒｅｇ The method according to any one of claims 18 to 25, wherein the cells are immunosuppressive in vitro and in vivo.

27. The method according to any one of claims 18 to 26, wherein the liquid culture medium comprises one or more single-chain chimeric polypeptides.

28. The method according to any one of claims 16 to 27, wherein the Treg cells contain a chimeric antigen receptor.

29. The method according to claim 28, wherein the chimeric antigen receptor includes an extracellular domain that specifically binds to tissue factor or CD36.

30. The method according to any one of claims 1 to 15, comprising administering a therapeutically effective amount of a Treg cell activator and / or a monoclonal antibody and / or an AGE inhibitor to the subject.

31. The method according to claim 30, wherein the Treg cell activating substance is one or more single-chain chimeric polypeptides.

32. The method according to claim 30, wherein the monoclonal antibody is an anti-tissue factor antibody and / or an anti-CD36 antibody, or both.

33. The method according to claim 30, wherein the AGE inhibitor is a soluble RAGE trap.

34. The method according to claim 30, wherein the Treg cell activator comprises one or more single-chain chimeric polypeptides, the monoclonal antibody comprises one or more anti-tissue factor antibodies and / or anti-CD36 antibodies, and the AGE inhibitor comprises one or more soluble RAGE traps.

35. The aforementioned polychain chimeric polypeptide (a) A first chimeric polypeptide, (i) First target binding domain, (ii) Soluble tissue factor domain, and (iii) The first domain of a pair of affinity domains The first chimeric polypeptide, comprising, (b) A second chimeric polypeptide, (i) the second domain of the pair of affinity domains, and (ii) Second target binding domain The second chimeric polypeptide, which includes the following Includes, The first chimeric polypeptide and the second chimeric polypeptide associate through the binding of the first domain and the second domain of the pair of affinity domains. The method according to claim 9, 13, or 15.

36. The single-chain chimeric polypeptide is (i) First target binding domain, (ii) Soluble tissue factor domain, and (iii) Second target binding domain The method according to claim 27, 31, or 34, including the following:

37. The method according to any one of claims 1 to 37, wherein the age-related disorder is selected from the group consisting of Alzheimer's disease, aneurysm, cystic fibrosis, fibrosis in pancreatitis, glaucoma, hypertension, idiopathic pulmonary fibrosis, inflammatory bowel disease, intervertebral disc degeneration, macular degeneration, osteoarthritis, type 2 diabetes mellitus, lipodystrophy, lipodystrophy, atherosclerosis, cataract, COPD, idiopathic pulmonary fibrosis, renal transplant failure, hepatic fibrosis, bone loss, myocardial infarction, sarcopenia, wound healing, alopecia, cardiomyocyte hypertrophy, osteoarthritis, Parkinson's disease, age-related loss of lung tissue elasticity, macular degeneration, cachexia, glomerulosclerosis, cirrhosis, NAFLD, osteoporosis, amyotrophic lateral sclerosis, Huntington's disease, spinocerebellar ataxia, multiple sclerosis, neurodegeneration, stroke, cancer, dementia, vascular disease, infection susceptibility, chronic inflammation, and renal dysfunction.

38. The method according to any one of claims 1 to 37, wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease, lupus erythematosus, lupus nephritis, diabetic nephropathy, CNS injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Crohn's disease, multiple sclerosis, Guillain-Barré syndrome, psoriasis, Graves' disease, ulcerative colitis, and non-alcoholic steatohepatitis.

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